Un-junking Junk DNA

Un-junking Junk DNA

Wednesday, 13 November 2013

A study led by researchers at the University of California, San Diego School of Medicine shines a new light on molecular tools our cells use to govern regulated gene expression. The study was published on line in advance of print November 10 in the journal Nature Structural and Molecular Biology.

"We uncovered a novel mechanism that allows proteins that direct pre-mRNA splicing – RNA-binding proteins – to induce a regulatory effect from greater distances than was thought possible," said first author Michael T. Lovci, of the Department of Cellular and Molecular Medicine, the Stem Cell Research Program and Institute for Genomic Medicine at UC San Diego.

Researchers from California, Oregon, Singapore and Brazil made this finding while working toward an understanding of the most basic signals that direct cell function. According to Lovci, the work broadens the scope that future studies on the topic must consider. More importantly, it expands potential targets of rationally designed therapies which could correct molecular defects through genetic material called antisense RNA oligonucleotides (ASOs).

"This study provides answers for a decade-old question in biology," explained principal investigator Gene Yeo, PhD, assistant professor of Cellular and Molecular Medicine, member of the Stem Cell Research Program and Institute for Genomic Medicine at UC San Diego, as well as with National University of Singapore.

"When the sequence of the human genome was fully assembled, under a decade ago, we learned that less than 3 percent of the entire genome contains information that encodes for proteins. This posed a difficult problem for genome scientists – what is the other 97 percent doing?"

The role of the rest of the genome was largely a mystery and was thus referred to as "junk DNA." Since then sequencing of other, non-human, genomes has allowed scientists to delineate the sequences in the genome that are remarkably preserved across hundreds of millions of years of evolution. It is widely accepted that this evidence of evolutionary constraint implies that, even without coding for protein, certain segments of the genome are vital for life and development.

Using this evolutionary conservation as a benchmark, scientists have described varied ways cells use these non-protein-coding regions. For instance, some exist to serve as DNA docking sites for proteins which activate or repress RNA transcription. Others, which were the focus of this study, regulate alternative mRNA splicing.

Eukaryotic cells use alternative pre-mRNA splicing to generate protein diversity in development and in response to the environment. By selectively including or excluding regions of pre-mRNAs, cells make on average ten versions of each of the more than 20,000 genes in the genome. RNA-binding proteins are the class of proteins most closely linked to these decisions, but very little is known about how they actually perform their roles in cells.

"For most genes, protein-coding space is distributed in segments on the scale of islands in an ocean," Lovci said.

"RNA processing machinery, including RNA-binding proteins, must pick out these small portions and accurately splice them together to make functional proteins. Our work shows that not only is the sequence space nearby these 'islands' important for gene regulation, but that evolutionarily conserved sequences very far away from these islands are important for coordinating splicing decisions."

Since this premise defies existing models for alternative splicing regulation, whereby regulation is enacted very close to protein-coding segments, the authors sought to define the mechanism by which long-range splicing regulation can occur. They identified RNA structures – RNA that is folded and base-paired upon itself – that exist between regulatory sites and far-away protein-coding "islands." Dubbing these types of interactions "RNA-bridges" for their capacity to link distant regulators to their targets, the authors show that this is likely a common and under-appreciated mechanism for regulation of alternative splicing.

These findings have foreseeable implications in the study of biomedicine, the researchers said, as the RNA-binding proteins on which they focused – RBFOX1 and RBFOX2 – show strong associations with neurodevelopmental disorders, such as autism and also certain cancers. Since these two proteins act upstream of a cascade of effects, understanding how they guide alternative splicing decisions may lead to advancements in targeted therapies which correct the inappropriate splicing decisions that underlie many diseases.

