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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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    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.

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  • 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

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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 

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    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.

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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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    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.

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  • 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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    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.

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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.

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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.

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  • richardmitnick 3:00 pm on September 18, 2015 Permalink | Reply
    Tags: , , , RNA   

    From MIT: “Extending super-resolution techniques” 


    MIT News

    1
    Department of Physics graduate student Takuma Inoue built the super-resolution microscopy set up in the Cissé lab at MIT to study single molecule behavior of enzyme clusters that enable gene copying and protein production within living cells. On the table, five different lasers excite different fluorescent proteins at different wavelengths and image their location.

    Photo: Denis Paiste/Materials Processing Center

    Overcoming limitations of super-resolution microscopy to optimize imaging of RNA in living cells is a key motivation for physics graduate student Takuma Inoue, who works in the lab of MIT assistant professor of physics Ibrahim Cissé.

    Inoue, 26, was the first student to join Cissé’s lab at MIT in January 2014, and he built the lab’s super-resolution microscopy setup to study enzyme clusters that enable gene copying and protein production within living cells. Inoue, who this September enters his fourth year toward his PhD, originally started his experimental work in an atomic physics lab, where he worked on an imaging setup to trap extremely cold atoms in a vacuum. He is studying biophysics, atomic physics, and condensed matter physics.

    After learning that Cissé needed someone to set up his super-resolution microscopy, Inoue switched to Cissé’s lab. Because he did not have a biology background, Inoue says, “I wasn’t very much familiar with that, but the tools that you use and the methods for imaging are very common with what I had previously done. By building the setup, I got used to what things we can do in the lab. Then I made the transition to actually targeting some biomolecules within the cell to image and for me that was RNA.”

    “Initially, I had to start with building the microscope, which took me several months, and then I tried doing the continuation of his previous work which is imaging some kind of protein inside of a living cell,” Inoue says. “But then he gave (me) this project of RNA imaging as my main project for my PhD, because that will be more challenging, and we thought no one has ever achieved this big goal.”

    “My project makes sure that we overcome as many limitations as possible because there are different aspects of this for all the projects that we do,” Inoue adds. “There is the setup and there is this data analysis software and also we need to label the target molecules of interest properly. Each of them is, of course, not perfect. There are many challenges and limitations. But if you have a final goal in your project, I think you need to care about all of those different aspects and try optimizing. I’ve been doing experiments for a long time, so overcoming such limitations in the lab was one of my interests.”

    Inoue is developing techniques for easily tagging and visualizing RNA directly in living cells. “For me as an experimentalist, it’s a very exciting challenge to achieve the imaging of RNA within a live cell and to bring it to the level of a single molecule. My goal is achieve a technique to image single molecules of RNA inside of a living cell. That can have very broad applications. I think it’s very transformative,” Inoue says.

    The common approach to such imaging is genetic modification that adds a derivative of green fluorescent protein to the target of study — for example, RNA polymerase II. Inoue says his approach is to avoid genetic modification by developing oligonucleotide probes, which are short strands of genetic material that can bind to the target. “I try to deliver these probes into these natural cells and try to see if the target molecules get this fluorescence. And then I bring those cells to the imaging room and then do imaging,” he says. The technique is called fluorescent in situ hybridization. The oligonucleotide and the RNA target both start out as single strand molecules, but when they bind they can form a double helix like DNA, Inoue explains.

    “There already are approaches for looking at RNA inside dead cells. That’s I think the easy part,” Takuma’s mentor, Cissé, explains. “A handful of labs have also reported on promising ways of labeling RNA in living cells, but those require extensive genetic modifications. Takuma’s whole point is actually bringing new techniques for easily tagging and visualizing any arbitrary RNA, without genetic modification, and directly inside the living cell. And his preliminary demonstrations also, I think, look very promising.”

