Scientists Finds Key to Making Neurons from Stem Cells

Pnky, a noncoding RNA found in brain stem cells, may have a broad range of clinical applications

Friday, 20 March 2015

A research team at UC San Francisco has discovered an RNA molecule called Pnky that can be manipulated to increase the production of neurons from neural stem cells.

The research, led by neurosurgeon Daniel A. Lim, MD, PhD, and published on March 19, 2015 in Cell Stem Cell, has possible applications in regenerative medicine, including treatments of such disorders as Alzheimer's disease, Parkinson's disease and traumatic brain injury, and in cancer treatment.

In this cluster of neurons, the greens cells have

been infected with a virus to reduce levels of the

RNA molecule called Pnky, resulting in

increased production of neurons. Someday this

finding could be important for regenerative

medicine and cancer treatment.

Pnky is one of a number of newly discovered long noncoding RNAs (lncRNAs), which are stretches of 200 or more nucleotides in the human genome that do not code for proteins, yet seem to have a biological function.

The name, pronounced "Pinky," was inspired by the popular American cartoon series Pinky and the Brain.

"Pnky is encoded near a gene called 'Brain,' so it sort of suggested itself to the students in my laboratory," said Lim. Pnky also appears only to be found in the brain, he noted.

Co-first authors Alex Ramos, PhD, and Rebecca Andersen, who are students in Lim's laboratory, first studied Pnky in neural stem cells found in mouse brains, and also identified the molecule in neural stem cells of the developing human brain. They found that when Pnky was removed from stem cells in a process called knockdown, neuron production increased three to four times.

"It is remarkable that when you take Pnky away, the stem cells produce many more neurons," said Lim, an assistant professor of neurological surgery and director of restorative surgery at UCSF.

"These findings suggest that Pnky, and perhaps lncRNAs in general, could eventually have important applications in regenerative medicine and cancer treatment."

Lim observed that Pnky has an intriguing possible connection with brain tumours.

Using an analytical technique called mass spectrometry, Ramos found that Pnky binds the protein PTBP1, which is also found in brain tumours and is known to be a driver of brain tumour growth. In neural stem cells, Pnky and PTBP1 appear to function together to suppress the production of neurons.

"Take away one or the other and the stem cells differentiate, making more neurons," said Lim.

"It is also possible that Pnky can regulate brain tumour growth, which means we may have identified a target for the treatment of brain tumours."

Lim said that the larger significance of the research is that it adds to a growing store of knowledge about lncRNAs, previously unknown sections of the genome that some biologists have referred to as the "dark matter" of the human genome.

"Recently, over fifty thousand human lncRNAs have been discovered. Thus, there may be more human lncRNAs than there are genes that code for proteins," said Lim.

"It is possible that not all lncRNAs have important biological functions, but we are making a start toward learning which ones do, and if so, how they function. It's a new world of experimental biology, and the students in my lab are right there on the frontier."

Lim had particular praise for Ramos, an MD-PhD student in the UCSF Medical Scientist Training Program, and Andersen, who has a fellowship from the prestigious National Science Foundation (NSF) Graduate Research Fellowship Program.

"They have been a great collaborative team and an inspiration to others in my lab," said Lim.

"I think they represent the pioneering, investigative spirit of the UCSF student body."

Source: UC San Francisco

Contact: Laura Kurtzman

Reference:

The Long Noncoding RNA Pnky Regulates Neuronal Differentiation of Embryonic and Postnatal Neural Stem Cells

Alexander D. Ramos, Rebecca E. Andersen, Siyuan John Liu, Tomasz Jan Nowakowski, Sung Jun Hong, Caitlyn C. Gertz, Ryan D. Salinas, Hosniya Zarabi, Arnold R. Kriegstein, Daniel A. Lim

Cell Stem Cell, March 19, 2015, DOI: http://dx.doi.org/10.1016/j.stem.2015.02.007

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Do Viruses Make Us Smarter?

Do Viruses Make Us Smarter?

Monday, 12 January 2015

A new study from Lund University in Sweden indicates that inherited viruses that are millions of years old play an important role in building up the complex networks that characterise the human brain.

Researchers have long been aware that endogenous retroviruses constitute around five per cent of our DNA. For many years, they were considered junk DNA of no real use, a side-effect of our evolutionary journey.

In the current study, Johan Jakobsson and his colleagues show that retroviruses seem to play a central role in the basic functions of the brain, more specifically in the regulation of which genes are to be expressed, and when. The findings indicate that, over the course of evolution, the viruses took an increasingly firm hold on the steering wheel in our cellular machinery. The reason the viruses are activated specifically in the brain is probably due to the fact that tumours cannot form in nerve cells, unlike in other tissues.

"We have been able to observe that these viruses are activated specifically in the brain cells and have an important regulatory role. We believe that the role of retroviruses can contribute to explaining why brain cells in particular are so dynamic and multifaceted in their function. It may also be the case that the viruses' more or less complex functions in various species can help us to understand why we are so different", says Johan Jakobsson, head of the research team for molecular neurogenetics at Lund University.