Source: University of California - San Diego 

Contact: Debra Kain

Reference:

Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges

Michael T Lovci, Dana Ghanem, Henry Marr, Justin Arnold, Sherry Gee, Marilyn Parra, Tiffany Y Liang, Thomas J Stark, Lauren T Gehman, Shawn Hoon, Katlin B Massirer, Gabriel A Pratt, Douglas L Black, Joe W Gray, John G Conboy & Gene W Yeo

Nature Structural & Molecular Biology, 10 November 2013 | doi:10.1038/nsmb.2699

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Scientists Identify Wide Variety of Gene Splicing in Embryonic Stem Cells

Scientists Identify Wide Variety of Gene Splicing in Embryonic Stem Cells Monday, 01 March 2010 Like homing in to an elusive radio frequency in a busy city, human embryonic stem cells must sort through a seemingly endless number of options to settle on the specific genetic message, or station, that instructs them to become more-specialized cells in the body (Easy Listening, maybe, for skin cells, and Techno for neurons?). Now researchers at the Stanford University School of Medicine have shown that this tuning process is accomplished in part by restricting the number of messages, called transcripts, produced from each gene. Most genes can yield a variety of transcripts through a process called splicing. Variations in the ways a gene is spliced can change the form and function of the final protein product. Nearly all our genes can be spliced in more than one way. This research is the first time, however, that splicing variety has been linked to the unprecedented developmental flexibility, or pluripotency, exhibited by embryonic stem cells. "The embryonic stem cells are loaded with many splicing variants for each gene," said Michael Snyder, PhD, chair of Stanford's genetics department. "But as the cells differentiate and become more specialized, the number of types of transcripts decreases." Snyder and his colleagues studied the changes in RNA transcript levels occurring as the embryonic stem cells were induced in a laboratory dish to differentiate into neural cells. (The creation of RNA transcripts is an intermediate step in the generation of proteins from DNA.) In the process they generated a unique "dictionary" of neural-specific splicing variants, or isoforms. "We've identified an extremely comprehensive suite of neural-specific transcripts that will be very powerful," said Snyder. "We can begin to study neural differentiation with a degree of precision that's never been dreamed of before." Snyder is the Stanford W. Ascherman, MD, FACS, Professor in Genetics and a member of Stanford's Cancer Center. He is the senior author of the research, which will be published online March 1 in the Proceedings of the National Academy of Sciences. The study's first author is postdoctoral scholar Jia Qian Wu, PhD. One way to understand gene splicing is to think of it like this: Genes are made up of several "words" of DNA called exons. Intervening bits of unexpressed DNA separate these exons from one another on the cell’s raw genetic material. By changing the way the exons are joined, or spliced, together in the final RNA transcript, the cell can generate several related, yet distinct, protein products, or "sentences" from each gene. These RNA variants are called RNA isoforms — and they are important in many biological processes, from generating antibodies to detoxifying drugs. Snyder and Wu used a method of RNA sequencing Snyder invented while at Yale University called RNA-Seq to track the many RNA isoforms found at varying levels in human embryonic stem cells. The technique can identify a much greater range of RNA transcript levels and is much more sensitive than more traditional methods like DNA microarray analysis. That means it is possible to more reliably detect rare isoforms, and, as a result, more accurately plumb the secret transcriptional life of an embryonic stem cell — that turns out to be richer than previously imagined. "The average human gene is known to have four or five transcripts," said Snyder. "But that number will likely go much higher now with this new technology. We are measuring these with a degree of specificity that's never been possible before." Choosing which genes to express, and then how to splice those genes, adds a layer of complexity that allows a cell to fine-tune its final protein profile. The researchers chose to study neural differentiation in a laboratory dish, rather than in the brain, because it is possible to start with and follow populations of purified cells. They monitored the variety of RNA isoforms found in the human embryonic stem cells and compared them to those found in the cells as they were coaxed through three stages of differentiation into neural cells called glia. At each stage, they found, the variety of isoforms in the cells decreased — a phenomenon they termed "isoform specialization" — as they settled into their chosen station. When the researchers looked more closely, they saw that the isoforms remaining were involved in key neural signalling pathways or cellular receptors. Furthermore, at the earliest stages of their differentiation, the nascent glial cells contain isoforms for receptors found on many other types of neural cells — suggesting they could be induced down several other developmental pathways. Finally, the value of the researcher's transcript "dictionary" is hinted at by the finding that the timing of expression of two genes important in neural differentiation — SOX1 and PAX6 — in humans is different from that observed in mice. Reference: Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing Jia Qian Wu, Lukas Habegger, Parinya Noisa, Anna Szekely, Caihong Qiu, Stephen Hutchison, Debasish Raha, Michael Egholm, Haifan Lin, Sherman Weissman, Wei Cui, Mark Gerstein, and Michael Snyder PNAS published online before print March 1, 2010, doi:10.1073/pnas.0914114107 ......... ZenMaster

