Molecular Structure Reveals How a Protein Reads RNAs to Preserve Stem Cell Identity

Knowing what to keep and what to trash: how an enzyme distinguishes cellular messages

Tuesday, 05 August 2014

Every once in a while, we are forced to sort that stack of papers on the kitchen counter. Interspersed between the expired coupons and dozens of takeout menus are important documents like your car insurance or electric bill. So it is not an option to simply drop it all in the trash at once – you need to read through the messages to be sure that you don’t lose vital information. 

Dis3l2 is protein that preserves the character of

stem cells by degrading specific messages. But

how does the enzyme know which messages to

destroy? CSHL researchers obtained a molecular

photograph (imaged on the left) of the protein in

complex with a “poly-U” chain (in orange) that

marks messages for degradation. The poly-U

chain is inserted into a funnel-like structure deep

within Dis3l2 (see cartoon on the right, poly-U

chain again in orange). Inside the funnel, there

are more than a dozen contacts that capture the

poly-U mark so that the entire message can be

destroyed. Credit: Leemor Joshua-Tor, Cold 

Spring Harbor Laboratory.

In the cell, proteins similarly read through messages to distinguish what needs to be saved and what needs to be discarded. But, here, the process takes on a much more important role. More than just clutter, messages that are marked for disposal can drastically alter the fate of a cell. In fact, stem cells use just such a mechanism to maintain their identity. So how does a protein detect the difference between two seemingly similar messages? Today, a team of Cold Spring Harbor Laboratory (CSHL) scientists, led by Professor and Howard Hughes Medical Institute Investigator Leemor Joshua-Tor, describe how the protein Dis3l2 uses numerous recognition sites to capture messages that are flagged for decay. 

Dis3l2 is a molecular machine that helps to preserve the character of stem cells. It serves as the executioner of an elegant pathway that prevents stem cells from changing into other cell types. The protein does this by acting like a garbage disposal for messages in the cell, cutting them up until they no longer encode useful information. But Dis3l2 is necessarily highly specific. While it must degrade messages that would alter the fate of the stem cell, discarding the wrong message could have devastating consequences.

Therefore, Dis3l2 only targets specific messages that have been marked with a molecular flag, known as a “poly-U” chain. The enzyme ignores the majority of messages in the cell – those that go on to encode proteins and other critical messages – whose ends are decorated with a different type of chain, called “poly-A” tail.

The CSHL scientists set out to understand how Dis3l2 is able to read and distinguish between these two chains. In work published today in Nature, they used a type of molecular photography, known as X-ray crystallography, to observe the structure of Dis3l2 while bound to a poly-U chain.

“We saw that the enzyme looks a lot like funnel, quite wide at the top and narrow at the base,” says Joshua-Tor.

“The poly-U chain inserts itself into the depths of this funnel while the rest of the bulky message can remain in the wide mouth at the top.”

But how does the enzyme “read” the poly-U chain? Christopher Faehnle, PhD and Jack Walleshauser, lead authors on the paper, found that the interior of the funnel contains more than a dozen contacts that interact specifically with the poly-U chain.

“Together, all of these points create a sticky web that holds the poly-U sequence deep within the enzyme,” says Faehnle.

“But other chains don’t interact – they can slide right out. It has helped us understand how an enzyme can differentiate between two sequences in the cell.”

More than that, the work provides insight into how a stem cell maintains its identity.

“Misregulation of any step in this pathway leads to developmental disorders and cancer,” says Joshua-Tor.

“We now have a much better appreciation of the terminal step, a critical point of control.” 

Source: Cold Spring Harbor Laboratory

Contact: Jaclyn Jansen

Reference:

Mechanism of Dis3l2 substrate recognition in the Lin28–let-7 pathway

Christopher Faehnle, Jack Walleshauser and Leemor Joshua-Tor

Nature, August 3, 2014, doi. 10.1038/nature13553

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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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Riboswitches in Action

Scientists at SISSA investigate a mechanism that switches genes on and off 

Monday, 04 November 2013

Riboswitch RNA. 

Riboswitches are RNA segments that switch genes on and off, either during DNA transcription or during protein translation, but little is known about the precise workings of this process. A study at SISSA uncovers some of the basic steps in this complex mechanism and paves the way for future research.

A cell is a complex environment in which substances (metabolites) must maintain a correct state of equilibrium, which may vary depending on specific needs. Cells can maintain the proper concentrations of metabolites by regulating gene protein encoding through specific “switches”, called riboswitches, which are able to block or activate protein synthesis. The precise mechanism by which these short strands of RNA carry out this function is still poorly understood. However, a study conducted by SISSA scientists Giovanni Bussi, Francesco Colizzi and Francesco De Palma and published in the journal RNA, now provides some important insights. 

