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  • richardmitnick 11:24 am on September 16, 2021 Permalink | Reply
    Tags: "New programmable gene editing proteins found outside of CRISPR systems", , , , , , IscB; IsrB; and TnpB are found in mobile genetic elements called transposons., IscBs and TnpBs appear to be predecessors of Cas9 and Cas12 CRISPR systems., , Programmable DNA modifying systems called OMEGAs (Obligate Mobile Element Guided Activity), Programmable enzymes-particularly those that use an RNA guide-can be rapidly adapted for different uses., RNA, The first hints that OMEGA proteins might be directed by RNA came from the genes for proteins called IscBs., Two other classes of small proteins known as IsrBs and TnpBs-one of the most abundant genes in bacteria-also use ωRNAs that act as guides to direct the cleavage of DNA.   

    From Massachusetts Institute of Technology (US) : “New programmable gene editing proteins found outside of CRISPR systems” 

    MIT News

    From Massachusetts Institute of Technology (US)

    September 15, 2021
    Jennifer Michalowski | McGovern Institute for Brain Research

    1
    Soumya Kannan is a 2021-22 Yang-Tan Center for Molecular Therapeutics Graduate Student Fellow in the lab of MIT Professor Feng Zhang and co-first author with Han Altae-Tran of a study reporting a new class of programmable DNA modifying systems known as OMEGAs. Credit: Caitlin Cunningham.

    Within the last decade, scientists have adapted CRISPR systems from microbes into gene editing technology, a precise and programmable system for modifying DNA. Now, scientists at MIT’s McGovern Institute for Brain Research and the Broad Institute of MIT and Harvard have discovered a new class of programmable DNA modifying systems called OMEGAs (Obligate Mobile Element Guided Activity), which may naturally be involved in shuffling small bits of DNA throughout bacterial genomes.

    These ancient DNA-cutting enzymes are guided to their targets by small pieces of RNA. While they originated in bacteria, they have now been engineered to work in human cells, suggesting they could be useful in the development of gene editing therapies, particularly as they are small (about 30 percent of the size of Cas9), making them easier to deliver to cells than bulkier enzymes. The discovery, reported Sept. 9 in the journal Science, provides evidence that natural RNA-guided enzymes are among the most abundant proteins on Earth, pointing toward a vast new area of biology that is poised to drive the next revolution in genome editing technology.

    The research was led by McGovern Investigator Feng Zhang, who is the James and Patricia Poitras Professor of Neuroscience at MIT, a Howard Hughes Medical Institute (US) investigator, and a Core Institute Member of the Broad Institute. Zhang’s team has been exploring natural diversity in search of new molecular systems that can be rationally programmed.

    “We are super excited about the discovery of these widespread programmable enzymes, which have been hiding under our noses all along,” says Zhang. “These results suggest the tantalizing possibility that there are many more programmable systems that await discovery and development as useful technologies.”

    Natural adaptation

    Programmable enzymes-particularly those that use an RNA guide-can be rapidly adapted for different uses. For example, CRISPR enzymes naturally use an RNA guide to target viral invaders, but biologists can direct Cas9 to any target by generating their own RNA guide. “It’s so easy to just change a guide sequence and set a new target,” says Soumya Kannan, MIT graduate student in biological engineering and co-first author of the paper. “So one of the broad questions that we’re interested in is trying to see if other natural systems use that same kind of mechanism.”

    The first hints that OMEGA proteins might be directed by RNA came from the genes for proteins called IscBs. The IscBs are not involved in CRISPR immunity and were not known to associate with RNA, but they looked like small, DNA-cutting enzymes. The team discovered that each IscB had a small RNA encoded nearby and it directed IscB enzymes to cut specific DNA sequences. They named these RNAs “ωRNAs.”

    The team’s experiments showed that two other classes of small proteins known as IsrBs and TnpBs-one of the most abundant genes in bacteria-also use ωRNAs that act as guides to direct the cleavage of DNA.

    IscB; IsrB; and TnpB are found in mobile genetic elements called transposons. Han Altae-Tran, MIT graduate student in biological engineering and co-first author on the paper, explains that each time these transposons move, they create a new guide RNA, allowing the enzyme they encode to cut somewhere else.

    It’s not clear how bacteria benefit from this genomic shuffling — or whether they do at all. Transposons are often thought of as selfish bits of DNA, concerned only with their own mobility and preservation, Kannan says. But if hosts can “co-opt” these systems and repurpose them, hosts may gain new abilities, as with CRISPR systems that confer adaptive immunity.

    IscBs and TnpBs appear to be predecessors of Cas9 and Cas12 CRISPR systems. The team suspects they, along with IsrB, likely gave rise to other RNA-guided enzymes, too — and they are eager to find them. They are curious about the range of functions that might be carried out in nature by RNA-guided enzymes, Kannan says, and suspect evolution likely already took advantage of OMEGA enzymes like IscBs and TnpBs to solve problems that biologists are keen to tackle.

    “A lot of the things that we have been thinking about may already exist naturally in some capacity,” says Altae-Tran. “Natural versions of these types of systems might be a good starting point to adapt for that particular task.”

    The team is also interested in tracing the evolution of RNA-guided systems further into the past. “Finding all these new systems sheds light on how RNA-guided systems have evolved, but we don’t know where RNA-guided activity itself comes from,” Altae-Tran says. Understanding those origins, he says, could pave the way to developing even more classes of programmable tools.

    This work was made possible with support from the Simons Center for the Social Brain at MIT, the National Institutes of Health and its Intramural Research Program, Howard Hughes Medical Institute, Open Philanthropy, G. Harold and Leila Y. Mathers Charitable Foundation, Edward Mallinckrodt, Jr. Foundation, Poitras Center for Psychiatric Disorders Research at MIT, Hock E. Tan and K. Lisa Yang Center for Autism Research at MIT, Yang-Tan Center for Molecular Therapeutics at MIT, Lisa Yang, Phillips family, R. Metcalfe, and J. and P. Poitras.

    See the full article here .


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    Please help promote STEM in your local schools.

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    MIT Campus

    Massachusetts Institute of Technology (US) is a private land-grant research university in Cambridge, Massachusetts. The institute has an urban campus that extends more than a mile (1.6 km) alongside the Charles River. The institute also encompasses a number of major off-campus facilities such as the MIT Lincoln Laboratory (US), the MIT Bates Research and Engineering Center (US), and the Haystack Observatory (US), as well as affiliated laboratories such as the Broad Institute of MIT and Harvard(US) and Whitehead Institute (US).

    Founded in 1861 in response to the increasing industrialization of the United States, Massachusetts Institute of Technology (US) adopted a European polytechnic university model and stressed laboratory instruction in applied science and engineering. It has since played a key role in the development of many aspects of modern science, engineering, mathematics, and technology, and is widely known for its innovation and academic strength. It is frequently regarded as one of the most prestigious universities in the world.

    As of December 2020, 97 Nobel laureates, 26 Turing Award winners, and 8 Fields Medalists have been affiliated with MIT as alumni, faculty members, or researchers. In addition, 58 National Medal of Science recipients, 29 National Medals of Technology and Innovation recipients, 50 MacArthur Fellows, 80 Marshall Scholars, 3 Mitchell Scholars, 22 Schwarzman Scholars, 41 astronauts, and 16 Chief Scientists of the U.S. Air Force have been affiliated with Massachusetts Institute of Technology (US) . The university also has a strong entrepreneurial culture and MIT alumni have founded or co-founded many notable companies. Massachusetts Institute of Technology (US) is a member of the Association of American Universities (AAU).

    Foundation and vision

    In 1859, a proposal was submitted to the Massachusetts General Court to use newly filled lands in Back Bay, Boston for a “Conservatory of Art and Science”, but the proposal failed. A charter for the incorporation of the Massachusetts Institute of Technology, proposed by William Barton Rogers, was signed by John Albion Andrew, the governor of Massachusetts, on April 10, 1861.

    Rogers, a professor from the University of Virginia (US), wanted to establish an institution to address rapid scientific and technological advances. He did not wish to found a professional school, but a combination with elements of both professional and liberal education, proposing that:

    “The true and only practicable object of a polytechnic school is, as I conceive, the teaching, not of the minute details and manipulations of the arts, which can be done only in the workshop, but the inculcation of those scientific principles which form the basis and explanation of them, and along with this, a full and methodical review of all their leading processes and operations in connection with physical laws.”

    The Rogers Plan reflected the German research university model, emphasizing an independent faculty engaged in research, as well as instruction oriented around seminars and laboratories.

    Early developments

    Two days after Massachusetts Institute of Technology (US) was chartered, the first battle of the Civil War broke out. After a long delay through the war years, MIT’s first classes were held in the Mercantile Building in Boston in 1865. The new institute was founded as part of the Morrill Land-Grant Colleges Act to fund institutions “to promote the liberal and practical education of the industrial classes” and was a land-grant school. In 1863 under the same act, the Commonwealth of Massachusetts founded the Massachusetts Agricultural College, which developed as the University of Massachusetts Amherst (US)). In 1866, the proceeds from land sales went toward new buildings in the Back Bay.

    Massachusetts Institute of Technology (US) was informally called “Boston Tech”. The institute adopted the European polytechnic university model and emphasized laboratory instruction from an early date. Despite chronic financial problems, the institute saw growth in the last two decades of the 19th century under President Francis Amasa Walker. Programs in electrical, chemical, marine, and sanitary engineering were introduced, new buildings were built, and the size of the student body increased to more than one thousand.

    The curriculum drifted to a vocational emphasis, with less focus on theoretical science. The fledgling school still suffered from chronic financial shortages which diverted the attention of the MIT leadership. During these “Boston Tech” years, Massachusetts Institute of Technology (US) faculty and alumni rebuffed Harvard University (US) president (and former MIT faculty) Charles W. Eliot’s repeated attempts to merge MIT with Harvard College’s Lawrence Scientific School. There would be at least six attempts to absorb MIT into Harvard. In its cramped Back Bay location, MIT could not afford to expand its overcrowded facilities, driving a desperate search for a new campus and funding. Eventually, the MIT Corporation approved a formal agreement to merge with Harvard, over the vehement objections of MIT faculty, students, and alumni. However, a 1917 decision by the Massachusetts Supreme Judicial Court effectively put an end to the merger scheme.

    In 1916, the Massachusetts Institute of Technology (US) administration and the MIT charter crossed the Charles River on the ceremonial barge Bucentaur built for the occasion, to signify MIT’s move to a spacious new campus largely consisting of filled land on a one-mile-long (1.6 km) tract along the Cambridge side of the Charles River. The neoclassical “New Technology” campus was designed by William W. Bosworth and had been funded largely by anonymous donations from a mysterious “Mr. Smith”, starting in 1912. In January 1920, the donor was revealed to be the industrialist George Eastman of Rochester, New York, who had invented methods of film production and processing, and founded Eastman Kodak. Between 1912 and 1920, Eastman donated $20 million ($236.6 million in 2015 dollars) in cash and Kodak stock to MIT.