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Human Genes Sing Different Tunes in Different Tissues

Biologists find almost all genes express multiple messenger RNAs Sunday, 02 November 2008 Scientists have long known that it's possible for one gene to produce slightly different forms of the same protein by skipping or including certain sequences from the messenger RNA. Now, an MIT team has shown that this phenomenon, known as alternative splicing, is both far more prevalent and varies more between tissues than was previously believed. Nearly all human genes, about 94 percent, generate more than one form of their protein products, the team reports in the Nov. 2 online edition of Nature. Scientists' previous estimates ranged from a few percent 10 years ago to 50-plus percent more recently. "A decade ago, alternative splicing of a gene was considered unusual, exotic … but it turns out that's not true at all — it's a nearly universal feature of human genes," said Christopher Burge, senior author of the paper and the Whitehead Career Development Associate Professor of Biology and Biological Engineering at MIT. Burge and his colleagues also found that in most cases the mRNA produced depends on the tissue where the gene is expressed. The work paves the way for future studies into the role of alternative proteins in specific tissues, including cancer cells. They also found that different people's brains often differ in their expression of alternative spliced mRNA isoforms. Human genes typically contain several "exons," or DNA sequences that code for amino acids, the building blocks of proteins. A single gene can produce multiple protein sequences, depending on which exons are included in the mRNA transcript, which carries instructions to the cell's protein-building machinery. Two different forms of the same protein, known as isoforms, can have different, even completely opposite functions. For example, one protein may activate cell death pathways while its close relative promotes cell survival. The researchers found that the type of isoform produced is often highly tissue-dependent. Certain protein isoforms that are common in heart tissue, for example, might be very rare in brain tissue, so that the alternative exon functions like a molecular switch. Scientists who study splicing have a general idea of how tissue-specificity may be achieved, but they have much less understanding of why isoforms display such tissue specificity, Burge said. Scientists have also observed that cells express different isoforms during embryonic development and at different stages of cellular differentiation. Burge's team is now studying cells at various stages of differentiation to see when different isoforms are expressed. Isoform switching also occurs in cancer cells. One such switch involves a metabolic enzyme and contributes to cancer cells burning large amounts of glucose and growing more rapidly. Learning more about such switches could lead to potential cancer therapies, Burge said. Until now, it has been difficult to study isoforms on a genome-wide scale because of the high cost of sequencing and technical issues in discriminating similar mRNA isoforms using microarrays. The team took mRNA samples from 10 types of tissue and five cell lines from a total of 20 individuals, and generated more than 13 billion base pairs of sequence, the equivalent of more than four entire human genomes. The sequencing was done by researchers at biotech firm Illumina, using a new high-throughput sequencing machine. Reference: Alternative isoform regulation in human tissue transcriptomes Eric T. Wang, Rickard Sandberg, Shujun Luo, Irina Khrebtukova, Lu Zhang, Christine Mayr, Stephen F. Kingsmore, Gary P. Schroth & Christopher B. Burge Nature advance online publication 2 November 2008, doi:10.1038/nature07509 See also: Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing Qun Pan, Ofer Shai, Leo J Lee, Brendan J Frey & Benjamin J Blencowe Nature Genetics, Published online: 2 November 2008, doi:10.1038/ng.259 ......... ZenMaster

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Panoramic View of Protein-RNA Interactions in Living Cells