    Inoue has high hopes for the project. “This project is about labeling arbitrary RNA that exist inside a living cell, and I am at the developing stage of these techniques,” he says. “I’m hoping that through this project I can contribute and help many researchers in studying their RNAs of interest and also I, myself, am interested in studying different kinds of RNA.”

    The Cissé lab’s single molecule studies of the role that enzymes, proteins, and RNA play in gene expression is funded under National Institutes of Health Project No. 1DP2CA195769-01 with additional funds from the National Cancer Institute.

    The super-resolution imaging setup captures images through the microscope onto an electron-multiplying charge-coupled device (EM-CCD). “It can detect very sensitive signals, even single photons, and also it’s a very fast camera,” Inoue explains. The EM-CCD has millisecond exposure times but overall it takes several minutes to get one super-resolution image made from about 10,000 images.

    A native of Yokohama, Japan, Inoue moved to the U.S. at age 18, three years after his father, Hiroshi Inoue, accepted a position in Maryland starting up a life sciences subsidiary for Canon. Now a resident of Rockville, Maryland, Takuma Inoue received his bachelor’s in physics with a minor in mathematics at the University of Maryland at College Park, where his father currently holds the title of Professor of the Practice in Bioengineering.

    “I’ve got a lot of influence from my dad because he was initially an engineer, but then it was a very big surprise to me that he switched to biology and started doing some kind of engineering that could help biologists or could help people. So, that was maybe one of the key events in my life,” he says.

    “I like to think about scientific challenges and also there are many engineering challenges, and I really like that I am doing that, and also that I am trying to solve the world’s most interesting problems in the field of biology using my physics background. I want to see first how this project goes, and if possible, I’d like to continue doing research, and I hope that my career becomes exciting,” Inoue says.

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  • richardmitnick 8:00 pm on August 27, 2015 Permalink | Reply
    Tags: , RNA,   

    From Rockefeller: “Research identifies a protein that helps determine the fate of RNA” 

    Rockefeller U bloc

    Rockefeller University

    August 27, 2015
    Wynne Parry | 212-327-7789

    1
    Ready to read: The newly identified protein, HNRNPA2B1 (green), which recognizes the m6A tag, is found within the nuclei (blue) of cells. Actin filaments, important elements of the cell’s structure, show up in red. Credit: Lisa Noble and Gloria Wu

    After it is transcribed from DNA, RNA can go on to many fates. While the most familiar path may lead directly to the production of protein, RNA molecules themselves can also become capable of altering the expression of genes. New research helps explain how the destiny of an RNA sequence is achieved.

    In a study published August 27 in Cell, Rockefeller University scientists and a colleague at Columbia University have identified a protein that recognizes a chemical instruction tag affixed to an RNA sequence, an important step in the decision-making process.

    “This tag, known as m6A, was identified more than four decades ago on RNA sequences. Since then, this abundant label has been implicated in several important processes that influence the production of proteins, as well as so-called microRNAs,” says study author Sohail Tavazoie, Leon Hess Associate Professor, and head of the Elizabeth and Vincent Meyer Laboratory of Systems Cancer Biology. (Micro-RNAs are small RNA molecules that do not code for proteins, but instead turn down the activity of genes.)

    “However,” Tavazoie says, “a lingering question remained: What reads this chemical tag within the nucleus of cells? Claudio Alarcón, a research associate in my lab, has identified one such “reader” protein. Because of the fundamental nature of the processes involved, this discovery has implications for cells’ normal function and for disease.”

    The tag, called m6A, is a methyl group attached to a particular part of an adenosine, a component of RNA’s sequence. In previous work, Alarcón and colleagues identified the m6A tag as an important regulator of the production of microRNAs. The “writer” protein that places this tag was already known to mark RNA molecules that need to be spliced before they are translated into proteins. Many genes contain sections that must be cut out, and the RNA splicing process is crucial to the function and identity of a cell.