The article, based on studies of neural stem cells, shows that these cells use a particular molecular mechanism to control the activation processes of the retroviruses. The findings provide us with a complex insight into the innermost workings of the most basal functions of the nerve cells. At the same time, the results open up potential for new research paths concerning brain diseases linked to genetic factors.

"I believe that this can lead to new, exciting studies on the diseases of the brain. Currently, when we look for genetic factors linked to various diseases, we usually look for the genes we are familiar with, which make up a mere two per cent of the genome. Now we are opening up the possibility of looking at a much larger part of the genetic material which was previously considered unimportant. The image of the brain becomes more complex, but the area in which to search for errors linked to diseases with a genetic component, such as neurodegenerative diseases, psychiatric illness and brain tumours, also increases".

Source: Lund University

Contact: Johan Jakobsson

Reference:

TRIM28 represses transcription of endogenous retroviruses in neural progenitor cells

Liana Fasching, Adamandia Kapopoulou, Rohit Sachdeva, Rebecca Petri, Marie E. Jönsson, Christian Männe, Priscilla Turelli, Patric Jern, Florence Cammas, Didier Trono, Johan Jakobsson

Cell Reports, 2015 Jan 6;10(1):20-8, doi: 10.1016/j.celrep.2014.12.004

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Induced Pluripotent Stem Cells Reveal Differences between Humans and Great Apes

Key differences in the regulation of jumping genes may have arisen relatively recently in evolution

Thursday, 24 October 2013

Researchers at the Salk Institute for Biological Studies have, for the first time, taken chimpanzee and bonobo skin cells and turned them into induced pluripotent stem cells (iPSCs), a type of cell that has the ability to form any other cell or tissue in the body.

This microscope image shows induced pluripotent

stem cells (iPSC) from our closest living relatives.

Skin cells from bonobos (pigmy chimps) were

reprogrammed to pluripotent stem cells, an

advance that allows scientists to study the

differences between the neurons of humans and

chimps. The colours show different aspects of the

cells' molecular components. Credit: Courtesy of

Carol Marchetto, Salk Institute for Biological

Studies. 

Mouse iPSCs were created in 2006 by Kazutoshi Takahashi and Shinya Yamanaka at Kyoto University in Japan, and human iPSCs soon followed – feats which earned Yamanaka the Nobel Prize in Physiology or Medicine last year. Now scientists regularly use iPSCs to model diseases using cells that would be otherwise difficult to obtain from a living person or animal. By adding a combination of four key factors, a skin cell can be made into an iPSC, which can then be coaxed into forming liver, lung and brain cells in a culture dish.

It's now possible to not only model disease using the cells, but also to compare iPSCs from humans to those of our closest living relatives – great apes, with which we share a majority of genes – for insight into what molecular and cellular features make us human.

"Comparing human, chimpanzee and bonobo cells can give us clues to understand biological processes, such as infection, diseases, brain evolution, adaptation or genetic diversity," says senior research associate Iñigo Narvaiza, who led the study with senior staff scientist Carol Marchetto at the Salk Institute in La Jolla.

"Until now, the sources for chimpanzee and bonobo cells were limited to post-mortem tissue or blood. Now you could generate neurons, for example, from the three different species and compare them to test hypotheses."

In the new study, published online October 23 in the journal Nature, scientists found disparities in the regulation of jumping genes or transposons – DNA elements that can copy and paste themselves into spots throughout the genome – between humans and non-human primate cells. Jumping genes provide a means to rapidly shuffle DNA and might be shaping the evolution of our genomes, the scientists say.

From left are Salk scientists Ahmet Denli, Carol

Marchetto, Iñigo Narvaiza and Fred Gage.

Credit: Courtesy of the Salk Institute for
Biological Studies.

Working in the lab of Salk's Fred Gage, the Vi and John Adler Chair for Research on Age-Related Neurodegenerative Disease, Narvaiza, Marchetto and their colleagues identified genes that are differentially expressed between iPSCs from humans and both chimpanzees and bonobos.

To the group's surprise, two of those genes code for proteins that restrict a jumping gene called long interspersed element-1or L1, for short. Compared with non-human primate cells, human iPSCs expressed higher levels of these restrictors, called APOBEC3B and PIWIL2.

"We weren't expecting that," Marchetto says.

"Those genes caught our eyes, so they were the first targets we focused on."

L1 and a handful of other jumping genes are abundant throughout our genomes. Where these bits of DNA insert themselves is hard to predict, and they can produce variable effects. For example, they might completely disrupt genes, modulate them, or cause them to be processed into entirely new proteins.

Using L1 tagged with a fluorescent marker, the group observed higher numbers of fluorescent iPSCs from non-human primates compared with humans. In separate experiments, they produced iPSCs with too much or too little APOBEC3B and PIWIL2, finding – as expected – that an excess of the two proteins dampened the mobility and reduced the appearance of newly inserted DNA in the non-human primate cells.