Panoramic View of Protein-RNA Interactions in Living Cells Sunday, 02 November 2008 DNA, it has turned out, is not all it was cracked up to be. In recent years we learned that the molecule of life, the discovery of the 20th century, did not – could not – by itself explain the huge differences in complexity between a human and a worm. Forced to look elsewhere, scientists turned to RNA, a direct yet more complex transcript of DNA. But methodological problems have historically plagued the study of RNA regulation in living cells, limiting not only the accuracy of results but also our understanding of RNA's role in human disease. But now, in research to appear in the November 2 advance online issue of Nature, Robert B. Darnell, head of the Laboratory of Molecular Neuro-oncology at Rockefeller University and a Howard Hughes Medical Institute investigator, and his team have changed all that. By adapting techniques mastered in the test tube and combining them with high throughput technology, the team has developed a genome-wide platform to study how specialized proteins regulate RNA in living, intact cells. The platform allows researchers to identify, in a single experiment, every sequence within every strand of RNA to which proteins bind. The result is an unbiased and unprecedented look at how differences in RNA can explain how a worm and a human can each have 25,000 genes yet be so different. "RNA offers a way to make the cell much more complex than what this limited set of genes can offer," says Darnell, who is Robert and Harriet Heilbrunn Professor at Rockefeller. "But how is RNA being regulated in different conditions and diseases, and in different cell types? With this platform, we now have a way to address all these questions." Traditional methods used molecules to extract protein-RNA complexes from living tissue. But often the molecule only extracted the RNA. Other times, the protein bound too weakly to survive the purification process, which involved stripping the complex of unwanted debris. To address the issue, Darnell and his team used a trick from test-tube biochemistry that molecularly cements these regulatory proteins to RNA at the moment they touch. The technique, when applied to high throughput sequencing, is called high throughput sequencing-cross linking immunoprecipitation, or HITS-CLIP for short. Since the RNA and RNA-binding protein are fused together, the researchers can really beat up the extract and rigorously purify the protein without fear of losing the RNA. At the end of the day, they are left with the RNA sequence to which the protein was bound. They can then take these sequences to Rockefeller's high throughput sequence facility, and with the help of Research Support Specialist Scott Dewell, overlay them onto the genome and see where they match. What they get is a map of every position on every transcribed RNA where the RNA binding protein is binding. When DNA is transcribed into RNA, the primary transcript is divided into many blocks called exons, which are separated by empty spaces. In order to convert the transcript into some sort of message, all the spaces need to be removed; but if an exon is dropped, a different version of that protein, which could carry a very different message, is created. "That's RNA splicing," says first author Donny Licatalosi, a postdoctoral associate in the lab. "It is what gives rise to this massive pool of diverse and complex tissues with a relatively small number of genes." In the past, the group used a sophisticated process of evidence and inference to make predictions of the points of regulation along the transcript. "Now, we have direct biochemical validation that these interactions occur in the brain to regulate splicing," says Licatalosi. "The observed map – and this was amazing – looked just like our predicted map," says Darnell. Darnell, Licatalosi and their colleagues Aldo Mele, a research assistant, John Fak, a research assistant, Sung-Wook Chi, a graduate fellow in computational biology and medicine, Xuning Wang, assistant director of biocomputing and Jennifer Darnell, a research associate professor, looked at an RNA-binding protein called Nova2 that is found exclusively in neurons. They found that depending on where Nova2 binds to RNA, they could predict and directly observe whether an exon would be included or excluded in the final transcript, and which protein version it created. "The cell seems to be going through great trouble to regulate these RNAs in different conditions and different cell types," says Darnell. "When RNA developed the ability to make a more stable copy of itself – DNA – it didn't write itself off as a relic for the textbooks. It stayed at the core of complex processes in the cell." Reference: HITS-CLIP yields genome-wide insights into brain alternative RNA processing Donny D. Licatalosi, Aldo Mele, John J. Fak, Jernej Ule, Melis Kayikci, Sung Wook Chi, Tyson A. Clark, Anthony C. Schweitzer, John E. Blume, Xuning Wang, Jennifer C. Darnell & Robert B. Darnell Nature advance online publication 2 November 2008, doi:10.1038/nature07488 ......... ZenMaster

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Study Finds Value in 'Junk' DNA