    Curricular reforms

    In the 1930s, President Karl Taylor Compton and Vice-President (effectively Provost) Vannevar Bush emphasized the importance of pure sciences like physics and chemistry and reduced the vocational practice required in shops and drafting studios. The Compton reforms “renewed confidence in the ability of the Institute to develop leadership in science as well as in engineering”. Unlike Ivy League schools, Massachusetts Institute of Technology (US) catered more to middle-class families, and depended more on tuition than on endowments or grants for its funding. The school was elected to the Association of American Universities (US)in 1934.

    Still, as late as 1949, the Lewis Committee lamented in its report on the state of education at Massachusetts Institute of Technology (US) that “the Institute is widely conceived as basically a vocational school”, a “partly unjustified” perception the committee sought to change. The report comprehensively reviewed the undergraduate curriculum, recommended offering a broader education, and warned against letting engineering and government-sponsored research detract from the sciences and humanities. The School of Humanities, Arts, and Social Sciences and the MIT Sloan School of Management were formed in 1950 to compete with the powerful Schools of Science and Engineering. Previously marginalized faculties in the areas of economics, management, political science, and linguistics emerged into cohesive and assertive departments by attracting respected professors and launching competitive graduate programs. The School of Humanities, Arts, and Social Sciences continued to develop under the successive terms of the more humanistically oriented presidents Howard W. Johnson and Jerome Wiesner between 1966 and 1980.

    Massachusetts Institute of Technology (US)‘s involvement in military science surged during World War II. In 1941, Vannevar Bush was appointed head of the federal Office of Scientific Research and Development and directed funding to only a select group of universities, including MIT. Engineers and scientists from across the country gathered at Massachusetts Institute of Technology (US)’s Radiation Laboratory, established in 1940 to assist the British military in developing microwave radar. The work done there significantly affected both the war and subsequent research in the area. Other defense projects included gyroscope-based and other complex control systems for gunsight, bombsight, and inertial navigation under Charles Stark Draper’s Instrumentation Laboratory; the development of a digital computer for flight simulations under Project Whirlwind; and high-speed and high-altitude photography under Harold Edgerton. By the end of the war, Massachusetts Institute of Technology (US) became the nation’s largest wartime R&D contractor (attracting some criticism of Bush), employing nearly 4000 in the Radiation Laboratory alone and receiving in excess of $100 million ($1.2 billion in 2015 dollars) before 1946. Work on defense projects continued even after then. Post-war government-sponsored research at MIT included SAGE and guidance systems for ballistic missiles and Project Apollo.

    These activities affected Massachusetts Institute of Technology (US) profoundly. A 1949 report noted the lack of “any great slackening in the pace of life at the Institute” to match the return to peacetime, remembering the “academic tranquility of the prewar years”, though acknowledging the significant contributions of military research to the increased emphasis on graduate education and rapid growth of personnel and facilities. The faculty doubled and the graduate student body quintupled during the terms of Karl Taylor Compton, president of Massachusetts Institute of Technology (US) between 1930 and 1948; James Rhyne Killian, president from 1948 to 1957; and Julius Adams Stratton, chancellor from 1952 to 1957, whose institution-building strategies shaped the expanding university. By the 1950s, Massachusetts Institute of Technology (US) no longer simply benefited the industries with which it had worked for three decades, and it had developed closer working relationships with new patrons, philanthropic foundations and the federal government.

    In late 1960s and early 1970s, student and faculty activists protested against the Vietnam War and Massachusetts Institute of Technology (US)’s defense research. In this period Massachusetts Institute of Technology (US)’s various departments were researching helicopters, smart bombs and counterinsurgency techniques for the war in Vietnam as well as guidance systems for nuclear missiles. The Union of Concerned Scientists was founded on March 4, 1969 during a meeting of faculty members and students seeking to shift the emphasis on military research toward environmental and social problems. Massachusetts Institute of Technology (US) ultimately divested itself from the Instrumentation Laboratory and moved all classified research off-campus to the MIT (US) Lincoln Laboratory facility in 1973 in response to the protests. The student body, faculty, and administration remained comparatively unpolarized during what was a tumultuous time for many other universities. Johnson was seen to be highly successful in leading his institution to “greater strength and unity” after these times of turmoil. However six Massachusetts Institute of Technology (US) students were sentenced to prison terms at this time and some former student leaders, such as Michael Albert and George Katsiaficas, are still indignant about MIT’s role in military research and its suppression of these protests. (Richard Leacock’s film, November Actions, records some of these tumultuous events.)

    In the 1980s, there was more controversy at Massachusetts Institute of Technology (US) over its involvement in SDI (space weaponry) and CBW (chemical and biological warfare) research. More recently, Massachusetts Institute of Technology (US)’s research for the military has included work on robots, drones and ‘battle suits’.

    Recent history

    Massachusetts Institute of Technology (US) has kept pace with and helped to advance the digital age. In addition to developing the predecessors to modern computing and networking technologies, students, staff, and faculty members at Project MAC, the Artificial Intelligence Laboratory, and the Tech Model Railroad Club wrote some of the earliest interactive computer video games like Spacewar! and created much of modern hacker slang and culture. Several major computer-related organizations have originated at MIT since the 1980s: Richard Stallman’s GNU Project and the subsequent Free Software Foundation were founded in the mid-1980s at the AI Lab; the MIT Media Lab was founded in 1985 by Nicholas Negroponte and Jerome Wiesner to promote research into novel uses of computer technology; the World Wide Web Consortium standards organization was founded at the Laboratory for Computer Science in 1994 by Tim Berners-Lee; the MIT OpenCourseWare project has made course materials for over 2,000 Massachusetts Institute of Technology (US) classes available online free of charge since 2002; and the One Laptop per Child initiative to expand computer education and connectivity to children worldwide was launched in 2005.

    Massachusetts Institute of Technology (US) was named a sea-grant college in 1976 to support its programs in oceanography and marine sciences and was named a space-grant college in 1989 to support its aeronautics and astronautics programs. Despite diminishing government financial support over the past quarter century, MIT launched several successful development campaigns to significantly expand the campus: new dormitories and athletics buildings on west campus; the Tang Center for Management Education; several buildings in the northeast corner of campus supporting research into biology, brain and cognitive sciences, genomics, biotechnology, and cancer research; and a number of new “backlot” buildings on Vassar Street including the Stata Center. Construction on campus in the 2000s included expansions of the Media Lab, the Sloan School’s eastern campus, and graduate residences in the northwest. In 2006, President Hockfield launched the MIT Energy Research Council to investigate the interdisciplinary challenges posed by increasing global energy consumption.

    In 2001, inspired by the open source and open access movements, Massachusetts Institute of Technology (US) launched OpenCourseWare to make the lecture notes, problem sets, syllabi, exams, and lectures from the great majority of its courses available online for no charge, though without any formal accreditation for coursework completed. While the cost of supporting and hosting the project is high, OCW expanded in 2005 to include other universities as a part of the OpenCourseWare Consortium, which currently includes more than 250 academic institutions with content available in at least six languages. In 2011, Massachusetts Institute of Technology (US) announced it would offer formal certification (but not credits or degrees) to online participants completing coursework in its “MITx” program, for a modest fee. The “edX” online platform supporting MITx was initially developed in partnership with Harvard and its analogous “Harvardx” initiative. The courseware platform is open source, and other universities have already joined and added their own course content. In March 2009 the Massachusetts Institute of Technology (US) faculty adopted an open-access policy to make its scholarship publicly accessible online.

    Massachusetts Institute of Technology (US) has its own police force. Three days after the Boston Marathon bombing of April 2013, MIT Police patrol officer Sean Collier was fatally shot by the suspects Dzhokhar and Tamerlan Tsarnaev, setting off a violent manhunt that shut down the campus and much of the Boston metropolitan area for a day. One week later, Collier’s memorial service was attended by more than 10,000 people, in a ceremony hosted by the Massachusetts Institute of Technology (US) community with thousands of police officers from the New England region and Canada. On November 25, 2013, Massachusetts Institute of Technology (US) announced the creation of the Collier Medal, to be awarded annually to “an individual or group that embodies the character and qualities that Officer Collier exhibited as a member of the Massachusetts Institute of Technology (US) community and in all aspects of his life”. The announcement further stated that “Future recipients of the award will include those whose contributions exceed the boundaries of their profession, those who have contributed to building bridges across the community, and those who consistently and selflessly perform acts of kindness”.

    In September 2017, the school announced the creation of an artificial intelligence research lab called the MIT-IBM Watson AI Lab. IBM will spend $240 million over the next decade, and the lab will be staffed by MIT and IBM scientists. In October 2018 MIT announced that it would open a new Schwarzman College of Computing dedicated to the study of artificial intelligence, named after lead donor and The Blackstone Group CEO Stephen Schwarzman. The focus of the new college is to study not just AI, but interdisciplinary AI education, and how AI can be used in fields as diverse as history and biology. The cost of buildings and new faculty for the new college is expected to be $1 billion upon completion.

    The Caltech/MIT Advanced aLIGO (US) was designed and constructed by a team of scientists from California Institute of Technology (US), Massachusetts Institute of Technology (US), and industrial contractors, and funded by the National Science Foundation (US) .

    MIT/Caltech Advanced aLigo .

    It was designed to open the field of gravitational-wave astronomy through the detection of gravitational waves predicted by general relativity. Gravitational waves were detected for the first time by the LIGO detector in 2015. For contributions to the LIGO detector and the observation of gravitational waves, two Caltech physicists, Kip Thorne and Barry Barish, and Massachusetts Institute of Technology (US) physicist Rainer Weiss won the Nobel Prize in physics in 2017. Weiss, who is also an Massachusetts Institute of Technology (US) graduate, designed the laser interferometric technique, which served as the essential blueprint for the LIGO.

    The mission of Massachusetts Institute of Technology (US) is to advance knowledge and educate students in science, technology, and other areas of scholarship that will best serve the nation and the world in the twenty-first century. We seek to develop in each member of the Massachusetts Institute of Technology (US) community the ability and passion to work wisely, creatively, and effectively for the betterment of humankind.

     
  • richardmitnick 12:38 pm on May 20, 2018 Permalink | Reply
    Tags: , , , , RNA   

    From Astrobiology Magazine: “How primordial life on Earth might have replicated itself” 

    Astrobiology Magazine

    From Astrobiology Magazine

    May 20, 2018

    1
    Liquid brine containing replicating RNA molecules is concentrated in the cracks between ice crystals, as seen with an electron microscope. Credit: Philipp Holliger, MRC LMB

    Scientists have created a new type of genetic replication system which demonstrates how the first life on Earth – in the form of RNA – could have replicated itself. The scientists from the Medical Research Council (MRC) Laboratory of Molecular Biology say the new RNA utilises a system of genetic replication unlike any known to naturally occur on Earth today.