These results suggested that L1 elements insert themselves less often throughout our genomes. Indeed, looking at genomes of humans and chimpanzees that had already been sequenced, the researchers found that the primates had more copies of L1 sequences than did humans.

The question that remains is what would be the impact of differences in L1 regulation?

"It could mean that we have gone, as humans, through one or more bottlenecks in evolution, that decrease the variability present in our genome," says Marchetto, though the hypothesis is admittedly hard to prove. It is known, however, that humans' genomes are less variable than chimpanzees'.

The new study provides proof of concept that the iPSC technology can be used to understand some of the evolutionary differences between humans and non-human primates, says Narvaiza. The group plans to make technology, and all the data, available to the broader research community – which is especially helpful now that great ape research is severely restricted in the United States and abroad – so that other scientists can learn about primates using non-invasive, ethically sound methods.

The team plans to differentiate the stem cells into other tissues, such as neurons, and comparing how the cells from different species behave. They will also use the iPSC technology to investigate how chimpanzees might differ from people in susceptibility to cancer, genetic diseases and viral infection.

Source: Salk Institute for Biological Studies

Contact: Kat Kearney

Reference:

Differential L1 regulation in pluripotent stem cells of humans and apes

Maria C. N. Marchetto, Iñigo Narvaiza, Ahmet M. Denli, Christopher Benner, Thomas A. Lazzarini, Jason L. Nathanson, Apuã C. M. Paquola, Keval N. Desai, Roberto H. Herai, Matthew D. Weitzman, Gene W. Yeo, Alysson R. Muotri, Fred H. Gage 

Nature (2013), doi:10.1038/nature12686

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Reading DNA, Backward and Forward

Reading DNA, Backward and Forward

Tuesday, 25 June 2013

MIT biologists have discovered a mechanism that allows cells to read their own DNA in the correct direction and prevents them from copying most of the so-called "junk DNA" that makes up long stretches of our genome.

Only about 15 percent of the human genome consists of protein-coding genes, but in recent years scientists have found that a surprising amount of the junk, or intergenic DNA, does get copied into RNA — the molecule that carries DNA's messages to the rest of the cell.

Scientists have been trying to figure out just what this RNA might be doing, if anything. In 2008, MIT researchers led by Institute Professor Phillip Sharp discovered that much of this RNA is generated through a process called divergent expression, through which cells read their DNA in both directions moving away from a given starting point.

In a new paper appearing in Nature on June 23, Sharp and colleagues describe how cells initiate but then halt the copying of RNA in the upstream, or non-protein-coding direction, while allowing it to continue in the direction in which genes are correctly read. The finding helps to explain the existence of many recently discovered types of short strands of RNA whose function is unknown.

"This is part of an RNA revolution where we're seeing different RNAs and new RNAs that we hadn't suspected were present in cells, and trying to understand what role they have in the health of the cell or the viability of the cell," says Sharp, who is a member of MIT's Koch Institute for Integrative Cancer Research.

"It gives us a whole new appreciation of the balance of the fundamental processes that allow cells to function."

Graduate students Albert Almada and Xuebing Wu are the lead authors of the paper. Christopher Burge, a professor of biology and biological engineering, and undergraduate Andrea Kriz are also authors.

Choosing direction

DNA, which is housed within the nucleus of cells, controls cellular activity by coding for the production of RNAs and proteins. To exert this control, the genetic information encoded by DNA must first be copied, or transcribed, into messenger RNA (mRNA).

When the DNA double helix unwinds to reveal its genetic messages, RNA transcription can proceed in either direction. To initiate this copying, an enzyme called RNA polymerase latches on to the DNA at a spot known as the promoter. The RNA polymerase then moves along the strand, building the mRNA chain as it goes.

When the RNA polymerase reaches a stop signal at the end of a gene, it halts transcription and adds to the mRNA a sequence of bases known as a poly-A tail, which consists of a long string of the genetic base adenine. This process, known as polyadenylation, helps to prepare the mRNA molecule to be exported from the cell's nucleus.

By sequencing the mRNA transcripts of mouse embryonic stem cells, the researchers discovered that polyadenylation also plays a major role in halting the transcription of upstream, noncoding DNA sequences. They found that these regions have a high density of signal sequences for polyadenylation, which prompts enzymes to chop up the RNA before it gets very long. Stretches of DNA that code for genes have a low density of these signal sequences.

The researchers also found another factor that influences whether transcription is allowed to continue. It has been recently shown that when a cellular factor known as U1 snRNP binds to RNA, polyadenylation is suppressed. The new MIT study found that genes have a higher concentration of binding sites for U1 snRNP than noncoding sequences, allowing gene transcription to continue uninterrupted.

A widespread phenomenon

The function of all of this upstream noncoding RNA is still a subject of much investigation.

"That transcriptional process could produce an RNA that has some function, or it could be a product of the nature of the biochemical reaction. This will be debated for a long time," Sharp says.