Significant number of exons created from junk DNA seem to play a role in gene regulation Friday, 17 October 2008 For about 15 years, scientists have known that certain "junk" DNA — repetitive DNA segments previously thought to have no function — could evolve into exons, which are the building blocks for protein-coding genes in higher organisms like animals and plants. Now, a University of Iowa study has found evidence that a significant number of exons created from junk DNA seem to play a role in gene regulation. The findings, which increase understanding of how humans differ from other animals, including non-human primates, appear Oct. 17 in the open-access journal PLoS Genetics. Nearly half of human DNA consists of repetitive DNA, including transposons, which can "transpose" or move around to different positions within the genome. A type of transposon called retrotransposons are transcribed into RNA and then reintegrated into the genomic DNA. The most common form of retrotransposons in the human genome are Alu elements, which have more than one million copies and occupy approximately 10 percent of the human genome. "Alu elements are a major source of new exons. Because Alu is a primate-specific retrotransposon, creation of new exons from Alu may contribute to unique traits of primates. We want to better understand this process," said the study's senior author Yi Xing, Ph.D., assistant professor of internal medicine and biomedical engineering, who holds a joint appointment in the University of Iowa Carver College of Medicine and the UI College of Engineering. To study the impact of Alu-derived exons on human gene expression, the researchers used a high-density exon microarray. The technology has nearly six million probes for monitoring the expression patterns of all human exons. Using data generated by these microarrays, the scientists analyzed 330 Alu-derived exons in 11 human tissues. The team then identified a number of exons with interesting expression and functional characteristics. "Hundreds of exons in the human genome were created from Alu elements. The whole-genome exon microarray allowed us to quickly identify exons that most likely contribute to the regulation of gene expression and function," said Lan Lin, Ph.D., University of Iowa postdoctoral fellow in internal medicine and the lead author of this study. Analysis of one human gene, SEPN1, which is known to be involved in a type of muscular dystrophy, along with comparative data from chimpanzee and macaque tissues, suggested that the presence of a muscle-specific Alu-derived exon resulted from a human-specific change that occurred after humans and chimpanzees diverged evolutionarily. "In this case, this exon is only expressed at a high level in the human muscle but not in any other human or non-human primate tissue, so this implies that the exon plays a functional role in muscle, and this role is human-specific," said Xing, who is also affiliated with University of Iowa Center for Bioinformatics and Computational Biology. Reference: Diverse Splicing Patterns of Exonized Alu Elements in Human Tissues Lan Lin, Shihao Shen, Anne Tye, James J. Cai, Peng Jiang, Beverly L. Davidson, Yi Xing PLoS Genet 4(10): e1000225. doi:10.1371/journal.pgen.1000225 See also: More 'Junk' DNA Proves Functional CellNEWS - Tuesday, 04 November 2008 ......... ZenMaster

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Deep Sequencing Reveals New Insights into Human Transcriptome

Joint project takes the first step towards a new picture of the mammalian genome annotation Tuesday, 08 July 2008 In collaborative project scientists from the Max-Planck-Institute for Molecular Genetics in Berlin (MPI MolGen), Germany and Genomatix with a business in Munich, Germany and Ann Arbor, MI, USA, applied next generation sequencing and analysis methods to generate an unprecedented view at the human transcriptome. Deep sequencing of transcripts from two human cell lines revealed so far unrecognized complexity and variability of the human transcriptome. They found that 34% of the polyadenylated transcriptome mapped to so far non-annotated genomic regions. Obviously a large number of novel gene candidates are active in the cell lines under study. In addition, a global survey of mRNA splicing events identified 94,241 splice junctions, of which 4,096 are novel, and showed that exon skipping is the most prevalent form of alternative splicing. Dr. Marie-Laure Yaspo, Group Leader at the MPI MolGen and head scientist of the study states: "Deep sequencing allows for the first time to explore directly the complexity and dynamics of the human transcriptome with a reasonable effort. This will lead to a new picture of the mammalian genome annotation far beyond the current state of the art. We provide here global features of alternative splicing events in human cell lines. Such a comparison of within-cell and between-cell alternative splicing events, combined with the simultaneous analysis of gene expression has never been presented before. It becomes clear that the so far available methods only delivered a part of the transcriptional landscape of mammalian cells, especially if gene regulation analysis is considered" Dr. Martin Seifert, Vice President Business Development and Consulting at Genomatix says: "The main biological impact is the observation of a new dimension in complexity and variability. Based on the method we could find a significant number of new transcriptional units and splice variants. Our analyses clearly show that transcription is a highly dynamic and variable process. We learned a lot by having access to such high quality data and co-developed necessary new analysis strategies with the MPI MolGen. Especially users of our brand new Genomatix Genome Analyzer will benefit from our experiences along the project, since they have access to all developed strategies." Annotation and data visualization is publicly available at http://www.genomatix.de/MPI.html. Reference: A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome Marc Sultan, Marcel H. Schulz, Hugues Richard, Alon Magen, Andreas Klingenhoff, Matthias Scherf, Martin Seifert, Tatjana Borodina, Aleksey Soldatov, Dmitri Parkhomchuk, Dominic Schmidt, Sean O’Keeffe, Stefan Haas, Martin Vingron, Hans Lehrach, Marie-Laure Yaspo Science Published Online July 3, 2008, DOI: 10.1126/science.1160342 ......... ZenMaster