    A popular theory for the earliest stages of life on Earth is that it was founded on strands of RNA, a chemical cousin of DNA. Like DNA, RNA strands can carry genetic information using a code of four molecular letters (bases), but RNA can be more than a simple ‘string’ of information. Some RNA strands can also fold up into three-dimensional shapes that can form enzymes, called ribozymes, and carry out chemical reactions.

    If a ribozyme could replicate folded RNA, it might be able to copy itself and support a simple living system.

    Previously, scientists had developed ribozymes that could replicate straight strands of RNA, but if the RNA was folded it blocked the ribozyme from copying it. Since ribozymes themselves are folded RNAs, their own replication is blocked.

    Now, in a paper published today in the journal eLife, the scientists have resolved this paradox by engineering the first ribozyme that is able to replicate folded RNAs, including itself.

    Normally when copying RNA, an enzyme would add single bases (C, G, A or U) one at a time, but the new ribozyme uses three bases joined together, as a ‘triplet’ (e.g. GAU). These triplet building blocks enable the ribozyme to copy folded RNA, because the triplets bind to the RNA much more strongly and cause it to unravel – so the new ribozyme can copy its own folded RNA strands.

    The scientists say that the ‘primordial soup’ could have contained a mixture of bases in many lengths – one, two, three, four or more bases joined together – but they found that using strings of bases longer than a triplet made copying the RNA less accurate.

    Dr Philipp Holliger, from the MRC Laboratory of Molecular Biology and senior author on the paper, said: “We found a solution to the RNA replication paradox by re-thinking how to approach the problem – we stopped trying to mimic existing biology and designed a completely new synthetic strategy. It is exciting that our RNA can now synthesise itself.

    “These triplets of bases seem to represent a sweet spot, where we get a nice opening up of the folded RNA structures, but accuracy is still high. Notably, although triplets are not used in present-day biology for replication, protein synthesis by the ribosome – an ancient RNA machine thought to be a relic of early RNA-based life – proceeds using a triplet code.

    “However, this is only a first step because our ribozyme still needs a lot of help from us to do replication. We provided a pure system, so the next step is to integrate this into the more complex substrate mixtures mimicking the primordial soup – this likely was a diverse chemical environment also containing a range of simple peptides and lipids that could have interacted with the RNA.”

    The experiments were conducted in ice at -7°C, because the researchers had previously discovered that freezing concentrates the RNA molecules in a liquid brine in tiny gaps between the ice crystals. This also is beneficial for the RNA enzymes, which are more stable and function better at cold temperatures.

    Dr Holliger added: “This is completely new synthetic biology and there are many aspects of the system that we have not yet explored. We hope in future, it will also have some biotechnology applications, such as adding chemical modifications at specific positions to RNA polymers to study RNA epigenetics or augment the function of RNA.”

    Dr Nathan Richardson, Head of Molecular and Cellular Medicine at the MRC, said: “This is a really exciting example of blue skies research that has revealed important insights into how the very beginnings of life may have emerged from the ‘primordial soup’ some 3.7 billion years ago. Not only is this fascinating science, but understanding the minimal requirements for RNA replication and how these systems can be manipulated could offer exciting new strategies for treating human disease.”

    See the full article here .

    Please help promote STEM in your local schools.

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  • richardmitnick 2:27 pm on December 19, 2017 Permalink | Reply
    Tags: 20 “loading” molecules called aminoacyl-tRNA synthetases, , Charles Carter, , Kurt Gödel's Theorem and the Chemistry of Life, Peter Wills, protein-like molecules rather than RNA may have been the planet’s first self-replicators, , RNA   

    From Quanta: “The End of the RNA World Is Near, Biochemists Argue” 

    Quanta Magazine
    Quanta Magazine

    December 19, 2017
    Jordana Cepelewicz

    1
    A popular theory holds that life emerged from a rich chemical soup in which RNA was the original self-replicator. But a combination of peptides and RNA might have been more effective.
    Novikov Aleksey

    Four billion years ago, the first molecular precursors to life emerged, swirling about in Earth’s primordial soup of chemicals. Although the identity of these molecules remains a subject of fractious debate, scientists agree that the molecules would have had to perform two major functions: storing information and catalyzing chemical reactions. The modern cell assigns these responsibilities to its DNA and its proteins, respectively — but according to the narrative that dominates origin-of-life research and biology-textbook descriptions today, RNA was the first to play that role, paving the way for DNA and proteins to take over later.

    This hypothesis, proposed in the 1960s and dubbed the “RNA world” two decades later, is usually viewed as the most likely explanation for how life got its start. Alternative “worlds” abound, but they’re often seen as fallback theories, flights of fancy or whimsical thought experiments.

    That’s mainly because, theorizing aside, the RNA world is fortified by much more experimental evidence than any of its competitors have accumulated. Last month, Quanta Magazine reported on an alternative theory suggesting that protein-like molecules, rather than RNA, may have been the planet’s first self-replicators. But its findings were purely computational; the researchers have only just begun experiments to seek support for their claims.

    Now, a pair of researchers has put forth another theory — this time involving the coevolution of RNA and peptides — that they hope will shake the RNA world’s hold.

    Recent papers published in Biosystems and Molecular Biology and Evolution delineated why the RNA world hypothesis does not provide a sufficient foundation for the evolutionary events that followed. Instead, said Charles Carter, a structural biologist at the University of North Carolina, Chapel Hill, who co-authored the papers, the model represents “an expedient proposal.” “There’s no way that a single polymer could carry out all of the necessary processes we now characterize as part of life,” he added.

    And that single polymer certainly couldn’t be RNA, according to his team’s studies. The main objection to the molecule concerns catalysis: Some research has shown that for life to take hold, the mystery polymer would have had to coordinate the rates of chemical reactions that could differ in speed by as much as 20 orders of magnitude. Even if RNA could somehow do this in the prebiotic world, its capabilities as a catalyst would have been adapted to the searing temperatures — around 100 degrees Celsius — that abounded on early Earth. Once the planet started to cool, Carter claims, RNA wouldn’t have been able to evolve and keep up the work of synchronization. Before long, the symphony of chemical reactions would have fallen into disarray.

    Perhaps most importantly, an RNA-only world could not explain the emergence of the genetic code, which nearly all living organisms today use to translate genetic information into proteins. The code takes each of the 64 possible three-nucleotide RNA sequences and maps them to one of the 20 amino acids used to build proteins. Finding a set of rules robust enough to do that would take far too long with RNA alone, said Peter Wills, Carter’s co-author at the University of Auckland in New Zealand — if the RNA world could even reach that point, which he deemed highly unlikely. In Wills’ view, RNA might have been able to catalyze its own formation, making it “chemically reflexive,” but it lacked what he called “computational reflexivity.”

    “A system that uses information the way organisms use genetic information — to synthesize their own components — must contain reflexive information,” Wills said. He defined reflexive information as information that, “when decoded by the system, makes the components that perform exactly that particular decoding.” The RNA of the RNA world hypothesis, he added, is just chemistry because it has no means of controlling its chemistry. “The RNA world doesn’t tell you anything about genetics,” he said.

    Nature had to find a different route, a better shortcut to the genetic code. Carter and Wills think they’ve uncovered that shortcut. It depends on a tight feedback loop — one that would not have developed from RNA alone but instead from a peptide-RNA complex.

    Bringing Peptides Into the Mix

    Carter found hints of that complex in the mid-1970s, when he learned in graduate school that certain structures seen in most proteins are “right-handed.” That is, the atoms in the structures could have two equivalent mirror-image arrangements, but the structures all use just one. Most of the nucleic acids and sugars that make up DNA and RNA are right-handed, too. Carter began to think of RNA and polypeptides as complementary structures, and he modeled a complex in which “they were made for each other, like a hand in a glove.”

    This implied an elementary kind of coding, a basis for the exchange of information between the RNA and the polypeptide. He was on his way to sketching what that might have looked like, working backward from the far more sophisticated modern genetic code. When the RNA world, coined in 1986, rose to prominence, Carter admitted, “I was pretty ticked off.” He felt that his peptide-RNA world, proposed a decade earlier, had been totally ignored.

    Since then, he, Wills and others have collaborated on a theory that circles back to that research. Their main goal was to figure out the very simple genetic code that preceded today’s more specific and complicated one. And so they turned not just to computation but also to genetics.

    At the center of their theory are 20 “loading” molecules called aminoacyl-tRNA synthetases. These catalytic enzymes allow RNA to bond with specific amino acids in keeping with the rules of the genetic code. “In a sense, the genetic code is ‘written’ in the specificity of the active sites” of those enzymes, said Jannie Hofmeyr, a biochemist at Stellenbosch University in South Africa, who was not involved in the study.

    3
    Lucy Reading-Ikkanda/Quanta Magazine

    Previous research showed that the 20 enzymes could be divided evenly into two groups of 10 based on their structure and sequence. These two enzyme classes, it turned out, have certain sequences that code for mutually exclusive amino acids — meaning that the enzymes had to have arisen from complementary strands of the same ancient gene. Carter, Wills and their colleagues found that in this scenario, RNA coded for peptides using a set of just two rules (or, in other words, using just two types of amino acids). The resulting peptide products ended up enforcing the very rules that governed the translation process, thus forming the tight feedback loop the researchers knew would be the linchpin of the theory.

    Gödel’s Theorem and the Chemistry of Life

    Carter sees strong parallels between this kind of loop and the mathematical one described by the philosopher and mathematician Kurt Gödel, whose “incompleteness” theorem states that in any logical system that can represent itself, statements will inevitably arise that cannot be shown to be true or false within that system. “I believe that the analogy to Gödel’s theorem furnishes a quite strong argument for inevitability,” Carter said.

    In their recent papers, Carter and Wills show that their peptide-RNA world solves gaps in origin-of-life history that RNA alone can’t explain. “They provide solid theoretical and experimental evidence that peptides and RNA were jointly involved in the origin of the genetic code right from the start,” Hofmeyr said, “and that metabolism, construction through transcription and translation, and replication must have coevolved.”

    Of course, the Carter-Wills model begins with the genetic code, the existence of which presupposes complex chemical reactions involving molecules like transfer RNA and the loading enzymes. The researchers claim that the events leading up to their proposed scenario involved RNA and peptides interacting (in the complex that Carter described in the 1970s, for example). Yet that suggestion still leaves many open questions about how that chemistry began and what it looked like.