His lab is now exploring the relationship between this transcription process and the observation of large numbers of so-called long noncoding RNAs (lncRNAs). He plans to investigate the mechanisms that control the synthesis of such RNAs and try to determine their functions.

"Once you see some data like this, it raises many more questions to be investigated, which I'm hoping will lead us to deeper insights into how our cells carry out their normal functions and how they change in malignancy," Sharp says.

Source: Massachusetts Institute of Technology 

Contact: Sarah McDonnell

Written by: Anne Trafton, MIT News Office

Reference:

Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Albert E. Almada, Xuebing Wu, Andrea J. Kriz, Christopher B. Burge & Phillip A. Sharp

Nature (2013) doi:10.1038/nature12349

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Paired Genes in Stem Cells Shed New Light on Gene Organization and Regulation

Paired Genes in Stem Cells Shed New Light on Gene Organization and Regulation

Tuesday, 05 February 2013

Whitehead Institute researchers have determined that DNA transcription, the process that produces messenger RNA (mRNA) templates used in protein production, also runs in the opposite direction along the DNA to create corresponding long noncoding RNAs (lncRNAs). Moreover, the mRNAs and lncRNAs are transcribed co-ordinately as stem cells differentiate into other cell types. This surprising finding could redefine our understanding of gene organization and its regulation.

In the transcription of protein-coding messenger RNA

(mRNA) and long noncoding RNA (lncRNA) genes

(shown as wide tan lines), the transcription machinery

(shown as green ovals) initiates transcription at an active

promoter and commonly runs opposite directions. This 

indicates that the long-established protein-coding genes

actually exist in the genome as gene pairs with lncRNA

genes and leads to coordinated production of both mRNAs

(shown in blue) and lncRNAs (shown in red). Credit: Image

by Alla Sigova.

"It's a surprise to me that genes come in pairs," says Whitehead Member Richard Young, who is also a professor of biology at MIT.

"At any one of the 20,000 protein-coding genes that are active in human stem cells, a lncRNA gene located upstream is also transcribed. So much effort has gone into studying protein-coding genes, and yet we have missed this concept that all protein-coding genes come in mRNA/lncRNA pairs. If you activate the mRNA gene, you're going to activate the lncRNA gene."

Young and his lab report their findings this week in the Proceedings of the National Academy of Sciences (PNAS).

Until now, scientists thought transcription machinery attaches to DNA at certain points called promoters and moves just in one direction along the DNA to create mRNAs from protein-coding genes. Other RNAs that are not protein templates, including lncRNAs, are also created by transcription, but despite their important roles in in regulation of gene expression, development and disease, scientists knew little about how lncRNA transcription is initiated or where most lncRNA genes reside in the genome.

By looking at human and mouse embryonic stem cells, researchers in the Young lab found something astonishing — most lncRNA genes are located adjacent mRNA genes, and the transcription of about 65% of lncRNAs originates at active promoters associated with these mRNAs' genes and runs "upstream" and in the opposite direction from the promoter.

When the transcription machinery attaches to a promoter, it seems that it is just as likely to move in one direction and transcribe the mRNA as it is to move in opposite direction and transcribe the neighbouring lncRNA.

The researchers also noticed that as embryonic stem cells begin differentiating into other cell types, the mRNA/lncRNA pairs are regulated in the same way — the transcription of paired mRNAs and lncRNAs is up regulated or down regulated together. This further confirms the relationship between the transcription of mRNAs and lncRNAs.

"I think it's quite a breakthrough," says Alla Sigova, who is a postdoctoral researcher in the Young lab and a co-author of the paper in PNAS.

"This provides a unifying principle for production of mRNAs and lncRNAs, and may lead to new insights into lncRNA mis-regulation in disease. For example, some mRNAs are up- or down regulated in cancer, so their lncRNA partners could potentially contribute to cancer."

Source: Whitehead Institute for Biomedical Research 

Contact: Nicole Rura

Reference:

Divergent transcription of long noncoding RNA/mRNA gene pairs in embryonic stem cells"

Alla A. Sigova, Alan C. Mullen, Benoit Molinie, Sumeet Gupta, David A. Orlando, Matthew G. Guenther, Albert E. Almada, Charles Lin, Phillip A. Sharp, Cosmas C. Giallourakis, and Richard A. Young

PNAS, February 4, 2013, doi:10.1073/pnas.1221904110 

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How Many Genes in the Human Genome? Human Genome Far More Active than Thought

GENCODE Consortium discovers far more genes than previously thought

Friday, 07 September 2012

The GENCODE Consortium expects the human genome has twice as many genes than previously thought, many of which might have a role in cellular control and could be important in human disease. This remarkable discovery comes from the GENCODE Consortium, which has done a painstaking and skilled review of available data on gene activity.

Among their discoveries, the team describes more than 10,000 novel genes; identify genes that have 'died' and others that are being resurrected. The GENCODE Consortium reference gene catalogue has been one of the underpinnings of the larger ENCODE Project and will be essential for the full understanding of the role of our genes in disease.

