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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” 

    UCLA bloc

    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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  • 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” 

    space-dot-com logo

    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.

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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.

    See the full article here .

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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.

    See the full article here .

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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.

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    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
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    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.

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  • richardmitnick 12:18 pm on April 10, 2015 Permalink | Reply
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    From LBL: “New target for anticancer drugs: RNA” 

    UC Berkeley

    UC Berkeley

    April 6, 2015
    Robert Sanders

    1
    DNA is transcribed into mRNA, which is then translated by ribosomes into proteins. UC Berkeley researchers demonstrated that dysregulation of the gene expression program governed by a ribosomal protein called eIF3 leads to increased cell growth and carcinogenesis. That makes this protein an ideal anticancer drug target. (Amy Lee graphic)

    Most of today’s anticancer drugs target the DNA or proteins in tumor cells, but a new discovery by University of California, Berkeley, scientists unveils a whole new set of potential targets: the RNA intermediaries between DNA and proteins.

    This RNA, called messenger RNA, is a blueprint for making proteins. Messenger RNA is created in the nucleus and shuttled to the cell cytoplasm to hook up with protein-making machinery, the ribosome. Most scientists have assumed that these mRNA molecules are, aside from their unique sequences, generic, with few distinguishing characteristics that could serve as an Achilles heel for targeted drugs.

    Jamie Cate, UC Berkeley professor of molecular and cell biology, and postdoctoral fellows Amy Lee and Philip Kranzusch have found, however, that a small subset of these mRNAs – most of them coding for proteins linked in some way to cancer – carry unique tags. These short RNA tags bind to a protein, eIF3 (eukaryotic initiation factor 3), that regulates translation at the ribosome, making the binding site a promising target.

    “We’ve discovered a new way that human cells control cancer gene expression, at the step where the genes are translated into proteins. This research puts on the radar that you could potentially target mRNA where these tags bind with eIF3,” Cate said. “These are brand new targets for trying to come up with small molecules that might disrupt or stabilize these interactions in such a way that we could control how cells grow.”

    These tagged mRNAs – fewer than 500 out of more than 10,000 mRNAs in a cell – seem to be special in that they carry information about specific proteins whose levels in the cell must be delicately balanced so as not to tip processes like cell growth into overdrive, potentially leading to cancer.

    Surprisingly, while some of the tags turn on the translation of mRNA into protein, others turn it off.

    “Our new results indicate that a number of key cancer-causing genes – genes that under normal circumstances keep cells under control – are held in check before the proteins are made,” Cate said. “This new control step, which no one knew about before, could be a great target for new anticancer drugs.

    “On the other hand,” he said, “the tags that turn on translation activate genes that cause cancer when too much of the protein is made. These could also be targeted by new anticancer drugs that block the activation step.”

    The new results will be reported April 6 in an advance online publication of the journal Nature. Cate directs the Center for RNA Systems Biology, a National Institutes of Health-funded group developing new tools to study RNA, a group of molecules increasingly recognized as key regulators of the cell.

    mRNA a messenger between DNA and ribosome

    While our genes reside inside the cell’s nucleus, the machinery for making proteins is in the cytoplasm, and mRNA is the messenger between the two. All the DNA of a gene is transcribed into RNA, after which nonfunctional pieces are snipped out to produce mRNA. The mRNA is then shuttled out of the nucleus to the cytoplasm, where a so-called initiation complex gloms onto mRNA and escorts it to the ribosome. The ribosome reads the sequence of nucleic acids in the mRNA and spits out a sequence of amino acids: a protein.

    “If something goes out of whack with a cell’s ability to know when and where to start protein synthesis, you are at risk of getting cancer, because you can get uncontrolled synthesis of proteins,” Cate said. “The proteins are active when they shouldn’t be, which over-stimulates cells.”

    The protein eIF3 is one component of the initiation complex, and is itself made up of 13 protein subunits. It was already known to regulate translation of the mRNA into protein in addition to its role in stabilizing the structure of the complex. Overexpression of eIF3 also is linked to cancers of the breast, prostate and esophagus.

    “I think eIF3 is able to drive multiple functions because it consists of a large complex of proteins,” Lee said. “This really highlights that it is a major regulator in translation rather than simply a scaffolding factor.”