    To answer these questions, theories abound that move far beyond the RNA world. In fact, some scientists take an approach precisely opposite to that of Carter and Wills: They think instead that the earliest stages of life did not need to begin with anything resembling the kind of chemistry seen today. Doron Lancet, a genomics researcher at the Weizmann Institute of Science in Israel, posits an alternative theory that rests on assemblies of lipids that catalyze the entrance and exit of various molecules. Information is carried not by genetic sequences, but rather by the lipid composition of such assemblies.

    Just like the model proposed by Carter and Wills, Lancet’s ideas involve not one type of molecule but a huge variety of them. “More and more bits of evidence are accumulating,” Lancet said, “that can make an alternative hypothesis be right.” The jury is still out on what actually transpired at life’s origins, but the tide seems to be turning away from a story dedicated solely to RNA.

    “We should put only a few of our eggs in the RNA world basket,” Hofmeyr said.

    See the full article here .

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    Formerly known as Simons Science News, Quanta Magazine is an editorially independent online publication launched by the Simons Foundation to enhance public understanding of science. Why Quanta? Albert Einstein called photons “quanta of light.” Our goal is to “illuminate science.” At Quanta Magazine, scientific accuracy is every bit as important as telling a good story. All of our articles are meticulously researched, reported, edited, copy-edited and fact-checked.

     
    • stewarthoughblog 2:02 am on December 20, 2017 Permalink | Reply

      It is about time that RNA nonsense comes to an end. There is admittedly considerable scientific knowledge that has and can still be gained by studying RNA macromolecules, but the desperation of naturalists proposing it either as the source of first life is intellectually insulting. RNA’s complexity may be assemblable in intelligent design highly managed labs environments, but ridiculous to consider possible in any geochemically relevant primordial environment. RNA is easily mutated, highly reactive, and only an intermediate macromolecule restricted by the protein catch-22.

      Frustratingly, the desperation continues with propositions of lipid collective assembly, but at least science appears to coming to its senses about RNA.
      .

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  • richardmitnick 10:24 am on July 31, 2017 Permalink | Reply
    Tags: , , , , , RNA   

    From HMS: “Making the Makers” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    July 21, 2017
    KEVIN JIANG

    1
    Rendering of the structure of the eukaryotic ribosome. Ribosomal RNA is represented as a grey tube. Proteins are shown in blue, orange and red. Image: Wikimedia Commons.

    Every living cell, whether a single bacterium or a human neuron, is a biological system as dynamic and complex as any city. Contained within cells are walls, highways, power plants, libraries, recycling centers and much more, all working together in unison to ensure the continuation of life.

    The vast majority of these myriad structures are made of and made by proteins, and those proteins are made by one uniquely important molecular machine, the ribosome.

    In a new study published in Nature on July 20, a team led by Johan Paulsson, professor of systems biology at Harvard Medical School, now reveals the likely origin of several previously mysterious characteristics of the ribosome.

    They mathematically demonstrated that ribosomes are precisely structured to produce additional ribosomes as quickly as possible, in order to support efficient cell growth and division.

    The study’s theoretical predictions accurately reflect observed large-scale features—revealing why are ribosomes made of an unusually large number of small, uniformly sized proteins and a few strands of RNA that vary greatly in size—and provide perspective on the evolution of an exceptional molecular machine.

    “The ribosome is one of the most important molecular complexes in all of life, and it’s been studied across scientific disciplines for decades,” Paulsson said.

    “I was always puzzled by the fact that it seemed like we could explain its finer details, but ribosomes have these bizarre features that have not often been addressed, or if so in an unsatisfying way,” he said.

    Mysterious features

    2
    Atomic structure of a ribosome subunit from an archaea, a type of microorganism. Proteins are shown in blue and RNA chains in orange and yellow. Animation: Wikimedia Commons/David Goodsell.

    Although scientists have unlocked how ribosomes turn genetic information into proteins at atomic resolution, revealing a molecular machine finely tuned for accuracy, speed and control, it hasn’t been clear what advantages lay in its several large-scale features.

    Ribosomes are composed of a puzzlingly large number of different structural proteins—anywhere from 55 to 80, depending on organism type. These proteins are not just more numerous than expected, they are unusually short and uniform in length. Ribosomes are also composed of two to three strands of RNA, which account for up to 70 percent of the total mass of the ribosome.

    “Without understanding why collective features exist, it is a bit like looking at a forest and understanding how chloroplasts and photosynthesis work, and not being able to explain why there are trees instead of grass,” Paulsson said.

    So Paulsson and his collaborators Shlomi Reuveni, an HMS postdoctoral fellow, and Måns Ehrenberg of Uppsala University in Sweden, decided to look at the ribosome in a different light.

    “Our breakthrough came by zooming out from the atomic and looking at the ribosome from a different perspective,” Reuveni said. “We didn’t think of the ribosome as a machine that produces proteins, but rather as the product of the protein production process.”

    Forest for the trees

    For a cell to divide, it must have two full sets of ribosomes to make all the proteins that the daughter cells will need. The speed at which ribosomes can make themselves, therefore, places a hard limit on how fast cell division occurs. Paulsson and his colleagues devised theoretical mathematical models for what the ribosome’s features should look like if speed was the primary selective pressure that drove its evolution.

    The team calculated that distributing the task of making a new ribosome among many ribosomes—each making a small piece of the final product—can increase the rate of production by as much as 30 percent, since each new ribosome helps make more ribosomes as soon as they are created, accelerating the process.

    This represents an enormous advantage for cells that need to divide quickly, such as bacteria. However, the protein production process takes time to initiate, and this overhead cost limits the number of proteins that a ribosome can be made of, according to the math.

    The team’s models predicted that, for maximum self-production efficacy, a ribosome should be made of between 40 and 80 proteins. Each of these proteins should be around three times smaller than an average cellular protein, and they should all be roughly similar in size.

    It turns out that the researchers’ theory, developed completely independently of the laboratory, accurately reflects the observed protein composition of the ribosome.

    “An analogy for our findings would be to think of ribosomes not as a group of carpenters who merely build a lot of houses, but as carpenters who also build other carpenters,” Paulsson said. “There is then an incentive to divide the job into many small pieces that can be done in parallel to more quickly assemble another complete carpenter to help in the process.”

    Theory and reality

    Paulsson and his colleagues also examined ribosomal RNA, which act as a structural component and carry out the ribosome’s enzymatic activity of linking amino acids together into proteins.

    Their analysis showed that, the more RNA a ribosome is made of, the more rapidly it can be produced. This is because cells can make RNA orders of magnitude faster than protein. Thus, while RNA enzymes are thought to be less efficient than protein enzymes, ribosomes have enormous pressure to use as much RNA as possible to maximize the rate at which more ribosomes can be made.

    “Any place the ribosome can get away with using RNA, it should use it because self-production speed can essentially be doubled or tripled,” Paulsson said. “Even if RNA were inferior compared to protein for enzymatic function, there is still a great advantage to using RNA if a cell is trying to produce ribosomes as fast as possible.”

    This observation was predicted to hold primarily for self-producing ribosomes, according to the team. Most other structures in the cell do not self-produce and can sacrifice production speed for the stability and efficacy provided by using protein instead of RNA.

    Taken together, the team’s theory accurately predicts large-scale features of the ribosome that are seen across domains of life. It explains why the fastest growing organisms, such as bacteria, have the shortest ribosomal proteins and the greatest amounts of RNA. At the opposite end of the spectrum are mitochondria—the power plants of eukaryotic cells, which are thought to have once been bacteria that entered a permanent symbiotic state. Mitochondria have their own ribosomes that do not produce themselves. Without this pressure, mitochondrial ribosomes are indeed made of larger proteins and far less RNA than cellular ribosomes.

    “When we started this project, we didn’t have a long list of features that we tried to explain through theory,” Reuveni said. “We started with the theory, and certain features emerged. When we looked at data to compare with what our math predicted, we found in most cases that they matched what is seen in nature.”

    Rather than being mere relics of an evolutionary past, the unusual features of ribosomes thus seem to reflect an additional layer of functional optimization acting on collective properties of its parts, the team writes.

    “While this study is basic science, we are addressing something that is shared by all life,” Paulsson said. “It is important that we understand where the constraints on structure and function come from, because like much of basic science, it is unpredictable what the consequences of new knowledge can unlock in the future.”

    See the full article here .

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    Established in 1782, Harvard Medical School began with a handful of students and a faculty of three. The first classes were held in Harvard Hall in Cambridge, long before the school’s iconic quadrangle was built in Boston. With each passing decade, the school’s faculty and trainees amassed knowledge and influence, shaping medicine in the United States and beyond. Some community members—and their accomplishments—have assumed the status of legend. We invite you to access the following resources to explore Harvard Medical School’s rich history.

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    Harvard is the oldest institution of higher education in the United States, established in 1636 by vote of the Great and General Court of the Massachusetts Bay Colony. It was named after the College’s first benefactor, the young minister John Harvard of Charlestown, who upon his death in 1638 left his library and half his estate to the institution. A statue of John Harvard stands today in front of University Hall in Harvard Yard, and is perhaps the University’s best known landmark.

    Harvard University has 12 degree-granting Schools in addition to the Radcliffe Institute for Advanced Study. The University has grown from nine students with a single master to an enrollment of more than 20,000 degree candidates including undergraduate, graduate, and professional students. There are more than 360,000 living alumni in the U.S. and over 190 other countries.

     
  • richardmitnick 12:51 pm on April 4, 2017 Permalink | Reply
    Tags: , RNA, Study reveals the multitasking secrets of an RNA-binding protein   

    From Princeton: “Study reveals the multitasking secrets of an RNA-binding protein” 

    Princeton University
    Princeton University

    April 4, 2017
    Staff, Department of Molecular Biology

    1
    Two views of one of Glo’s RNA-binding domains highlight the amino acids required for binding G-tract RNA (left) and U-A stem structures (right). Courtesy of Cell Reports.

    Researchers from Princeton University and the National Institute of Environmental Health Sciences have discovered how a fruit fly protein binds and regulates two different types of RNA target sequence. The study, which will be published April 4 in the journal Cell Reports, may help explain how various RNA-binding proteins, many of which are implicated in cancer and neurodegenerative disease, perform so many different functions in the cell.

    There are hundreds of RNA-binding proteins in the human genome that together regulate the processing, turnover and localization of the many thousands of RNA molecules expressed in cells. These proteins also control the translation of RNA into proteins. RNA-binding proteins are crucial for maintaining normal cellular function, and defects in this family of proteins can lead to disease. For example, RNA-binding proteins are overexpressed in many human cancers, and mutations in some of these proteins have been linked to neurological and neurodegenerative disorders such as amyotrophic lateral sclerosis. “Understanding the fundamental properties of this class of proteins is very relevant,” said Elizabeth Gavis, the Damon B. Pfeiffer Professor in the Life Sciences and a professor of molecular biology.