The GENCODE Consortium is part of the ENCODE Project that, today, publishes 30 research papers describing findings from their nearly decade-long effort to describe comprehensively all the active regions of our human genome. ENCODE was launched in 2003 after the completion of the Human Genome Project, and brought together an international group of scientists tasked with identifying and describing all functional regions of the human genome sequence.

"We have uncovered a staggering array of genes in our genome, simply because we can examine many genomes in a detail that was not possible a decade ago," says Dr. Jennifer Harrow, GENCODE principle investigator from the Wellcome Sanger Institute. 

"As sequencing technology improves, so we have much more data to explore.

"But our work remains a skilled effort to annotate correctly our human genome - or, more precisely, our human genomes, for each of us differ. These vast texts of genetic information will not give up their secrets easily. GENCODE has made amazing strides to enable immediate access of its reference gene set by other researchers."

The team more accurately described the genes that contain the genetic code to make proteins: they found 20,687 such protein-coding genes, a value that has not changed greatly from previous work. The new set captures far more of the alternative forms of these genes found in different cell types.

More significant are their findings on genes that do not contain genetic code to make proteins - non-coding genes - and the graveyard of supposedly 'dead' genes from which some are emerging, resurrected from the catalogue of pseudogenes.

They mapped and described 9,277 long non-coding genes, a relatively new type that acts, not through producing a protein, but directly through its RNA messenger. Long non-coding RNAs derived from these genes can play a significant part in human biology and disease, but they remain only poorly understood.

"If the Human Genome Project was the baseline for genetics, ENCODE is the baseline for biology, and GENCODE are the parts that make the human biological machine work. Our list is essential to all those who would fix the human machine," said Dr. Tim Hubbard.

The new map of such genetic components gives researchers more avenues to explore in their quest to understand human biology and human disease. Remarkably, the team thinks their job is not complete and believes that there may be another 10,000 of these genes yet to be uncovered.

"Our initial work from the Human Genome Project suggested there were around 20,000 protein-coding genes and that value has not changed greatly," says Professor Roderic Guigo, GENCODE principle investigator from Centre for Genomic Regulation, Barcelona.

"However GENCODE has shown that long non-coding RNAs are far more numerous and important than previously thought"

"The limited knowledge we have of the class of long non-coding RNAs suggests they might play a major role in regulating the activity of other genes. If this is generally true of this group, we have much more to explore than we imagined."

As dramatic, GENCODE has catalogued for the first time a set of more than 11,000 pseudogenes by examining the entire human genome. There is some emerging evidence that many of these genes, too, might have some biological activity.

The GENCODE team predicts that at least 9% of pseudogenes may be active with some controlling the activity of other genes. Pseudogenes have been implicated in many biological activities, such as the prevention of certain elements known to be involved in the development of cancer.

"At the announcement of the Human Genome Project draft sequence, we emphasized this was the end of the beginning, that 'at present most genes - probably tens of thousands - remain a mystery'", says Dr. Tim Hubbard, lead principle investigator of GENCODE from the Wellcome Trust Sanger Institute. 

"Today, we describe many thousands of genes for the first time."

"If the Human Genome Project was the baseline for genetics, ENCODE is the baseline for biology, and GENCODE are the parts that make the human biological machine work. Our list is essential to all those who would fix the human machine."

The GENCODE human reference set will be updated every three months to ensure that models are continually refined and assessed based on new experimental data deposited in the public databases.

Source: Wellcome Trust Sanger Institute 

Contact: Don Powell

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lincRNAs are Directing Embryonic Stem Cells Differentiation

New roles emerge for non-coding RNAs in directing embryonic development
Monday, 29 August 2011

Scientists at the Broad Institute of MIT and Harvard have discovered that a mysterious class of large RNAs plays a central role in embryonic development, contrary to the dogma that proteins alone are the master regulators of this process. The research, published online August 28 in the journal Nature, reveals that these RNAs orchestrate the fate of embryonic stem (ES) cells by keeping them in their fledgling state or directing them along the path to cell specialization.

lincRNAs orchestrate the fate of embryonic

stem cells (shown here) by keeping them in

their fledgling state or directing them along

the path to cell specialization.
Image courtesy of Alex Meissner.

Broad scientists discovered several years ago that the human and mouse genomes encode thousands of unusual RNAs — termed large, intergenic non-coding RNAs (lincRNAs) — but their role was almost entirely unknown. By studying more than 100 lincRNAs in ES cells, the researchers now show that these RNAs help regulate development by physically interacting with proteins to coordinate gene expression and suggest that lincRNAs may play similar roles in most cells.

“There’s been a lot of debate about what lincRNAs are doing,” said Eric Lander, director of the Broad Institute and the senior author of the paper.

“It’s now clear that they play critical roles in regulating developmental decisions — that is, cell fate. This was a big surprise, because specific types of proteins have been thought to be the master controls of development.”

“This is the first global study of lincRNAs,” said Mitchell Guttman, first author of the paper and a graduate student at MIT and the Broad Institute.

