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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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    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?

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    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

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    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

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    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.

     
  • richardmitnick 8:43 pm on October 3, 2014 Permalink | Reply
    Tags: , , , RNA   

    From LBL: “News Center RCas9: A Programmable RNA Editing Tool” 

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    Berkeley Lab

    October 3, 2014
    Lynn Yarris (510) 486-5375

    A powerful scientific tool for editing the DNA instructions in a genome can now also be applied to RNA, the molecule that translates DNA’s genetic instructions into the production of proteins. A team of researchers with Berkeley Lab and the University of California (UC) Berkeley has demonstrated a means by which the CRISPR/Cas9 protein complex can be programmed to recognize and cleave RNA at sequence-specific target sites. This finding has the potential to transform the study of RNA function by paving the way for direct RNA transcript detection, analysis and manipulation.

    sch
    Schematic shows how RNA-guided Cas9 working with PAMmer can target ssRNA for programmable, sequence-specific cleavage.

    Led by Jennifer Doudna, biochemist and leading authority on the CRISPR/Cas9 complex, the Berkeley team showed how the Cas9 enzyme can work with short DNA sequences known as “PAM,” for protospacer adjacent motif, to identify and bind with specific site of single-stranded RNA (ssRNA). The team is designating this RNA-targeting CRISPR/Cas9 complex as RCas9.

    “Using specially designed PAM-presenting oligonucleotides, or PAMmers, RCas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, or it can be used to isolate specific endogenous messenger RNA from cells,” says Doudna, who holds joint appointments with Berkeley Lab’s Physical Biosciences Division and UC Berkeley’s Department of Molecular and Cell Biology and Department of Chemistry, and is also an investigator with the Howard Hughes Medical Institute (HHMI). “Our results reveal a fundamental connection between PAM binding and substrate selection by RCas9, and highlight the utility of RCas9 for programmable RNA transcript recognition without the need for genetically introduced tags.”

    jd
    Biochemist Jennifer Doudna is leading authority on the CRISPR/Cas9 complex (Photo by Roy Kaltschmidt)

    From safer, more effective medicines and clean, green, renewable fuels, to the clean-up and restoration of our air, water and land, the potential is there for genetically engineered bacteria and other microbes to produce valuable goods and perform critical services. To exploit the vast potential of microbes, scientists must be able to precisely edit their genetic information.

    In recent years, the CRISPR/Cas complex has emerged as one of the most effective tools for doing this. CRISPR, which stands for Clustered Regularly Interspaced Short Palindromic Repeats, is a central part of the bacterial immune system and handles sequence recognition. Cas9 – Cas stands for CRISPR-assisted – is an RNA-guided enzyme that handles the sniping of DNA strands at the specified sequence site.

    Together, CRISPR and Cas9 can be used to precisely edit the DNA instructions in a targeted genome for making desired types of proteins. The DNA is cut at a specific location so that old DNA instructions can be removed and/or new instructions inserted.

    Until now, it was thought that Cas9 could not be used on the RNA molecules that transcribe those DNA instructions into the desired proteins.

    “Just as Cas9 can be used to cut or bind DNA in a sequence-specific manner, RCas9 can cut or bind RNA in a sequence-specific manner,” says Mitchell O’Connell, a member of Doudna’s research group and the lead author of a paper in Nature that describes this research titled Programmable RNA recognition and cleavage by CRISPR/Cas9. Doudna is the corresponding author. Other co-authors are Benjamin Oakes, Samuel Sternberg, Alexandra East Seletsky and Matias Kaplan.

    two
    Benjamin Oakes and Mitch O’Connell are part of the collaboration led by Jennifer Doudna that showed how the CRISPR/Cas9 complex can serve as a programmable RNA editor. (Photo by Roy Kaltschmidt)

    In an earlier study, Doudna and her group showed that the genome editing ability of Cas9 is made possible by presence of PAM, which marks where cutting is to commence and activates the enzyme’s strand-cleaving activity. In this latest study, Doudna, Mitchell and their collaborators show that PAMmers, in a similar manner, can also stimulate site-specific endonucleolytic cleavage of ssRNA targets. They used Cas9 enzymes from the bacterium Streptococcus pyogenes to perform a variety of in vitro cleavage experiments using a panel of RNA and DNA targets.

    “While RNA interference has proven useful for manipulating gene regulation in certain organisms, there has been a strong motivation to develop an orthogonal nucleic-acid-based RNA-recognition system such as RCas9,” Doudna says. “The molecular basis for RNA recognition by RCas9 is now clear and requires only the design and synthesis of a matching guide RNA and complementary PAMmer.”