    The recent experiments show that the newly discovered reader, a protein known as HNRNPA2B1, recognizes m6A tags on RNA destined for two separate fates: Trimming to become microRNAs or splicing for proper production into protein. After recognizing m6A tags on microRNA precursors, HNRNPA2B1 then recruits the cutting machinery responsible for further trimming and processing those RNA molecules. Future work is required to understand how HNRNPA2B1 interacts with the proteins involved in the splicing of RNA.

    The researchers suspected that the HNRNPA2B1 protein acts as a reader because they found it frequently binds to the same sites on the RNA molecule where the m6A tag attaches. To determine what the alleged reader was doing there, the team reduced its presence in cells.

    In cells with reduced HNRNPA2B1 levels, they found a shift in the expression of microRNAs overall, with many microRNAs reduced. They also looked at the effects on RNA destined for splicing. Here too, they found telling changes in the splicing of different RNA molecules that are dependent on m6A tags.

    HNRNPA2B1 is the first m6A nuclear reader to be identified, and evidence from the experiments suggests the existence of additional readers within the nucleus that also recognize this tag.

    “The discovery of this new m6A reader has ramifications for a broad range of processes,” Alarcón says. “RNA splicing establishes the repertoire of proteins available in cells. Meanwhile, abnormalities in microRNAs have been associated with several diseases, including cancer. This work also contributes to growing evidence that information beyond the sequence of RNA, such as chemical modifications, can determine the ultimate fate and function of RNA molecules.”

    See the full article here.

    Please help promote STEM in your local schools.

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    Rockefeller U Campus

    The Rockefeller University is a world-renowned center for research and graduate education in the biomedical sciences, chemistry, bioinformatics and physics. The university’s 76 laboratories conduct both clinical and basic research and study a diverse range of biological and biomedical problems with the mission of improving the understanding of life for the benefit of humanity.

    Founded in 1901 by John D. Rockefeller, the Rockefeller Institute for Medical Research was the country’s first institution devoted exclusively to biomedical research. The Rockefeller University Hospital was founded in 1910 as the first hospital devoted exclusively to clinical research. In the 1950s, the institute expanded its mission to include graduate education and began training new generations of scientists to become research leaders around the world. In 1965, it was renamed The Rockefeller University.

     
  • richardmitnick 9:31 am on July 30, 2015 Permalink | Reply
    Tags: , , , , , RNA   

    From livescience: “Origin-of-Life Story May Have Found Its Missing Link” 

    Livescience

    June 06, 2015
    Jesse Emspak

    1
    A field of geysers called El Tatio located in northern Chile’s Andes Mountains. Credit: Gerald Prins

    How did life on Earth begin? It’s been one of modern biology’s greatest mysteries: How did the chemical soup that existed on the early Earth lead to the complex molecules needed to create living, breathing organisms? Now, researchers say they’ve found the missing link.

    Between 4.6 billion and 4.0 billion years ago, there was probably no life on Earth. The planet’s surface was at first molten and even as it cooled, it was getting pulverized by asteroids and comets. All that existed were simple chemicals. But about 3.8 billion years ago, the bombardment stopped, and life arose. Most scientists think the “last universal common ancestor” — the creature from which everything on the planet descends — appeared about 3.6 billion years ago.

    But exactly how that creature arose has long puzzled scientists. For instance, how did the chemistry of simple carbon-based molecules lead to the information storage of ribonucleic acid, or RNA?

    2
    A hairpin loop from a pre-mRNA. Highlighted are the nucleobases (green) and the ribose-phosphate backbone (blue). Note that this is a single strand of RNA that folds back upon itself.

    The RNA molecule must store information to code for proteins. (Proteins in biology do more than build muscle — they also regulate a host of processes in the body.)

    The new research — which involves two studies, one led by Charles Carter and one led by Richard Wolfenden, both of the University of North Carolina — suggests a way for RNA to control the production of proteins by working with simple amino acids that does not require the more complex enzymes that exist today. [7 Theories on the Origin of Life on Earth]

    Missing RNA link

    This link would bridge this gap in knowledge between the primordial chemical soup and the complex molecules needed to build life. Current theories say life on Earth started in an “RNA world,” in which the RNA molecule guided the formation of life, only later taking a backseat to DNA, which could more efficiently achieve the same end result.