“We picked embryonic stem cells in particular because they are so important to development and so well understood. This allowed us to dissect the role of lincRNAs within the circuitry of a cell.”

The researchers used genetic tools to inhibit more than 100 lincRNAs and found that the vast majority — more than 90 percent — had a significant impact on embryonic stem cells, indicating that the RNAs play a key role in the cells’ circuitry.

Embryonic stem cells can follow one of two main routes. They can either differentiate, becoming cells of a specific lineage such as blood cells or neurons, or they can stay in a pluripotent state, duplicating themselves without losing the ability to become any cell in the body. When the researchers turned off each lincRNA in turn, they found dozens that suppress genes that are important only in specific kinds of cells. They also found dozens of lincRNAs that cause the stem cells to exit the pluripotent state.

“It’s a balancing act,” said Guttman.

“To maintain the pluripotent state, you need to repress differentiation genes.”

The researchers also uncovered a critical clue about how lincRNAs carry out their important job. Through biochemical analysis, they found that lincRNAs physically interact with key proteins involved in influencing cell fate to coordinate their responses.

“The lincRNAs appear to play an organizing role, acting as a scaffold to assemble a diverse group of proteins into functional units,” said John Rinn, an author on the paper, an assistant professor at Harvard University and Medical School, and a senior associate member of the Broad Institute.

“lincRNAs are like team captains, bringing together the right players to get a job done.”

“By understanding how these interactions form, we may be able to engineer these RNAs to do what we want them to do,” said Guttman.

“This could make it possible to target key genes that are improperly regulated in disease.”

Aviv Regev, an author on the paper, a core member of the Broad Institute, and associate professor at MIT, sees the team’s approach to studying the lincRNAs as important for the field.

“Many people are interested in lincRNAs, but they need a comprehensive view of the whole collection of lincRNAs,” said Regev.

“The large-scale data and technology from this study will be useful for scientists worldwide in studying both lincRNAs as well as many other RNAs in the cell.”

This project marks a collaborative effort involving experts in embryonic stem cells and lincRNAs as well as computational biologists and researchers in the Broad’s RNAi Platform, which developed the tools needed to systematically silence lincRNAs. Other researchers who contributed to this work include Julie Donaghey, Bryce W. Carey, Manuel Garber, Jennifer K. Grenier, Glen Munson, Geneva Young, Anne Bergstrom Lucas, Robert Ach, Xiaoping Yang, Ido Amit, Alexander Meissner, and David E. Root. This work was funded by the National Human Genome Research Institute, the Richard Merkin Foundation for Stem Cell Research at the Broad Institute, and funds from the Broad Institute of MIT and Harvard.

Source: Broad Communications

Contact: Haley Bridger

Reference:

lincRNAs act in the circuitry controlling pluripotency and differentiation
Mitchell Guttman, Julie Donaghey, Bryce W. Carey, Manuel Garber, Jennifer K. Grenier, Glen Munson, Geneva Young, Anne Bergstrom Lucas, Robert Ach, Laurakay Bruhn, Xiaoping Yang, Ido Amit, Alexander Meissner, Aviv Regev, John L. Rinn, David E. Root & Eric S. Lander
Nature. August 28, 2011 DOI: 10.1038/nature10398

See also:

LincRNAs Serve as Genetic Air-traffic Controllers
CellNEWS - Tuesday, 14 July 2009
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Gene Regulation Mechanism Unique to Primates Discovered

Study finds long-known, but little-understood DNA elements serve important purpose
Thursday, 10 February 2011

Scientists have discovered a new way genes are regulated that is unique to primates, including humans and monkeys. Though the human genome – all the genes that an individual possesses – was sequenced 10 years ago, greater understanding of how genes function and are regulated is needed to make advances in medicine, including changing the way we diagnose, treat and prevent a wide range of diseases.

"It's extremely valuable that we've sequenced a large bulk of the human genome, but sequence without function doesn't get us very far, which is why our finding is so important," said Lynne E. Maquat, Ph.D., lead author of the new study published today in the journal Nature.

When our genes go awry, many diseases, such as cancer, Alzheimer's and cystic fibrosis can result. The study introduces a unique regulatory mechanism that could prove to be a valuable treatment target as researchers seek to manipulate gene expression – the conversion of genetic information into proteins that make up the body and perform most life functions – to improve human health.

The newly identified mechanism involves Alu elements, repetitive DNA elements that spread throughout the genome as primates evolved. While scientists have known about the existence of Alu elements for many years, their function, if any, was largely unknown.

Maquat discovered that Alu elements team up with molecules called long noncoding RNAs (lncRNAs) to regulate protein production. They do this by ensuring messenger RNAs (mRNAs), which take genetic instructions from DNA and use it to create proteins, stay on track and create the right number of proteins. If left unchecked, protein production can spiral out of control, leading to the proliferation or multiplication of cells, which is characteristic of diseases such as cancer.