    Gavis and colleagues are particularly interested in a protein called Glorund (Glo), a type of RNA-binding protein that performs several functions in fruit fly development. This protein was originally identified due to its ability to repress the translation of an RNA molecule called nanos to protein in fly eggs. By binding to a stem structure formed by uracil and adenine nucleotides in the nanos RNA, Glo prevents the production of Nanos protein at the front of the embryo, a step that enables the fly’s head to form properly.

    Like many other RNA-binding proteins, however, Glo is multifunctional. It regulates several other steps in fly development, apparently by binding to RNAs other than nanos. The mammalian counterparts of Glo, known as heterogeneous nuclear ribonucleoprotein (hnRNP) F/H proteins, bind to RNAs containing stretches of guanine nucleotides known as G-tracts, and, rather than repressing translation, mammalian hnRNP F/H proteins regulate processes such as RNA splicing, in which RNAs are rearranged to produce alternative versions of the proteins they encode.

    To understand how Glo might bind to diverse RNAs and regulate them in different ways, Gavis and graduate student Joel Tamayo collaborated with Traci Tanaka Hall and Takamasa Teramoto from the National Institute of Environmental Health Sciences to generate X-ray crystallographic structures of Glo’s three RNA-binding domains. As expected, the three domains were almost identical to the corresponding domains of mammalian hnRNP F/H proteins. They retained, for example, the amino acid residues that bind to G-tract RNA, and the researchers confirmed that, like their mammalian counterparts, each RNA-binding domain of Glo can bind to this type of RNA sequence.

    However, the researchers also saw something new. “When we looked at the structures, we realized that there were also some basic amino acids that projected from a different part of the RNA-binding domains that could be involved in contacting RNA,” Gavis explained.

    The researchers found that these basic amino acids mediate binding to uracil-adenine (U-A) stem structures like the one found in nanos RNA. Each of Glo’s RNA-binding domains therefore contains two distinct binding surfaces that interact with different types of RNA target sequence. “While there have been examples previously of RNA-binding proteins that carry more than one binding domain, each with a different specificity, this represents the first example of a single domain harboring two different specificities,” said Howard Lipshitz, a professor of molecular genetics at the University of Toronto who was not involved in the study.

    To investigate which of Glo’s two RNA-binding modes was required for its different functions in flies, Gavis and colleagues generated insects carrying mutant versions of the RNA-binding protein. Glo’s ability to repress nanos translation during egg development required both of the protein’s RNA-binding modes. The researchers discovered that, as well as binding the U-A stem in the nanos RNA, Glo also recognized a nearby G-tract sequence. But Glo’s ability to regulate other RNAs at different developmental stages only depended on the protein’s capacity to bind G-tracts.

    “We think that the binding mode may correlate with Glo’s activity towards a particular RNA,” said Gavis. “If it binds to a G-tract, Glo might promote RNA splicing. If it simultaneously binds to both a G-tract and a U-A stem, Glo acts as a translational repressor.”

    The RNA-binding domains of mammalian hnRNP F/H proteins probably have a similar ability to bind two different types of RNA, allowing them to regulate diverse target RNAs within the cell. “This paper represents an exciting advance in a field that has become increasingly important with the discovery that defects in RNA-binding proteins contribute to human diseases such as metabolic disorders, cancer and neurodegeneration,” Lipshitz said. “Since these proteins are evolutionarily conserved from fruit flies to humans, experiments of this type tell us a lot about how their human versions normally work or can go wrong.”

    The research was supported in part by a National Science Foundation Graduate Research Fellowship (DGE 1148900), a Japan Society for the Promotion of Science fellowship, the National Institutes of Health (R01 GM061107) and the Intramural Research Program of the National Institute of Environmental Health Sciences. The Advanced Photon Source used for this study is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38.

    The study, “The Drosophila hnRNP F/H Homolog Glorund Uses Two Distinct RNA-binding Modes to Diversify Target Recognition,” by Joel Tamayo, Takamasa Teramoto, Seema Chatterjee, Traci Tanaka Hall, and Elizabeth Gavis, was published in the journal Cell Reports on April 4, 2017. http://dx.doi.org/10.1016/j.celrep.2017.03.022

    See the full article here .

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    About Princeton: Overview

    Princeton University is a vibrant community of scholarship and learning that stands in the nation’s service and in the service of all nations. Chartered in 1746, Princeton is the fourth-oldest college in the United States. Princeton is an independent, coeducational, nondenominational institution that provides undergraduate and graduate instruction in the humanities, social sciences, natural sciences and engineering.

    As a world-renowned research university, Princeton seeks to achieve the highest levels of distinction in the discovery and transmission of knowledge and understanding. At the same time, Princeton is distinctive among research universities in its commitment to undergraduate teaching.

    Today, more than 1,100 faculty members instruct approximately 5,200 undergraduate students and 2,600 graduate students. The University’s generous financial aid program ensures that talented students from all economic backgrounds can afford a Princeton education.

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  • richardmitnick 9:44 am on March 29, 2017 Permalink | Reply
    Tags: , , , , RNA,   

    From MIT: “Progress toward a Zika vaccine” A lot of Zika News Lately 

    MIT News

    MIT Widget

    MIT News

    March 29, 2017
    Anne Trafton

    1
    MIT researchers have devised a new vaccine candidate for the Zika virus. “It functions almost like a synthetic virus, except it’s not pathogenic and it doesn’t spread,” says postdoc Omar Khan. Image: Jose-Luis Olivares/MIT

    Researchers program RNA nanoparticles that could protect against the virus.

    Using a new strategy that can rapidly generate customized RNA vaccines, MIT researchers have devised a new vaccine candidate for the Zika virus.

    The vaccine consists of strands of genetic material known as messenger RNA, which are packaged into a nanoparticle that delivers the RNA into cells. Once inside cells, the RNA is translated into proteins that provoke an immune response from the host, but the RNA does not integrate itself into the host genome, making it potentially safer than a DNA vaccine or vaccinating with the virus itself.

    “It functions almost like a synthetic virus, except it’s not pathogenic and it doesn’t spread,” says Omar Khan, a postdoc at MIT’s Koch Institute for Integrative Cancer Research and an author of the new study. “We can control how long it’s expressed, and it’s RNA so it will never integrate into the host genome.”

    This research also yielded a new benchmark for evaluating the effectiveness of other Zika vaccine candidates, which could help others who are working toward the same goal.

    Jasdave Chahal, a postdoc at MIT’s Whitehead Institute for Biomedical Research, is the first author of the paper, which appears in Scientific Reports. The paper’s senior author is Hidde Ploegh, a former MIT biology professor and Whitehead Institute member who is now a senior investigator in the Program in Cellular and Molecular Medicine at Boston Children’s Hospital.

    Other authors of the paper are Tao Fang and Andrew Woodham, both former Whitehead Institute postdocs in the Ploegh lab; Jingjing Ling, an MIT graduate student; and Daniel Anderson, an associate professor in MIT’s Department of Chemical Engineering and a member of the Koch Institute and MIT’s Institute for Medical Engineering and Science (IMES).

    Programmable vaccines

    The MIT team first reported its new approach to programmable RNA vaccines last year. RNA vaccines are appealing because they induce host cells to produce many copies of the proteins encoded by the RNA. This provokes a stronger immune reaction than if the proteins were administered on their own. However, finding a safe and effective way to deliver these vaccines has proven challenging.

    The researchers devised an approach in which they package RNA sequences into a nanoparticle made from a branched molecule that is based on fractal-patterned dendrimers. This modified-dendrimer-RNA structure can be induced to fold over itself many times, producing a spherical particle about 150 nanometers in diameter. This is similar in size to a typical virus, allowing the particles to enter cells through the same viral entry mechanisms. In their 2016 paper, the researchers used this nanoparticle approach to generate experimental vaccines for Ebola, H1N1 influenza, and the parasite Toxoplasma gondii.

    In the new study, the researchers tackled Zika virus, which emerged as an epidemic centered in Brazil in 2015 and has since spread around the world, causing serious birth defects in babies born to infected mothers. Since the MIT method does not require working with the virus itself, the researchers believe they might be able to explore potential vaccines more rapidly than scientists pursuing a more traditional approach.

    Instead of using viral proteins or weakened forms of the virus as vaccines, which are the most common strategies, the researchers simply programmed their RNA nanoparticles with the sequences that encode Zika virus proteins. Once injected into the body, these molecules replicate themselves inside cells and instruct cells to produce the viral proteins.

    The entire process of designing, producing, and testing the vaccine in mice took less time than it took for the researchers to obtain permission to work with samples of the Zika virus, which they eventually did get.

    “That’s the beauty of it,” Chahal says. “Once we decided to do it, in two weeks we were ready to vaccinate mice. Access to virus itself was not necessary.”

    Measuring response

    When developing a vaccine, researchers usually aim to generate a response from both arms of the immune system — the adaptive arm, mediated by T cells and antibodies, and the innate arm, which is necessary to amplify the adaptive response. To measure whether an experimental vaccine has generated a strong T cell response, researchers can remove T cells from the body and then measure how they respond to fragments of the viral protein.

    Until now, researchers working on Zika vaccines have had to buy libraries of different protein fragments and then test T cells on them, which is an expensive and time-consuming process. Because the MIT researchers could generate so many T cells from their vaccinated mice, they were able to rapidly screen them against this library. They identified a sequence of eight amino acids that the activated T cells in the mouse respond to. Now that this sequence, also called an epitope, is known, other researchers can use it to test their own experimental Zika vaccines in the appropriate mouse models.

    “We can synthetically make these vaccines that are almost like infecting someone with the actual virus, and then generate an immune response and use the data from that response to help other people predict if their vaccines would work, if they bind to the same epitopes,” Khan says. The researchers hope to eventually move their Zika vaccine into tests in humans.

    “The identification and characterization of CD8 T cell epitopes in mice immunized with a Zika RNA vaccine is a very useful reference for all those working in the field of Zika vaccine development,” says Katja Fink, a principal investigator at the A*STAR Singapore Immunology Network. “RNA vaccines have received much attention in the last few years, and while the big breakthrough in humans has not been achieved yet, the technology holds promise to become a flexible platform that could provide rapid solutions for emerging viruses.”

    Fink, who was not involved in the research, added that the “initial data are promising but the Zika RNA vaccine approach described needs further testing to prove efficacy.”

    Another major area of focus for the researchers is cancer vaccines. Many scientists are working on vaccines that could program a patient’s immune system to attack tumor cells, but in order to do that, they need to know what the vaccine should target. The new MIT strategy could allow scientists to quickly generate personalized RNA vaccines based on the genetic sequence of an individual patient’s tumor cells.