    Lee zeroed in on mRNAs that bind to eIF3, and found a way to pluck them out of the 10,000+ mRNAs in a typical human cell, sequenced the entire set and looked for eIF3 binding sites. She discovered 479 mRNAS – about 3 percent of the mRNAs in the cell – that bind to eIF3, and many of them seem to share similar roles in the cell.

    “When we look at the biological functions of these mRNAs, we see that there is an emphasis on processes that become dysregulated in cancer,” Lee said. These involve the cell cycle, the cytoskeleton, and programmed cell death (apoptosis), along with cell growth and differentiation.

    “Therapeutically, one could screen for increased expression of eIF3 in a cancer tissue and then target the pathways that we have identified as being eIF3-regulated,” she said.

    Lee actually demonstrated that she could tweak the mRNA of two cancer-related genes, both of which control cell growth, to stop cells from becoming invasive.

    “We showed that we could put a damper on invasive growth by manipulating these interactions, so clearly this opens the door to another layer of possible anticancer therapeutics that could target these RNA-binding regions,” Cate said.

    The work was funded by a grant from NIH’s National Institute of General Medical Sciences to the Center for RNA Systems Biology.

    “A goal of systems biology is to map entire biological networks, such as genes and their regulatory mechanisms, to better understand how those complex networks function and can contribute to disease,” said Peter Preusch, chief of the biophysics branch of NIGMS. “This center is using cutting-edge technology to interrogate the structure and function of many RNAs at a time, which is helping piece together RNA’s regulatory components.”

    Lee is supported through the American Cancer Society Postdoctoral Fellowship Program.

    See the full article here.

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  • richardmitnick 11:37 am on March 24, 2015 Permalink | Reply
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    From Rockefeller: “Chemical tag marks future microRNAs for processing, study shows” 

    Rockefeller U bloc

    Rockefeller University

    March 24, 2015
    Zach Veilleux | 212-327-8982

    1
    Molecular sorting machine: Scientists found the enzyme METTL3 (green) tags a particular sequence within RNA molecules destined to become gene-regulating microRNAs. While this happens within cells’ nuclei (blue), METTL3 is also found outside the nucleus in cells’ cytoplasm, as shown above.

    Just as two DNA strands naturally arrange themselves into a helix, DNA’s molecular cousin RNA can form hairpin-like loops. But unlike DNA, which has a single job, RNA can play many parts — including acting as a precursor for small molecules that block the activity of genes. These small RNA molecules must be trimmed from long hairpin-loop structures, raising a question: How do cells know which RNA loops need to be processed this way and which don’t?

    New research at Rockefeller University, published March 18 in Nature, reveals how cells sort out the loops meant to encode small RNAs, known as microRNAs, by tagging them with a chemical group. Because microRNAs help control processes throughout the body, this discovery has wide-ranging implications for development, health and disease, including cancer, the entry point for this research.

    “Work in our lab and elsewhere has shown changes in levels of microRNAs in a number of cancers. To better understand how and why this happens, we needed to first answer a more basic question and take a closer look at how cells normally identify and process microRNAs,” says study author Sohail Tavazoie, Leon Hess Associate Professor, Senior Attending Physician and head of the Elizabeth and Vincent Meyer Laboratory of Systems Cancer Biology. “Claudio Alarcón, a research associate in my lab, has discovered that cells can increase or decrease microRNAs by using a specific chemical tag.”

    Long known as the intermediary between DNA and proteins, RNA has turned out to be a versatile molecule. Scientists have discovered a number of RNA molecules, including microRNAs that regulate gene expression. MicroRNAs are encoded into the genome as DNA, then transcribed into hairpin loop RNA molecules, known as primary microRNAs. These loops are then clipped to generate microRNA precursors.

    To figure out how cells know which hairpin loops to start trimming, Alarcón set out to look for modifications cells might make to the RNA molecules that are destined to become microRNAs. Using bioinformatics software, he scanned for unusual patterns in the unprocessed RNA sequences. The sequence GGAC, code for the bases guanine-guanine-adenine-cytosine, stood out because it appeared with surprising frequency in the unprocessed primary microRNAs. GGAC, in turn, led the researchers to an enzyme known as METTL3, which tags the GGAC segments with a chemical marker, a methyl group, at a particular spot on the adenine.

    “Once we arrived at METTL3, everything made sense. The methyl in adenosines (m6A tag) is the most common known RNA modification. METTL3 is known to contribute to stabilizing and processing messenger RNA, which is transcribed from DNA, but it is suspected of doing much more,” Alarcón says. “Now, we have evidence for a third role: the processing of primary microRNAs.”