"Previously, no one knew what Alu elements and long noncoding RNAs did, whether they were junk or if they had any purpose. Now, we've shown that they actually have important roles in regulating protein production," said Maquat, the J. Lowell Orbison Chair, professor of Biochemistry and Biophysics and director of the Center for RNA Biology at the University of Rochester Medical Center.

The expression of genes that call for the development of proteins involves numerous steps, all of which are required to occur in a precise order to achieve the appropriate timing and amount of protein production. Each of these steps is regulated, and the pathway discovered is one of only a few pathways known to regulate mRNAs directly in the midst of the protein production process.

Regulating mRNAs is one of several ways cells control gene expression, and researchers from institutions and companies around the world are honing in on this regulatory landscape in search of new ways to manage and treat disease.

"This new mechanism is really a surprise. We continue to be amazed by all the different ways mRNAs can be regulated," according to Maquat.

Maquat and the study's first author, Chenguang Gong, a graduate student in the Department of Biochemistry and Biophysics at the Medical Center, found that long noncoding RNAs and Alu elements work together to trigger a process known as SMD (Staufen 1-mediated mRNA decay). SMD conditionally destroys mRNAs after they orchestrate the production of a certain amount of proteins, preventing the creation of excessive, unwanted proteins in the body that can disrupt normal processes and initiate disease.

Specifically, long noncoding RNAs and Alu elements recruit the protein Staufen-1 to bind to numerous mRNAs. Once an mRNA finishes directing a round of protein production, Staufen-1 works with another regulatory protein previously identified by Maquat, UPF1, to initiate the degradation or decay of the mRNA so that it cannot create any more proteins.

While the research fills in a piece of the puzzle as to how our genes operate, it also accentuates the overwhelming complexity of how our DNA shapes us and the many known and unknown players involved. Maquat and Gong plan on exploring the newly identified pathway in future research.

Source: University of Rochester Medical Center
Contact: Emily Boynton

Reference:
lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements
Chenguang Gong & Lynne E. Maquat
Nature Vol. 470, 284–288, (10 February 2011), doi:10.1038/nature09701
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LincRNAs Serve as Genetic Air-traffic Controllers

Study helps answer the question, 'What does this new class of RNA genes do?'
Tuesday, 14 July 2009

Earlier this year, a scientific team from Beth Israel Deaconess Medical Center (BIDMC) and the Broad Institute of Harvard and MIT identified a class of RNA genes known as large intervening non-coding RNAs or "lincRNAs". The discovery has pushed the field forward in understanding the roles of these molecules in many biological processes, including stem cell pluripotency, cell cycle regulation, and the innate immune response.
Nevertheless, even as one question was being answered, another was close on its heels: What, exactly, were these mysterious molecules doing? They now appear to have found an important clue. Described in the July 14 issue of the Proceedings of the National Academy of Sciences (PNAS) the scientific teams shows that lincRNAs – once dismissed as "genomic junk" – have a global role in genome regulation, ferrying proteins to assist their regulation at specific regions of the genome.
"I like to think of them as genetic air-traffic controllers," explains co-senior author John Rinn, PhD, a Harvard Medical School Assistant Professor of Pathology at BIDMC and Associate Member of the Broad Institute.
"It has long been a mystery as to how widely expressed proteins shape the fate of cells. How does the same protein know to regulate one genomic location in a brain cell and regulate a different genomic region in a liver cell? Our study suggests that in the same way that air traffic controllers organize planes in the air, lincRNAs may be organizing key chromatin complexes in the cell."
Inspired by a lincRNA called HOTAIR – which is known to bind key chromatin modifier proteins and to assist in getting these proteins to the proper location in the genome – the researchers hypothesized that other lincRNA molecules might be playing similar roles.
"DNA wraps around partner proteins to form a structure called chromatin, which affects which genes are 'turned on' and which are 'turned off'," explains first author Ahmad Khalil, PhD, a scientist in the Department of Pathology at BIDMC and the Broad Institute.
"Chromatin does this through a process of compaction; by determining which areas to compact and which to leave open, chromatin successfully determines which genes are accessible for transcription."
But he adds, it has been a mystery as to how this chromatin structure is so precisely targeted by specific enzymes – and not others.
"By utilizing a technology known as RIP-Chip we were able to examine RNA-protein interaction on a large scale and determine which lincRNAs are associated with each enzyme we examined," he adds.
To analyze this tremendous amount of data, co-author Mitchell Guttman, PhD, a bioinformatician at BIDMC and the Broad Institute, used a mathematical algorithm that identified which lincRNAs are bound by chromatin-modifying enzymes and which are not.
"This analysis revealed that 20 to 30 percent of lincRNAs are bound by three distinct chromatin-modifying complexes," adds Khalil.
"By depleting several of these lincRNAs from cells, we were able to show a significant overlap between the genes which become affected by the depletion of lincRNAs and the depletion of the enzymes. This provided us with the evidence that these proteins work together with lincRNAs to target specific regions of the genome."
Standard "textbook" genes encode RNAs that are translated into proteins, and mammalian genomes contain about 20,000 such protein-coding genes. Some genes, however, encode functional RNAs that are never translated into proteins. These include a handful of classical examples known for decades and some recently discovered classes of tiny RNAs, such as microRNAs. By contrast, lincRNAs are thousands of bases long. Because only about 10 examples of functional RNAs were previously identified, they seemed more like genomic oddities than key players. With these latest findings, which also uncovered an additional 1,500 lincRNAs, it is clear these RNA molecules are no mere messengers – they have demonstrated that they can and do play a leading role.
"Much work still remains to be done but we could one day envision utilizing RNA to guide personalized stem cells to specific cell fates to restore diseased and degenerative disease tissues," notes Rinn.