    The researchers envision a wide range of potential applications for RCas9. For example, an RCas9 tethered to a protein translation initiation factor and targeted to a specific mRNA could essentially act as a designer translation factor to “up-” or “down-” regulate protein synthesis from that mRNA.

    “Tethering RCas9 to beads could be used to isolate RNA or native RNA–protein complexes of interest from cells for downstream analysis or assays,” Mitchell says. “RCsa9 fused to select protein domains could promote or exclude specific introns or exons, and RCas9 tethered to a fluorescent proteins could be used to observe RNA localization and transport in living cells.”

    This research was primarily supported by the NIH-funded Center for RNA Systems Biology.

    See the full article here.

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  • richardmitnick 8:50 am on September 1, 2014 Permalink | Reply
    Tags: , , , , RNA   

    From Astrobio: “DNA May Have Had Humble Beginnings As Nutrient Carrier” 

    Astrobiology Magazine

    Astrobiology Magazine

    Sep 1, 2014
    Adam Hadhazy

    New research intriguingly suggests that DNA, the genetic information carrier for humans and other complex life, might have had a rather humbler origin. In some microbes, a study shows, DNA pulls double duty as a storage site for phosphate. This all-important biomolecule contains phosphorus, a sometimes hard-to-get nutrient.

    dna

    Maintaining an in-house source of phosphate is a newfound tactic for enabling microorganisms to eke out a living in harsh environments, according to a new study published in the open-access, peer reviewed scientific journal PLOS ONE. The finding bodes well for life finding a way, as it were, in extreme conditions on worlds less hospitable than Earth.

    The results also support a second insight: DNA might have come onto the biological scene merely as a means of keeping phosphate handy. Only later on in evolutionary history did the mighty molecule perhaps take on the more advanced role of genetic carrier.

    “DNA might have initially evolved for the purpose of storing phosphate, and the various genetic benefits evolved later,” said Joerg Soppa, senior author of the paper and a molecular biologist at Goethe University in Frankfurt, Germany.

    Unraveling life’s origins

    Scientists continue to investigate the development of self-replicating, intricate sets of chemistry — in other words, life — from the chemical compounds thought available on early Earth. Out of this mixture of prebiotic chemicals, two nucleic acids — RNA and DNA — emerged as champions.

    early
    Early Earth, in an artist’s impression, where somehow complex, self-replicating chemistry (in other words, life) emerged. Credit: Peter Sawyer / Smithsonian Institution

    Today, these two types of biomolecules serve as the genetic information carriers for all Earthly biota. RNA on its own suffices for the business of life for simpler creatures, such as some viruses. Complex life, like humans, however, relies on DNA as its genetic carrier.

    Astrobiologists want to understand the origin of DNA and its genetic cousin, RNA, because figuring out how life got started here on Earth is key for gauging if it might ever develop on alien planets.

    Many researchers think RNA must have preceded DNA as the genetic molecule of choice. RNA is more versatile, acting as both genetic code and a catalyst for chemical reactions. Explicating the rise of DNA as a genetic material directly from RNA, however, is tricky. Compared to RNA, DNA needs significantly more supporting players for it to work well in a biological setting.

    “The switch from RNA to DNA is not easy because many additional enzymes are required for DNA genomes,” said Soppa.

    This unclear transition from RNA to DNA opens the door for a precursor to DNA possibly having a more mundane job. The new study offers an attractive explanation: that DNA was a fancy way to store nutrients in cells.

    Phosphate depot?

    DNA is chock-full of phosphate. Cells depend on phosphate to form not only DNA and RNA, but also related genetic machinery, such as the ribosome. Phosphate, furthermore, is a must for building the molecule ATP, life’s energy carrier, as well as fatty membrane molecules, certain phospho-proteins and phospho-sugars, and more.

    ds
    The shores of the Dead Sea, which borders Jordan, Palestine and Israel. As the lowest and saltiest lake in the world, it is home to some extreme creatures. Image Credit: Aaron L. Gronstal

    “Phosphate is important for an immense set of biomolecules,” said Soppa.

    Unfortunately for some microbes, ample phosphate is not always available. For example, in salty, nutrient-poor habitats, such as the Dead Sea in the Middle East, an organism called Haloferax volcanii must regularly “eat” ambient DNA to obtain phosphate (plus some other nutritional goodies, such as nitrogen).

    Notably, H. volcanii can still survive and reproduce when phosphorus, the element needed to make phosphate, is lacking. Somehow, then, the microbe must turn to an inner source of phosphate, for otherwise it should cease to grow.