    The research was funded by the National Institutes of Health, a Fujifilm/MediVector grant, the Lustgarten Foundation, a Koch Institute and Dana-Farber/Harvard Center Center Bridge Project award, the Department of Defense Office of Congressionally Directed Medical Research’s Joint Warfighter Medical Research Program, and the Cancer Center Support Grant from the National Cancer Institute.

    See the full article here .

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    The mission of MIT is to advance knowledge and educate students in science, technology, and other areas of scholarship that will best serve the nation and the world in the twenty-first century. We seek to develop in each member of the MIT community the ability and passion to work wisely, creatively, and effectively for the betterment of humankind.

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  • richardmitnick 9:08 am on August 3, 2016 Permalink | Reply
    Tags: , , RNA,   

    From UCLA: “Scientists develop new way to measure important chemical modification on RNA” 

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    UCLA

    August 02, 2016
    Mirabai Vogt-James

    A team of scientists including researchers from UCLA has developed an RNA sequencing technique that provides detailed information about a chemical modification that occurs on RNA and plays an important role in pluripotent stem cells’ ability to turn into other types of cells. The method could advance scientists’ use of stem cells in regenerative medicine, since pluripotent stem cells can turn into any cell type in the body.

    The study, published in the journal Nature Methods, outlines the new sequencing technique, which measures the percentage of RNA that is methylated, or chemically modified, for each gene in the genome.

    RNA serves an important purpose inside cells; it carries genetic messages from DNA. These messages direct cells to make the proteins that play many critical roles in the body, but errors in how those messages are produced or regulated can lead to a variety of diseases, including cancer and neurological disorders.

    Until recently, little was known about how RNA activity is regulated by methylation of the RNA molecules. The new study looks at a specific type of RNA methylation known as m6A or N6-methyladenosine, which is a chemical modification that has a variety of functions, such as controlling how long the RNA will live in the cell and how much protein it will produce. The m6A modification is the most abundant type of RNA methylation on protein-producing RNAs.

    The data analyses were led by co-senior author Yi Xing, a professor of microbiology, immunology and molecular genetics in the UCLA College and a member of the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research at UCLA. Dr. Cosmas Giallourakis, co-senior author and an assistant professor of medicine at Harvard Medical School and Massachusetts General Hospital, led the development of the new sequencing technique. First authors were Benoit Molinie at Harvard Medical School and Jinkai Wang, a UCLA postdoctoral fellow.

    “Previously, we were only able to determine the location of m6A on the RNA, but not the amount,” said Xing, who is also a member of the UCLA Institute for Quantitative and Computational Biosciences and director of UCLA’s bioinformatics doctoral program.

    The ability to determine the percentage of m6A on RNA gives researchers information that could potentially help detect disease, Xing said, since m6A levels on RNA may be different in diseased cells than in healthy cells. Researchers can also use information about m6A levels to gain insights into a pluripotent stem cell’s ability to turn into other types of cells.

    Pluripotent stem cells have two unique abilities. They can turn into any specialized cell in the body, such as skin, bone, blood or brain cells; this process is called “differentiation.” They can also create copies of themselves. These abilities hold great promise for advances in regenerative medicine. But scientists are particularly interested in understanding how to control the process through which pluripotent stem cells differentiate into specialized cell types that are safe and fully capable of regenerating aging or diseased tissue. Another challenge is maintaining pluripotent stem cells in the lab, since they have a tendency to spontaneously differentiate, at which point scientists lose the ability to control the cell’s fate.

    Previous research by a team led by Xing and Giallourakis showed that blocking m6A prevents pluripotent stem cells from differentiating into specialized cell types, while allowing them to retain their critical pluripotent flexibility.

    The new sequencing technique, called m6A-LAIC-seq, is a novel method that scientists can use to obtain valuable data about RNA methylation using specialized machines that produce hundreds of millions of RNA sequences and provide insights into the molecular signature of a cell.

    “We are very excited about the promising data and the new tool that is now available to study m6A in a wide range of cell types including pluripotent stem cells,” Xing said. “We anticipate that our research will improve the understanding and use of pluripotent stem cells in regenerative medicine.”

    The study was supported by grants from Massachusetts General Hospital, the National Institutes of Health (GM088342, DK090122, ES002109 and ES024615) and the National Science Foundation (CHE-1308839); an Alfred Sloan Research Fellowship; the National Research Foundation of Singapore through the Singapore–MIT Alliance for Research and Technology; and by the UCLA Broad Stem Cell Research Center–Rose Hills Foundation Research Award.

    See the full article here .

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    For nearly 100 years, UCLA has been a pioneer, persevering through impossibility, turning the futile into the attainable.

    We doubt the critics, reject the status quo and see opportunity in dissatisfaction. Our campus, faculty and students are driven by optimism. It is not naïve; it is essential. And it has fueled every accomplishment, allowing us to redefine what’s possible, time after time.

    This can-do perspective has brought us 12 Nobel Prizes, 12 Rhodes Scholarships, more NCAA titles than any university and more Olympic medals than most nations. Our faculty and alumni helped create the Internet and pioneered reverse osmosis. And more than 100 companies have been created based on technology developed at UCLA.

     
  • richardmitnick 2:56 pm on March 29, 2016 Permalink | Reply
    Tags: , , , Life's Building Blocks Form In Replicated Deep Sea Vents, RNA,   

    From SPACE.com: “Life’s Building Blocks Form In Replicated Deep Sea Vents” 

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    SPACE.com

    March 28, 2016
    Charles Q. Choi

    1
    Alkaline hydrothermal vents may have played a role in the origin of life.
    Credit: NOAA

    Chimney-like mineral structures on the seafloor could have helped create the RNA molecules that gave rise to life on Earth and hold promise to the emergence of life on distant planets.

    Scientists think Earth was born roughly 4.54 billion years ago. Life on Earth may be nearly that old with recent findings suggesting that life might have emerged only about 440 million years after the planet formed.

    However, it remains a mystery how life might have first arisen. The main building blocks of life now are DNA, which can store genetic data, and proteins, which include enzymes that can direct chemical reactions. However, DNA requires proteins in order to form, and proteins need DNA to form, raising the chicken-and-egg question of how protein and DNA could have formed without each other.

    To resolve this conundrum, scientists have suggested that life may have first primarily depended on compounds known as RNA. These molecules can store genetic data like DNA, serve as enzymes like proteins, and help create both DNA and proteins. Later DNA and proteins replaced this “RNA world” because they are more efficient at their respective functions, although RNA still exists and serves vital roles in biology.

    However, it remains uncertain how RNA might have arisen from simpler precursors in the primordial soup that existed on Earth before life originated. Like DNA, RNA is complex and made of helix-shaped chains of smaller molecules known as nucleotides.

    One way that RNA might have first formed is with the help of minerals, such as metal hydrides. These minerals can serve as catalysts, helping create small organic compounds from inorganic building blocks. Such minerals are found at alkaline hydrothermal vents on the seafloor.

    Alkaline hydrothermal vents are also home to large chimney-like structures rich in iron and sulfur. Prior studies suggested that ancient counterparts of these chimneys might have isolated and concentrated organic molecules together, spurring the origin of life on Earth.

    To see how well these chimneys support the formation of strings of RNA, researchers synthesized chimneys by slowly injecting solutions containing iron, sulfur and silicon into glass jars. Depending on the concentrations of the different chemicals used to grow these structures, the chimneys were either mounds with single hollow centers or, more often, spires and “chemical gardens” with multiple hollow tubes.

    2
    Chimney-like mineral structures created in the lab created from solutions containing iron, sulfur and silicon under a) low concentrations and b) high concentrations. Structures in a) represent mound (left) and spindle (right) formations, while those in b) represent chemical garden formations.
    Credit: Bradley Burcar et al., Astrobiology.

    “Being able to perform our experiments in chimney structures that looked like something one might encounter in the darker regions of Tolkien’s Middle Earth gave these studies a geologic context that sparked the imagination,” said study co-author Linda McGown, an analytical chemist and astrobiologist at Rensselaer Polytechnic Institute in Troy, N.Y.

    The chimneys were grown in liquids and gases resembling the oceans and atmosphere of early Earth. The liquids were acidic and enriched with iron, while the gases were rich in nitrogen and had no oxygen. The scientists then poked syringes up the chimneys to pump alkaline solutions containing a variety of chemicals into the model oceans. This simulated ancient vent fluid seeping into primordial seas.

    Sometimes the researchers added montmorillonite clay to their glass jars. Clays are produced by interactions between water and rock, and would likely have been common on the early Earth, McGown said.

    The kind of nucleotides making up RNA are known as ribonucleotides, since they are made with the sugar ribose. The scientists found that unmodified ribonuclotides could form strings of two nucleotides. In addition, ribonucleotides “activated” with a compound known as imidazole — a molecule created during chemical reactions that synthesize nucleotides — could form RNA strings or polymers up to four ribonucleotides long.

    “In order to observe significant RNA polymerization on the time scale of laboratory experiments, it is generally necessary to activate the nucleotides,” McGown said. “Imidazole is commonly used for nucleotide activation in these types of experiments.”

    The scientists found that not only was the chemical composition of the chimneys important when it came to forming RNA, but the physical structure of the chimneys was key too. When the researchers mixed iron, sulfur and silicon solutions into their simulated oceans, instead of slowly injecting them into the seawater to form chimneys, the resulting blend could not trigger RNA formation.

    “The chimneys, and not just their constituents, are responsible for the polymerization,” McGown said.

    These experiments for the first time demonstrate that RNAs can form in alkaline hydrothermal chimneys, albeit synthetic ones.

    “Our goal from the start of our RNA polymerization research has been to place the RNA polymerization experiments as closely as possible in the context of the most likely early Earth environments,” McGown said. “Most previous RNA polymerization research has focused on surface environments, and the exploration of deep-ocean hydrothermal vents could yield exciting new possibilities for the emergence of an RNA world.”

    One concern about these findings is that the experiments were performed at room temperature. Hydrothermal vents are much hotter, and such temperatures could destroy RNA. [Video: The Search For Another Earth]

    “Keep in mind, however, that hydrothermal vents are dynamic systems with gradients of chemical and physical conditions, including temperature,” McGown said.

    In principle, cooler sections of hydrothermal vents might have nurtured RNA and its precursor molecules, she said.

    In the future, McGown and her colleagues will perform experiments investigating what effects variables such as pressure, temperature and mineralogy might have on the formation of RNA molecules, focusing primarily on conditions mimicking deep-ocean environments on an early Earth and those that may also have existed on Mars and elsewhere, McGown said.

    The scientists detailed their findings in the July 22 issue of the journal Astrobiology.