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Unravelling new complexity in the genome

Unravelling new complexity in the genome Tuesday, 14 August 2007

A major surprise emerging from genome sequencing projects is that humans have a comparable number of protein-coding genes as significantly less complex organisms such as the minute nematode worm Caenorhabditis elegans. Clearly something other than gene count is behind the genetic differences between simpler and more complex life forms.

Increased functional and cellular complexity can be explained, in large part, by how genes and the products of genes are regulated. A University of Toronto-led study published in the latest issue of Genome Biology reveals that a step in gene expression (referred to as alternative splicing) is more highly regulated in a cell and tissue-specific manner than previously appreciated and much of this additional regulation occurs in the nervous system. The alternative splicing step allows a single gene to specify multiple protein products by processing the RNA transcripts made from genes (which are translated to make protein).

“We are finding that a significant number of genes operating in the same biological processes and pathways are regulated by alternative splicing differently in nervous system tissues compared to other mammalian tissues,” says lead investigator Professor Benjamin Blencowe of the Banting and Best Department of Medical Research and Centre for Cellular and Biomolecular Research (CCBR) at the University of Toronto

According to Blencowe, it is particularly interesting that many of the genes have important and specific functions in the nervous system, including roles associated with memory and learning. However, in most cases the investigators working on these genes were not aware that their favourite genes are regulated at the level of splicing. Blencowe believes that the data his group has generated provides a valuable basis for understanding molecular mechanisms by which genes can function differently in different parts of the body.

Blencowe attributes these new findings in part to the power of a new tool that he, together with his colleagues including Profs. Brendan Frey (Department of Electrical and Computer Engineering) and Timothy Hughes (Banting and Best, CCBR), developed a few years ago. This tool, which comprises tailored designed microarrays or “gene chips” and computer algorithms, allows the simultaneous measurement of thousands of alternative splicing events in cells and tissues.

“Until recently researchers studied splicing regulation on a gene by gene basis. Now we can obtain a picture of what is happening on a global scale, which provides a fascinating new perspective on how genes are regulated,” Blencowe explains.

A challenge now is to figure out how the alternative splicing process is regulated in a cell and tissue-specific manner. In their new paper in Genome Biology, Dr. Yoseph Barash, a postdoctoral fellow working jointly with Blencowe and Frey, has provided what is likely part of the answer. By applying computational methods to the gene chip data generated by Matthew Fagnani (an MSc student) and other members of the Blencowe lab, Barash has uncovered what appears to be part of a “regulatory code” that controls alternative splicing patterns in the brain.

One outcome of these new studies is that the alternative splicing process appears to provide a largely separate layer of gene regulation that works in parallel with other important steps in gene regulation.

“The number of genes and coordinated regulatory events involved in specifying cell and tissue type characteristics appear to be considerably more extensive than appreciated in previous studies,” says Blencowe.

“These findings also have implications for understanding human diseases such as cancers, since we can anticipate a more extensive role for altered regulation of splicing events that similarly went unnoticed due to the lack of the appropriate technology allowing their detection.” .........

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