Reference:
Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression
Ahmad M. Khalil, Mitchell Guttman, Maite Huarte, Manuel Garber, Arjun Raj, Dianali Rivea Morales, Kelly Thomas, Aviva Presser, Bradley E. Bernstein, Alexander van Oudenaarden, Aviv Regev, Eric S. Lander, and John L. Rinn
PNAS 2009 106:11667-11672, doi:10.1073/pnas.0904715106
See also: New Group of Long Non-coding RNA Discovered CellNEWS - Sunday, 01 February 2009

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New Group of Long Non-coding RNA Discovered

Scientists uncover new class of non-protein coding genes in mammals with key functions Sunday, 01 February 2009 A research team at the Broad Institute of Harvard and MIT and Beth Israel Deaconess Medical Center has uncovered a vast new class of previously unrecognized mammalian genes that do not encode proteins, but instead function as long RNA molecules. Their findings, presented in the February 1st advance online issue of the journal Nature, demonstrate that this novel class of "large intervening non-coding RNAs" or "lincRNAs" plays critical roles in both health and disease, including cancer, immune signalling and stem cell biology. "We've known that the human genome still has many tricks up its sleeve," said Eric Lander, founding director of the Broad Institute and co-senior author of the Nature paper. "But, it is astounding to realize that there is a huge class of RNA-based genes that we have almost entirely missed until now." Standard "textbook" genes encode RNAs that are translated into proteins, and mammalian genomes harbour about 20,000 such protein-coding genes. Some genes, however, encode functional RNAs that are never translated into proteins. These include a handful of classical examples known for decades and some recently discovered classes of tiny RNAs, such as microRNAs. By contrast, the newly discovered lincRNAs are thousands of bases long. Because only about ten examples of functional lincRNAs were known previously, they seemed more like genomic oddities than critical components. The new Nature study shows that there are actually thousands of such genes and that they have been conserved across mammalian evolution. "The challenge in finding these lincRNAs is that they have been hiding in plain sight," said John Rinn, a Harvard Medical School assistant professor at Beth Israel Deaconess Medical Center and an associate member of the Broad Institute of Harvard and MIT. "The human and mouse genomes are already known to produce many large RNA molecules, but the vast majority show no evolutionary conservation across species, suggesting that they may simply be 'genomic noise' without any biological function." To uncover this large collection of new genes, the Broad scientific team looked not at the RNA molecules themselves but at telltale signs in the DNA called chromatin modifications or epigenomic marks. They searched for genomic regions that have the same chromatin patterns as protein-coding genes, but do not encode proteins. By surveying the genomes of four different types of mouse cells (including embryonic stem cells and cells from various tissue types), they found an astounding 1,586 such loci that had not been previously described. The researchers also found that the vast majority of these genomic regions are transcribed into lincRNAs, and that these are conserved across mammals. "The epigenomic marks revealed where these genes were hiding," said Mitch Guttman, a MIT graduate student working at the Broad Institute. "Analysis of their sequence then revealed that the genes are highly conserved in mammalian genomes, which strongly suggested that these genes play critical biological functions." By correlating the expression patterns of lincRNAs in various cell types with the expression patterns of known critical protein-coding genes in those same cells, the scientists observed that lincRNAs likely play critical roles in helping to regulate a variety of different cellular processes, including cell proliferation, immune surveillance, maintenance of embryonic stem cell pluripotency, neuronal and muscle development, and gametogenesis. Further experimental evidence from several of the identified lincRNAs verified these observations. Because of the stringent experimental conditions imposed by the researchers in identifying the 1,600 lincRNAs in the Nature study, it is likely that there are many more lincRNA genes hiding in plain sight in the genome, as well as other RNA-encoding genes that are as important to genome function as their better-recognized protein-coding counterparts. Reference: Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals Mitchell Guttman, Ido Amit, Manuel Garber, Courtney French, Michael F. Lin, David Feldser, Maite Huarte, Or Zuk, Bryce W. Carey, John P. Cassady, Moran N. Cabili, Rudolf Jaenisch, Tarjei S. Mikkelsen, Tyler Jacks, Nir Hacohen, Bradley E. Bernstein, Manolis Kellis, Aviv Regev, John L. Rinn & Eric S. Lander Nature advance online publication 1 February 2009, doi:10.1038/nature07672 ......... ZenMaster

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