    3
    The structure of the DNA double helix. The atoms in the structure are colour-coded by element and the detailed structure of two base pairs are shown in the bottom right.

    Like DNA, RNA is a helix-shaped molecule that can store or pass on information. (DNA is a double-stranded helix, whereas RNA is single-stranded.) Many scientists think the first RNA molecules existed in a primordial chemical soup — probably pools of water on the surface of Earth billions of years ago. [Photo Timeline: How the Earth Formed]

    The idea was that the very first RNA molecules formed from collections of three chemicals: a sugar (called a ribose); a phosphate group, which is a phosphorus atom connected to oxygen atoms; and a base, which is a ring-shaped molecule of carbon, nitrogen, oxygen and hydrogen atoms. RNA also needed nucleotides, made of phosphates and sugars.

    The question: How did the nucleotides come together within the soupy chemicals to make RNA? John Sutherland, a chemist at the University of Cambridge in England, published a study in May in the journal Nature Chemistry that showed that a cyanide-based chemistry could make two of the four nucleotides in RNA and many amino acids.

    That still left questions, though. There wasn’t a good mechanism for putting nucleotides together to make RNA. Nor did there seem to be a natural way for amino acids to string together and form proteins. Today, adenosine triphosphate (ATP) does the job of linking amino acids into proteins, activated by an enzyme called aminoacyl tRNA synthetase. But there’s no reason to assume there were any such chemicals around billions of years ago.

    Also, proteins have to be shaped a certain way in order to function properly. That means RNA has to be able to guide their formation — it has to “code” for them, like a computer running a program to do a task.

    Carter noted that it wasn’t until the past decade or two that scientists were able to duplicate the chemistry that makes RNA build proteins in the lab. “Basically, the only way to get RNA was to evolve humans first,” he said. “It doesn’t do it on its own.”

    Perfect sizes

    In one of the new studies, Carter looked at the way a molecule called “transfer RNA,” or tRNA, reacts with different amino acids.

    They found that one end of the tRNA could help sort amino acids according to their shape and size, while the other end could link up with amino acids of a certain polarity. In that way, this tRNA molecule could dictate how amino acids come together to make proteins, as well as determine the final protein shape. That’s similar to what the ATP enzyme does today, activating the process that strings together amino acids to form proteins.

    Carter told Live Science that the ability to discriminate according to size and shape makes a kind of “code” for proteins called peptides, which help to preserve the helix shape of RNA.

    “It’s an intermediate step in the development of genetic coding,” he said.

    In the other study, Wolfenden and colleagues tested the way proteins fold in response to temperature, since life somehow arose from a proverbial boiling pot of chemicals on early Earth. They looked at life’s building blocks, amino acids, and how they distribute in water and oil — a quality called hydrophobicity. They found that the amino acids’ relationships were consistent even at high temperatures — the shape, size and polarity of the amino acids are what mattered when they strung together to form proteins, which have particular structures.

    “What we’re asking here is, ‘Would the rules of folding have been different?'” Wolfenden said. At higher temperatures, some chemical relationships change because there is more thermal energy. But that wasn’t the case here.

    By showing that it’s possible for tRNA to discriminate between molecules, and that the links can work without “help,” Carter thinks he’s found a way for the information storage of chemical structures like tRNA to have arisen — a crucial piece of passing on genetic traits. Combined with the work on amino acids and temperature, it offers insight into how early life might have evolved.

    This work still doesn’t answer the ultimate question of how life began, but it does show a mechanism for the appearance of the genetic codes that pass on inherited traits, which got evolution rolling.

    The two studies are published in the June 1 issue of the journal Proceedings of the National Academy of Sciences.

    See the full article here.

    Please help promote STEM in your local schools.

    STEM Icon

    Stem Education Coalition

     
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