    In series of experiments, the researchers confirmed the importance of methyl tagging, finding high levels of it near all types of unprocessed microRNAs, suggesting it is a generic mark associated with these molecules. When they reduced expression of METTL3, unprocessed primary microRNAs accumulated, indicating that the enzyme’s tagging action was important to the process. And, working in cell culture and in biochemical systems, they found primary microRNAs were processed much more efficiently in the presence of the methyl tags than without them.

    “Cells can remove these tags, as well as add them, so these experiments have identified a switch that can be used to ramp up or tamp down microRNA levels, and as a result, alter gene expression,” Tavazoie says. “Not only do we see abnormalities in microRNAs in cancer, levels of METTL3 can be altered as well, which suggests this pathway is could govern cancer progression.”

    See the full article here.

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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 6:48 pm on November 26, 2014 Permalink | Reply
    Tags: , , , , RNA   

    From Quanta: “New Twist Found in the Story of Life’s Start” 

    Quanta Magazine
    Quanta Magazine

    November 26, 2014
    Emily Singer

    All life on Earth is made of molecules that twist in the same direction. New research reveals that this may not always have been so.

    t
    The mirror-image asymmetry of life is one of the biggest mysteries in biology.
    Brendan Monroe for Quanta Magazine

    For 30 years, Gerald Joyce has been trying to create life. As a graduate student in the 1980s, he studied how the first RNA molecules — chemical cousins to DNA that can both store and transmit genetic information — might have assembled themselves out of simpler units, a process that many scientists believe led to the first living things.

    Unfortunately, he had a problem. At a chemical level, a deep bias permeates all of biology. The molecules that make up DNA and other nucleic acids such as RNA have an inherent “handedness.” These molecules can exist in two mirror image forms, but only the right-handed version is found in living organisms. Handedness serves an essential function in living beings; many of the chemical reactions that drive our cells only work with molecules of the correct handedness. But the pre-biological building blocks of life didn’t exhibit such an overwhelming bias. Some were left-handed and some right. So how did right-handed RNA emerge from a mix of molecules?

    Joyce was able to build RNA out of right-handed building blocks, as others had done before him. But when he added in left-handed molecules, mimicking the conditions on the early Earth, everything came to a halt. “Our paper said if you have [both] forms in the same place at the same time, you can’t even get started,” Joyce said.

    His findings, published in Nature in 1984, suggested that in order for life to emerge, something first had to crack the symmetry between left-handed and right-handed molecules, an event biochemists call “breaking the mirror.” Since then, scientists have largely focused their search for the origin of life’s handedness in the prebiotic worlds of physics and chemistry, not biology.

    m
    Olena Shmahalo / Quanta Magazine
    Many molecules come in mirror-image forms, known as left-handed and right-handed. A chemical process will create both forms of a given molecule, but a biological processes will produce just one.

    Three decades later, Joyce’s latest research has shown that perhaps life came first after all. Joyce, now at the Scripps Research Institute in La Jolla, Calif., and Jonathan Sczepanski, a postdoctoral researcher, created an RNA enzyme — a substance that copies RNA — that can function in a soup of left- and right-handed building blocks, providing a potential mechanism for how some of the first biological molecules might have evolved in a symmetrical world. The new experiment, published in the November 20 issue of Nature, is reinvigorating the discussion over how life first arose. “They have really opened up a new realm of possible roads,” said Niles Lehman, a biochemist at Portland State University in Oregon who wasn’t involved in the study.

    Even more intriguing, Joyce and Sczepanski’s enzyme works differently from other RNA-copying molecules, a discovery that may have profound implications for how life originated. The enzyme is much more efficient and flexible than other RNA-based enzymes developed to date, and it may provide the key to Joyce’s ultimate goal — making life from scratch.

    A Crack in the Mirror

    Louis Pasteur, the famous 19th-century French chemist, was the first to describe chemical handedness, or “chirality.” He was puzzled by the fact that crystals derived from the dregs of wine twisted light in a specific direction, but the same crystal synthesized in the lab did not. Examining the crystals under a microscope, he discovered that the synthetic chemical came in two mirror-image forms, which canceled out the polarizing effect. The crystal derived from wine had only one.