    Science team:

    Bradley T. Burcar,1,2 Laura M. Barge,3,4 Dustin Trail,1,5,* E. Bruce Watson,1,5 Michael J. Russell,3,4 and Linda B. McGown1,2
    1 New York Center for Astrobiology, Rensselaer Polytechnic Institute, Troy, New York.
    2 Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York.
    3 NASA Jet Propulsion Laboratory, California Institute of Technology, Pasadena, California.
    4 NASA Astrobiology Institute, Icy Worlds.
    5 Department of Earth and Environmental Sciences, Rensselaer Polytechnic Institute School of Science, Troy, New York.

    See the full article here .

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  • richardmitnick 5:14 pm on December 28, 2015 Permalink | Reply
    Tags: , , , RNA   

    From NOVA: “The Man Who Rewrote the Tree of Life” 2014 but Interesting and Important 

    PBS NOVA

    NOVA

    30 Apr 2014
    Carrie Arnold

    Carl Woese may be the greatest scientist you’ve never heard of. “Woese is to biology what [Albert] Einstein is to physics,” says Norman Pace, a microbiologist at the University of Colorado, Boulder. A physicist-turned-microbiologist, Woese specialized in the fundamental molecules of life—nucleic acids—but his ambitions were hardly microscopic. He wanted to create a family tree of all life on Earth.

    Woese certainly wasn’t the first person with this ambition. The desire to classify every living thing is ageless. The Ancient Greeks and Romans worked to develop a system of classifying life. The Jewish people, in writing the Book of Genesis, set Adam to the task of naming all the animals in the Garden of Eden. And in the mid-1700s, Swedish botanist Carl von Linné published Systema Naturae, introducing the world to a system of Latin binomials—Genus species—that scientists use to this day.

    Temp 1
    Carl Woese in his later years. Photo credits: Jason Lindsey/University of Illinois, Tim Bocek/Flickr (CC BY-NC-SA)

    What Woese was proposing wasn’t to replace Linnaean classification, but to refine it. During the late 1960s, when Woese first started thinking about this problem as a young professor at the University of Illinois, biologists were relying a lot on guesswork to determine how organisms were related to each other, especially microbes. At the time, researchers used the shapes of microbes—their morphologies—and how they turned food into energy—their metabolisms—to sort them into bins. Woese was underwhelmed. To him, the morphology-metabolism approach was like trying to create a genealogical history using only photographs and drawings. Are people with dimples on their right cheeks and long ring fingers all members of the same family? Maybe, but probably not.

    “If you wanted to build a tree of life prior to what Woese did, there was no way to put something together that was based upon actual data,” says Jonathan Eisen, an evolutionary microbiologist at the University of California Davis.

    Just as outward appearances aren’t the best way to determine family relations, Woese believed that morphology and metabolism were inadequate classifiers for life on Earth. Instead, he figured that DNA could sketch a much more accurate picture. Today, that approach may seem like common sense. But in the late 60s and early 70s, this was no easy task. Gene sequencing was a time-consuming, tedious task. Entire PhDs were granted for sequencing just one gene. To create his tree of life, Woese would need to sequence the same gene in hundreds, if not thousands, of different species.

    So Woese toiled in his lab, sometimes with his postdoc George Fox but often alone, hunched over a light box with a magnifying glass, sequencing genes nucleotide by nucleotide. It took more than a decade. “When Woese first announced his results, I thought he was exaggerating at first,” Fox recalls. “Carl liked to think big, and I thought this was just another of his crazy ideas. But then I looked at the data and the enormity of what we had discovered hit me.”

    Woese and Fox published their results in 1977 in a well-respected journal, the Proceedings of the National Academy of Science. They had essentially rewritten the tree of life. But Woese still had a problem: few scientists believed him. He would spend the rest of his life working to convince the biological community that his work was correct.

    Animal, Vegetable, Mineral

    Following the publication of Linnaeus’s treatise in the 18th century, taxonomy progressed incrementally. The Swedish botanist had originally sorted things into three “kingdoms” of the natural world: animal, vegetable, and mineral. He placed organisms in their appropriate cubbyholes by looking at similarities in appearance. Plants with the same number of pollen-producing stamens were all lumped together, animals with the same number of teeth per jaw were grouped, and so on. With no knowledge of evolution and natural selection, he didn’t have a better way to comprehend the genealogy of life on Earth.

    The publication of [Charles]Darwin’s On the Origin of Species in 1859, combined with advances in microscopy, forced scientists to revise Linnaeus’s original three kingdoms to include the tiniest critters, including newly visible ones like amoebae and E. coli. Scientists wrestled with how to integrate microbial wildlife into the tree of life for the next 100 years. By the mid-20th century, however, biologists and taxonomists had mostly settled on a tree with five major branches: protists, fungi, plants, animals, and bacteria. It’s the classification system that many people learned in high school biology class.

    Woese and other biologists weren’t convinced, though. Originally a physics major at Amherst College in Massachusetts and having received a PhD in biophysics from Yale in 1953, Woese believed that there had to be a more objective, data-driven way to classify life. Woese was particularly interested in how microbes fit into the classification of life, which had escaped a rigorous genealogy up until that point.

    He arrived at the University of Illinois Urbana-Champaign as a microbiologist in the mid-1960s, shortly after James Watson and Francis Crick won the Nobel prize for their characterization of DNA’s double-helix form. It was the heyday of DNA. Woese was enthralled. He believed that DNA could unlock the hidden relationships between different organisms. In 1969, Woese wrote a letter to Crick, stating that:

    ” …this can be done by using the cell’s ‘internal fossil record’—i.e., the primary structures of various genes. Therefore, what I want to do is to determine primary structures for a number of genes in a very diverse group of organisms, on the hope that by deducing rather ancient ancestor sequences for these genes, one will eventually be in the position of being able to see features of the cell’s evolution….”

    This type of thinking was “radically new,” says Norman Pace, a microbiologist at the University of Colorado, Boulder. “No one else was thinking in this direction at the time, to look for sequence-based evidence of life’s diversity.”

    Evolution’s Timekeeper

    Although the field of genetics was still quite young, biologists had already figured out some of the basics of how evolution worked at the molecular level. When a cell copies its DNA before dividing in two, the copies aren’t perfectly identical. Mistakes inevitably creep in. Over time, this can lead to significant changes in the sequence of nucleotides and the proteins they code for. By finding genes with sites that mutate at a known rate—say 4 mutations per site per million years—scientists could use them as an evolutionary clock that would give biologists an idea of how much time had passed since two species last shared a common ancestor.

    To create his evolutionary tree of life, then, Woese would need to choose a gene that was present in every known organism, one that was copied from generation to generation with a high degree of precision and mutated very slowly, so he would be able to track it over billions of years of evolution.

    “This would let him make a direct measure of evolutionary history,” Pace says. “By tracking these gene sequences over time, he could calculate the evolutionary distance between two organisms and make a map of how life on Earth may have evolved.”

    Some of the most ancient genes are those coding for molecules known as ribosomal RNAs. In ribosomes, parts of the cell that float around the soupy cytoplasm, proteins and ribosomal RNA, or rRNA, work together to crank out proteins. Each ribosome is composed of large and small subunits, which are similar in both simple, single-celled prokaryotes and more complex eukaryotes. Woese had several different rRNA molecules to choose from in the various subunits, which are classified based on their length. At around 120 nucleotides long, 5S rRNA wasn’t big enough to use to compare lots of different organisms. On the other end of the spectrum, 23S rRNA was more than 2300 nucleotides long, making it far too difficult for Woese to sequence using the technologies of the time. The Goldilocks molecule—long enough to allow for meaningful comparisons but not too long and difficult to sequence—was 16S rRNA in prokaryotes and its slightly longer eukaryotic equivalent, 18S rRNA. Woese decided to use these to create his quantitative tree of life.

    His choice was especially fortuitous, Eisen says, because of several factors inherent in 16S rRNA that Woese couldn’t have been aware of at the time, including its ability to measure evolutionary time on several different time scales. Certain parts of the 16S rRNA molecule mutate at different speeds. Changes to 16S rRNA are, on the whole, still extremely slow (humans share about 50% of their 16S rRNA sequence with the bacterium E. coli), but one portion mutates much more slowly than the other. It’s as if the 16S rRNA clock has both an hour hand and a minute hand. The very slowly evolving “hour hand” lets biologists study the long-term changes to the molecule, whereas the more quickly evolving “minute hand” provides a more recent history. “This gives this gene an advantage because it lets use ask questions about deep evolutionary history and more recent history at the same time,” Eisen says.

    Letter by letter

    Selecting the gene was just Woese’s first challenge. Now he had to sequence it in a variety of different organisms. In the late 60s and early 70s, when Woese began his work, DNA sequencing was far from automated. Everything, down to the last nucleotide, had to be done by hand. Woese used a method to catalog short pieces of RNA developed in 1965 by British scientist Frederick Sanger, which used enzymes to chop RNA into small pieces. These small pieces were sequenced, and then scientists had to reassemble the overlapping pieces to determine the overall sequence of the entire molecule—a process that was tedious, expensive, and time-consuming, but that was seen as a minor annoyance to a workhorse like Woese, Fox says. “All he cared about was getting the answer.”

    Woese started with prokaryotes, the single-celled organisms that were his primary area of interest. He and his lab started by growing bacteria in a solution of radioactive phosphate, which the cells incorporated into backbones of their RNA molecules. This made the 16S rRNA radioactive. Then, Woese and Fox extracted the RNA from the cells and chopped it into smaller pieces using enzymes that acted like scissors. The enzymatic scissors would only cut at certain sequences. If a sequence was present in one organism but missing in a second, the scissors would pass over the second one’s sequence. Its fragment would be longer.

    Since RNA’s sugar-phosphate backbone is negatively charged, the researchers could use a process known as electrophoresis to separate the different length pieces. As electricity coursed through gels containing samples, it pulled the smaller, lighter bits farther through the gels than the longer, heavier chunks. The result was distinct bands of different lengths of RNA. Woese and Fox then exposed each gel to photographic paper over several days. The radioactive bands in the gel transferred marks to the paper. This created a Piet Mondrian-esque masterpiece of black bands on a white background. Each different organism left its own mark. “To Carl, each spot was a puzzle that he would solve,” Fox says.

    After developing each image, Woese and Fox returned to the gel and neatly cut out each individual blotch that contained fragments of a certain length. They then chopped up these fragments with another set of enzymes until they were about five to 15 nucleotides long, a length that made sequencing easier. For some of the longer fragments, it took several iterations of the process before they were successfully sequenced. The sequences were then recorded on a set of 80-column IBM punch cards. The cards were then run through a large computer to compare band patterns and RNA sequences among different organisms to determine evolutionary relationships. At the beginning, it took Woese and Fox months to obtain a single 16S rRNA fingerprint.