    In their study, Soppa and colleagues from Germany, the United States and Israel sought out this source. The nature of H. volcanii provided some clues. The organism is classified as archaea, one of the three domains of life, in addition to bacteria and eukarya, the latter encompassing all multicellular organisms, from fungi to fruit flies. Many archaea and bacteria — collectively, “prokaryotes”— have just one, circular chromosome. Eukaryotes, like us, on the other hand, can have any number of the chunky pieces of DNA, RNA and proteins. (Humans have 23 pairs of different chromosomes, for the record.) H. volcanii is unusual. It has 20 copies of the same chromosome when it’s growing happily under favorable conditions, and 10 when nutrients are exhausted and it reaches a stationary phase.

    Strength in numbers

    Lots of chromosome copies are good to have in a pinch. So-called polyploidal organisms like H. volcanii use their copious chromosomes to tough it out through bad situations, such as high radiation exposure or total dry-outs, called desiccation. Either scenario causes the strands in chromosomal DNA to break. For single-chromosome species, only a few breaks lead to death because it is impossible to repair a chromosome scattered into fragments.

    But if there are multiple copies of the cracked chromosomes, fragments can fortuitously line up. Rather like how a jigsaw puzzle is easier to put together if there are numerous duplicates of each necessary piece, the chromosome shards can sync up and restore a functional chromosome.

    hv
    H. Volcanii grown in culture. Credit: Yejineun/Wikipedia

    “In polyploid species, the fragments generated from different copies of the chromosome overlap, and it is possible to regenerate an intact chromosome from overlapping fragments,” said Soppa.

    Desperate times, desperate measures

    To investigate if H. volcanii‘s extra chromosomes might help the archaeon survive low phosphate conditions, Soppa and colleagues starved the organism in the lab of the critical substance. The microbe continued to reproduce by splitting one cell apart into two. Interestingly, chromosome counts diminished in the “parent” and the “daughter” cells.

    “From quantifying the number of chromosomes prior to and after growth in the absence of phosphate, we have found that about 30 percent of the chromosomes are ‘missing’ afterwards,” said Soppa.

    The numbers for another potential in-house source of phosphate, H. volcanii‘s ribosomes, however, remained steady. The most likely explanation, then, of the microorganism’s hardiness when facing a phosphate nutrient shortage: H. volcanii simply cannibalizes some of its own chromosomes.

    As further verification, Soppa and colleagues tested the survival skills of H. volcanii cells that contained varying numbers of chromosome copies. Those archaea with just two copies of their chromosome turned out to be more than five times as sensitive to desiccation as those H. volcanii with a hefty complement of 20 chromosomes.

    Life, undaunted

    This newly described benefit of polyploidy in H. volcanii is a fresh demonstration of how life can make do in severe environments. So-called extremophiles have been discovered in recent decades thriving in strongly acidic hot springs, within liquid asphalt, and in other eyebrow-raising niches. Salt-tolerant bacteria and archaea, like H. volcanii, have been found to survive in deserts, simulated Mars conditions, and even the rigors of a space flight. We should not be surprised, perhaps, if life has managed to take hold on formidable worlds.

    rw
    Extremophile microbes have been found that can survive in the polluted Rio Tinto River in Spain. Mining in the river’s vicinity has led to its waters having a high heavy metal content and very low pH, though the bacteria themselves, through their metabolism, also likely contribute to the intense acidity. Image credit: Leslie Mullen

    “The understanding of how harsh the conditions can be that can be survived by some archaea and bacteria helps us to be more optimistic that life could have evolved at very rough and unsuitable places on early Earth or on other planets,” said Soppa.

    The new role ascribed to DNA, as phosphate storage, might help to explain how a completely RNA-dominated primordial era began sharing genetic duties with DNA. Life did not leap from RNA to DNA. Rather, DNA, slowly but surely, learned new tricks.

    “The hypothesis that DNA might have evolved as a storage polymer and became genetic material later, makes the step from RNA to DNA as genetic material easier, because it then would be a two-step and not a one-step process,” said Soppa. “DNA would have been around, and during long time spans additional roles could have been evolved.”

    See the full article here.
    Astrobiology Magazine is a NASA-sponsored online popular science magazine. Our stories profile the latest and most exciting news across the wide and interdisciplinary field of astrobiology — the study of life in the universe. In addition to original content, Astrobiology Magazine also runs content from non-NASA sources in order to provide our readers with a broad knowledge of developments in astrobiology, and from institutions both nationally and internationally. Publication of press-releases or other out-sourced content does not signify endorsement or affiliation of any kind.
    Established in the year 2000, Astrobiology Magazine now has a vast archive of stories covering a broad array of topics.

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