    Scientists later discovered that this bias encompasses the entire living world. Synthetic chemical processes will generate both left- and right-handed molecules. But when nature makes a molecule, the product is either left- or right-handed. For example, all amino acids that are used to make proteins twist light to the left.

    Indeed, chirality is an essential component of biochemistry. “It provides a form of molecular recognition,” said Donna Blackmond, a chemical engineer at Scripps and a colleague of Joyce’s. The chirality of a molecule affects how it interacts with other components of the cell. Molecular locks can only be opened with a key of the correct handedness.

    Some scientists look to the heavens to explain how this biological bias first arose. Some meteorites show a slight predominance of left-handed amino acids, the building blocks of proteins, suggesting that the influence came from outer space. An alternative cosmic origin story proposes that circularly polarized light coming from a supernova triggered a bias. In addition, radioactive decays produce electrons that are slightly more likely to be left-handed. Such electrons raining down on Earth’s surface might have changed its early chemistry.

    Yet most biologists and chemists are skeptical of these astrophysical theories. The bias they create is just too minute. The theories create “a beautiful union between life and nonlife,” said Marcelo Gleiser, a theoretical physicist at Dartmouth College. “But the problem is that those interactions are very weak and short-range.” According to Joyce, the effect of these physical forces would be lost in the noise of chemical reactions. “Such a small asymmetry in the universe is not enough to move the needle,” he said.

    Biochemists have tended to favor an alternative proposal, that a chance occurrence of prebiotic chemistry triggered an initial disequilibrium. Perhaps a slight excess of right-handed nucleotides was trapped and amplified in a shallow pool or some other prebiotic test tube. Eventually the bias reached a tipping point, breaking the chemical mirror and setting the stage for the emergence of life. Blackmond has done extensive work showing how to transform a small asymmetry to a nearly complete one using purely physical and chemical means.

    Shaking Both Hands

    When Joyce entered the field 30 years ago, researchers were already trying to test some of the astrophysical theories. But Joyce was skeptical. “I thought, why are you trying so hard to find a universal explanation when it’s probably chance?” he said.
    Gerald Joyce (right), a biochemist at the Scripps Research Institute, and postdoc Jonathan Sczepanski created an RNA enzyme that can replicate in an entirely new way.

    2
    Gerald Joyce (right), a biochemist at the Scripps Research Institute, and postdoc Jonathan Sczepanski created an RNA enzyme that can replicate in an entirely new way.
    Courtesy of The Scripps Research Institute.

    Around the same time, scientists were trying to figure out how the building blocks of life — amino acids and nucleic acids — could have spontaneously formed into more complex molecules such as proteins, DNA and RNA. Joyce thought that this assembly process might generate a crack in the mirror. A reaction that selectively plucked right-handed building blocks from the primordial soup would quickly start to create only right-handed molecules, just as a machine that selects only red or only blue Legos from a mixed box would create single-colored towers.

    Such a process would simultaneously solve two problems in the origins of life: It would create complex biological molecules while breaking the mirror. Joyce’s experiment in the 1980s set out to test that idea, but its failure called into question how right-handed RNA molecules could form from the ingredients of the primordial soup. “It was a mess,” Joyce said. “The left-handed building block poisons the growing chain.”

    The findings were particularly problematic for the nascent “RNA world” theory, which proposed that life began with an RNA molecule capable of replicating itself. RNA is the best candidate for the first biological molecule because it shares characteristics of both DNA and proteins. Like DNA, it carries information in its sequence of bases. And like an enzyme, it can catalyze chemical reactions. (RNA enzymes are known as ribozymes.)

    But if a ribozyme that copies RNA can’t function in a chemically symmetrical world, how could RNA-based life have emerged? “It’s kind of a showstopper,” said Peter Unrau, a biochemist at Simon Fraser University in Canada. In the decades since Joyce’s 1984 experiment, scientists have proposed myriad ways around the problem, from physical and chemical theories to RNA precursors that lack chirality.

    Given the known limitations, Joyce began to focus on creating a simple ribozyme that could copy RNA when only right-handed blocks were around. His group had some success, but none that fulfilled the requirements of the RNA world theory.

    So last year, Joyce and Sczepanski decided to start from scratch. They unleashed a pool of random right-handed RNA molecules and let them react in a test tube with left-handed building blocks. They hoped that within that random pool of RNA molecules was a ribozyme capable of stringing the building blocks together. They then isolated the best candidates — ribozymes that could copy RNA of the opposite handedness — replicated them, and subjected the new pool to the same trial over and over again.