    “This process was a huge breakthrough,” says Peter Moore, an RNA chemist at Yale University who worked with Woese on other research relating to RNA’s structure. “It gave biologists a tool for sorting through microorganisms and giving them a conceptual way to understand the relationship between them. At the time, the field was just a total disaster area. Nobody knew what the hell was going on.”

    RNA is so fundamental to life that some scientists think it’s the spark that started it all. To learn more about RNA, visit NOVA’s RNA Lab.

    By the spring of 1976, Woese and Fox had created fingerprints of a variety of bacterial species when they turned to an oddball group of prokaryotes: methanogens. These microbes produce methane when they break down food for energy. Because even tiny amounts of oxygen are toxic to these prokaryotes, Woese and Fox had to grow them under special conditions.

    After months of trial and error, the two scientists were finally able to obtain an RNA fingerprint of one type of methanogen. When they finally analyzed its fingerprint, however, it looked nothing like any of the other bacteria Woese and Fox had previously analyzed. All of the previous bacterial gels contained two large splotches at the bottom. They were entirely absent from these new gels. Woese knew instantly what this meant.

    To fellow microbiologist Ralph Wolfe, who worked in the lab next door, Woese announced, “I don’t even think these are bacteria, Wolfe.”

    He dropped the full bombshell on Fox. “The methanogens didn’t have any of the spots he was expecting to see. When he realized this wasn’t a mistake, he just went nuts. He ran into my lab and told me we had discovered a new form of life,” Fox recalls.

    The New Kingdom

    The methanogens Woese and Fox had analyzed looked superficially like other bacteria, yet their RNA told a different story, sharing more in common with nucleus-containing eukaryotes than with other bacteria. After more analysis of his RNA data, Woese concluded that what he was tentatively calling Archaea (from Latin, meaning primitive) wasn’t a minor twig on the tree of life, but a new main branch. It wasn’t just Bacteria and Eukarya any more .

    To prove to their critics that these prokaryotes really were a separate domain on the tree of life, Woese and Fox knew the branch needed more than just methanogens. Fox knew enough about methanogen biology to know that their unique RNA fingerprint wasn’t the only thing that made them strange. For one thing, their cell walls lacked a mesh-like outer layer made of peptidoglycan. Nearly every other bacterium Fox could think of contained peptidoglycan in its cell wall—until he recalled a strange fact he had learned as a graduate student—another group of prokaryotes, the salt-loving halophiles, also lacked peptidoglycan.

    2
    Grand Prismatic Spring in Yellowstone National Park is home to many species of thermophilic archaea.

    Fox turned to the research literature to search for other references to prokaryotes that lack peptidoglycan. He found two additional examples: Thermoplasma and Sulfolobus. Other than the missing peptidoglycan, these organisms and the methanogens seemed nothing alike. Methanogens were found everywhere from wetlands to the digestive tracts, halophiles flourished in salt, Thermoplasma liked things really hot, and Sulfolobus are often found in volcanoes and hot, acidic springs.

    Despite their apparent differences, they all metabolized food in the same, unusual way—unlike anything seen in other bacteria—and the fats in the cell membrane were alike, too. When Woese and Fox sequenced the 16S rRNA of these organisms, they found that these prokaryotes were most similar to the methanogens.

    “Once we had the fingerprints, it all fell together,” Fox says.

    Woese believed his findings were going to revolutionize biology, so he organized a press conference when the paper was published in PNAS in 1977. It landed Woese on the front page of the New York Times, and created animosity among many biologists. “The write-ups were ludicrous and the reporters got it all wrong,” Wolfe says. “No biologists wanted anything to do with him.”

    It wasn’t just distaste for what looked like a publicity stunt that was working against Woese. He had spent most of the last decade holed up in his third floor lab, poring over RNA fingerprints. His reclusive nature had given him the reputation of a crank. It also didn’t help that he had single-handedly demoted many biologists’ favorite species. Thanks to Woese, Wolfe says, “Microbes occupy nearly all of the tree. Then you have one branch at the very end where all the animals and plants were. And the biologists just couldn’t believe that all the plants and all the animals were really just one tiny twig on one branch.”

    Although some specialists were quick to adopt Woese’s new scheme, the rest of biology remained openly hostile to the idea. It wasn’t until the mid-1980s that other microbiologists began to warm to the idea, and it took well over another decade for other areas of biology to follow suit. Woese had grown increasingly bitter that so many other scientists were so quick to reject his claims. He knew his research and ideas were solid. But he was left to respond to what seemed like an endless stream of criticism. Shying from these attacks, Woese retreated to his office for the next two decades.

    “He was a brash, iconoclastic outsider, and his message did not go down well,” says Moore, the Yale RNA chemist.

    Woese’s cause wasn’t helped by his inability to engage critics in dialogue and discussion. Both reticent and abrupt, he preferred his lab over conferences and presentations. In place of public appearances to address his detractors, he sent salvos of op-eds and letters to the editor. Still, nothing seemed to help. The task of publicly supporting this new tree of life fell to Woese’s close colleagues, especially Norman Pace.

    But as technology improved, scientists began to obtain the sequences of an increasing number of 16S rRNAs from different organisms. More and more of their analyses supported Woese’s hypothesis. As sequencing data poured in from around the world, it became clear to nearly everyone in biology that Woese’s initial tree was, in fact, been correct.

    Now, when scientists try to discover unknown microbial species, the first gene they sequence is 16S rRNA. “It’s become one of the fundamentals of biology,” Wolfe says. “After more than 20 years, Woese was finally vindicated.”

    Woese died on December 30, 2012, at the age of 84 of complications from pancreatic cancer. At the time of his death, he had won some of biology’s most prestigious awards and had become one of the field’s most respected scientists. Thanks to Woese’s legacy, we now know that most of the world’s biodiversity is hidden from view, among the tiny microbes that live unseen in and around us, and in them, the story of how life first evolved on this planet.

    See the full article here .

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  • richardmitnick 1:08 pm on October 22, 2015 Permalink | Reply
    Tags: , , , RNA   

    From MIT: “Biologists unravel drug-resistance mechanism in tumor cells” 


    MIT News

    October 22, 2015
    Anne Trafton | MIT News Office

    1
    P53, which helps healthy cells prevent genetic mutations, is missing from about half of all tumors. Researchers have found that a backup system takes over when p53 is disabled and encourages cancer cells to continue dividing. In the background of this illustration are crystal structures of p53 DNA-binding domains. Image: Jose-Luis Olivares/MIT (p53 illustration by Richard Wheeler/Wikimedia Commons)

    Targeting the RNA-binding protein that promotes resistance could lead to better cancer therapies.

    About half of all tumors are missing a gene called p53, which helps healthy cells prevent genetic mutations. Many of these tumors develop resistance to chemotherapy drugs that kill cells by damaging their DNA.

    MIT cancer biologists have now discovered how this happens: A backup system that takes over when p53 is disabled encourages cancer cells to continue dividing even when they have suffered extensive DNA damage. The researchers also discovered that an RNA-binding protein called hnRNPA0 is a key player in this pathway.

    “I would argue that this particular RNA-binding protein is really what makes tumor cells resistant to being killed by chemotherapy when p53 is not around,” says Michael Yaffe, the David H. Koch Professor in Science, a member of the Koch Institute for Integrative Cancer Research, and the senior author of the study, which appears in the Oct. 22 issue of Cancer Cell.

    The findings suggest that shutting off this backup system could make p53-deficient tumors much more susceptible to chemotherapy. It may also be possible to predict which patients are most likely to benefit from chemotherapy and which will not, by measuring how active this system is in patients’ tumors.

    Rewired for resistance

    In healthy cells, p53 oversees the cell division process, halting division if necessary to repair damaged DNA. If the damage is too great, p53 induces the cell to undergo programmed cell death.

    In many cancer cells, if p53 is lost, cells undergo a rewiring process in which a backup system, known as the MK2 pathway, takes over part of p53’s function. The MK2 pathway allows cells to repair DNA damage and continue dividing, but does not force cells to undergo cell suicide if the damage is too great. This allows cancer cells to continue growing unchecked after chemotherapy treatment.

    “It only rescues the bad parts of p53’s function, but it doesn’t rescue the part of p53’s function that you would want, which is killing the tumor cells,” says Yaffe, who first discovered this backup system in 2013.

    In the new study, the researchers delved further into the pathway and found that the MK2 protein exerts control by activating the hnRNPA0 RNA-binding protein.

    RNA-binding proteins are proteins that bind to RNA and help control many aspects of gene expression. For example, some RNA-binding proteins bind to messenger RNA (mRNA), which carries genetic information copied from DNA. This binding stabilizes the mRNA and helps it stick around longer so the protein it codes for will be produced in larger quantities.

    “RNA-binding proteins, as a class, are becoming more appreciated as something that’s important for response to cancer therapy. But the mechanistic details of how those function at the molecular level are not known at all, apart from this one,” says Ian Cannell, a research scientist at the Koch Institute and the lead author of the Cancer Cell paper.

    In this paper, Cannell found that hnRNPA0 takes charge at two different checkpoints in the cell division process. In healthy cells, these checkpoints allow the cell to pause to repair genetic abnormalities that may have been introduced during the copying of chromosomes.

    One of these checkpoints, known as G2/M, is controlled by a protein called Gadd45, which is normally activated by p53. In lung cancer cells without p53, hnRNPA0 stabilizes mRNA coding for Gadd45. At another checkpoint called G1/S, p53 normally turns on a protein called p21. When p53 is missing, hnRNPA0 stabilizes mRNA for a protein called p27, a backup to p21. Together, Gadd45 and p27 help cancer cells to pause the cell cycle and repair DNA so they can continue dividing.

    Personalized medicine

    The researchers also found that measuring the levels of mRNA for Gadd45 and p27 could help predict patients’ response to chemotherapy. In a clinical trial of patients with stage 2 lung tumors, they found that patients who responded best had low levels of both of those mRNAs. Those with high levels did not benefit from chemotherapy.

    “You could measure the RNAs that this pathway controls, in patient samples, and use that as a surrogate for the presence or absence of this pathway,” Yaffe says. “In this trial, it was very good at predicting which patients responded to chemotherapy and which patients didn’t.”

    “The most exciting thing about this study is that it not only fills in gaps in our understanding of how p53-deficient lung cancer cells become resistant to chemotherapy, it also identifies actionable events to target and could help us to identify which patients will respond best to cisplatin, which is a very toxic and harsh drug,” says Daniel Durocher, a senior investigator at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital in Toronto, who was not part of the research team.

    The MK2 pathway could also be a good target for new drugs that could make tumors more susceptible to DNA-damaging chemotherapy drugs. Yaffe’s lab is now testing potential drugs in mice, including nanoparticle-based sponges that would soak up all of the RNA binding protein so it could no longer promote cell survival.

    See the full article here .

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