    In just a few short months, they had a surprisingly effective ribozyme. The right-handed version binds to a left-handed RNA template and produces a left-handed copy. The left-handed copy can then go on to produce a right-handed version. “It’s amazing what they did,” said John Chaput, a biochemist at Arizona State University in Tempe. “It really does get to the heart of the question of the origins of chirality and provides some solid evidence to move things forward.”

    Perhaps even more exciting is how well the enzyme works. Other ribozymes created to date are too finicky to have spawned life; they replicate only certain RNA sequences, like soil that will grow potatoes but not carrots or peas. But Joyce’s ribozyme could produce a range of sequences — including its own. And it’s still getting better. The ribozyme in the paper emerged after just 16 rounds of evolution, a shockingly short run for this kind of experiment. Further rounds of evolution have already boosted its abilities, though these findings are not yet published. “The beautiful thing is that this is still a young enzyme,” Lehman said. “There’s lots of room for improvement.”

    The new ribozyme nearly fulfills the most basic properties of life — the ability to replicate and to evolve.

    The reason the new ribozyme works so well lies in the unusual way it operates. A regular ribozyme binds to its target according to its sequence of letters, like two sides of a zipper coming together. Sometimes it works too well, and the targets get stuck. This kind of binding only works with two molecules of the same handedness, which means Joyce’s ribozyme can’t bind this way.

    Instead, it binds based on the molecule’s shape rather than its sequence, an approach that turns out to be much more flexible. “They found something completely novel,” Lehman said. “It goes to show there’s a lot out there we don’t know.”

    Scientists now have an enzyme that doesn’t need a chiral world. Researchers, including Joyce himself, are still trying to understand the implications. The findings open the possibility that chirality emerged after life first evolved. “Maybe we didn’t need to break symmetry,” said Blackmond.

    Jack Szostak, a biochemist at Harvard University and one of Joyce’s collaborators, is excited by the findings, particularly because the ribozyme is so much more flexible than earlier versions. But, he said, “I am skeptical that life began in this way.” Szostak argues that this scenario would require both left-handed and right-handed RNA enzymes to have emerged at the same time and in the same place, which would be highly unlikely.

    Right-Handed Reign

    If chirality emerged sometime after the origins of life, the question remains: Why did right-handed RNA win? Left- and right-handed molecules have chemically identical properties, so there’s no obvious reason for one to triumph.

    Joyce and others suspect it’s simply chance. Say a ribozyme capable of transforming a pool of mixed nucleic acids into left- and right-handed RNAs appeared on the early Earth. It would produce two distinct groups, lefties and righties, which in turn might have functioned like competing populations. “If the right hand stumbles on useful mutations and runs away with the game, then the other side of the mirror can go dark,” Joyce said. For example, the right-handed group of RNAs might have developed some kind of competitive advantage, such as producing proteins, eventually overtaking the left-handed group and generating the bias we see today.

    There is only one way to truly determine whether one hand is superior: Build life forms that twist in each direction and evaluate them side by side. George Church and collaborators at Harvard are aiming to do just that. If they can make mirror versions of all the cells’ parts, they can construct synthetic cells and compare otherwise identical left- and right-handed versions of life.

    To create mirror-image RNAs, Church and his collaborators first need to make mirror enzymes capable of stitching together mirror building blocks. Michael Kay’s team at the University of Utah has almost finished developing a method for chemically synthesizing an ordinary version of one such enzyme. Once completed, the two teams will apply the same approach to make a mirror enzyme capable of assembling mirror RNAs. Church and others are also building tools to detect mirror life, which could prove important when searching for signs of life on other planets.

    Joyce remains interested in making life from scratch. Everything else, including the chirality problem, is just a hurdle toward that larger prize, he said.

    The new ribozyme may provide the best shot yet. It nearly fulfills the most basic properties of life — the ability to replicate and to evolve. “They went so far as to show the mirror image can copy itself,” Chaput said. “That gets very close to replication.” The next step will be to make that happen iteratively. “If you look in the mirror, make a copy, then put yourself in the mirror, and make a copy of the person in the mirror, then you have replication,” Chaput said.

    That iterative process opens the possibility for evolution, as mistakes made during copying will allow the molecule to evolve new traits. “The real key to all of it has been setting up a system in the lab capable of evolution on its own,” Unrau said. “Jerry is close.”

    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.

     
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