A riboswitch is contained in a strand of messenger RNA, an RNA fragment that acts as a sort of template that “prints” the proteins that are needed for cell metabolism. However, unlike the rest of the messenger RNA unit, riboswitches don’t actually encode any portion of the protein (i.e., they form part of what geneticists call non-coding RNA) but they serve to activate or deactivate the protein printing process and are located, in bacteria, in a stretch preceding the coding sequence. Scientists know that this switching action is made possible by a change in shape, which occurs when a part of the riboswitch (the aptamer) binds to a molecule in the cell environment which acts as a signal. Bussi and colleagues used computer simulations to reproduce the dynamics of the process and understand how binding to the metabolite brings about the change in shape.

More specifically, Bussi and colleagues simulated the riboswitch that uses an adenine molecule as a signal, to regulate the gene expressing a protein involved in the metabolism of adenine itself. Their findings clarify how adenine stabilizes the active form of the riboswitch (the one triggering protein synthesis) to the detriment of the inactive conformation.

“We used molecular dynamics as a kind of ‘virtual microscope’ with which we observed the workings of the process”, explained Bussi.

“It’s very important to understand these regulatory mechanisms since they are present in many bacteria – as well as in multicellular organisms – and may be useful for developing new antibiotics in the future.”

The computer-based simulations carried out in the study relied on PLUMED software, the latest release of which was presented in another recent paper published by Bussi in Computer Physics Communications. PLUMED is a software program devised to create and analyse simulations.

“This more advanced version of the program was developed as a team of five young researchers from international institutions”.

It’s no coincidence that the researchers involved in this work are all young: the research project was in part funded by a special grant provided for young, non-permanent research fellows at SISSA and awarded to Bussi in 2011.

The research was carried out within the framework of a European Research Council (ERC)-funded project coordinated by  Bussi.

Source: Sissa Medialab

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Stem Cells Engineered to Become Targeted Drug Factories

Stem Cells Engineered to Become Targeted Drug Factories

Saturday, 05 October 2013

A group of Brigham and Women's Hospital, and Harvard Stem Cell Institute researchers and collaborators at MIT and MGH have found a way to use stem cells as drug delivery vehicles.

Engineered mesenchymal stem cells are 

targeted to a site of inflammation to secrete 

anti-inflammatory interleukin-10 proteins. 

Credit: Courtesy by Jeffrey Karp. 

The researchers inserted modified strands of messenger RNA into connective tissue stem cells — called mesenchymal stem cells — which stimulated the cells to produce adhesive surface proteins and secrete interleukin-10, an anti-inflammatory molecule. When injected into the bloodstream of a mouse, these modified human stem cells were able to target and stick to sites of inflammation and release biological agents that successfully reduced the swelling.

"If you think of a cell as a drug factory, what we're doing is targeting cell-based, drug factories to damaged or diseased tissues, where the cells can produce drugs at high enough levels to have a therapeutic effect," said research leader Jeffrey Karp, PhD, a Harvard Stem Cell Institute principal faculty member and Associate Professor at the Brigham and Women's Hospital, Harvard Medical School and Affiliate faculty at MIT.

Karp's proof of concept study, published in the journal Blood, is drawing early interest from biopharmaceutical companies for its potential to target biological drugs to disease sites. While ranked as the top sellers in the drug industry, biological drugs are still challenging to use, and Karp's approach may improve their clinical application as well as improve the historically mixed, clinical trial results of mesenchymal stem cell-based treatments.

This is Jeffrey Karp, Ph.D., a Harvard Stem

Cell Institute principal faculty member and

associate professor at the Brigham and

Women's Hospital, Harvard Medical School

and Affiliate faculty at MIT. Credit: 

Brigham and Women's Hospital. 

Mesenchymal stem cells have become cell therapy researchers' tool of choice because they can evade the immune system, and thus are safe to use even if they are derived from another person. The researchers' method of engineering the cells with messenger RNA is also harmless, as it cannot integrate into a cell's genome, which can be a problem when DNA is used (via viruses) to manipulate gene expression.

"This opens the door to thinking of messenger RNA transfection of cell populations as next generation therapeutics in the clinic, as they get around some of the delivery challenges that have been encountered with biological agents", said Oren Levy, co-lead author of the study and Instructor of Medicine in Karp's lab. The study was also co-led by Professor Weian Zhao at University of California, Irvine who was previously a postdoctoral fellow in Karp's lab.

One such challenge with using mesenchymal stem cells is they have a "hit-and-run" effect, since they are rapidly cleared after entering the bloodstream, typically within a few hours or days. The Harvard/MIT team demonstrated that rapid targeting of the cells to the inflamed tissue produced a therapeutic effect despite the cells being rapidly cleared. The scientists want to extend cell lifespan even further and are experimenting with how to use messenger RNA to make the stem cells produce pro-survival factors.

"We're interested to explore the platform nature of this approach and see what potential limitations it may have or how far we can actually push it," Zhao said. 

"Potentially, we can simultaneously deliver proteins that have synergistic therapeutic impacts."

Source: Harvard University 

Contact: B. D. Colen

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Scientists Discover the Origins of Genomic 'Dark Matter'

Scientists Discover the Origins of Genomic 'Dark Matter'

Wednesday, 18 September 2013

A duo of scientists at Penn State University has achieved a major milestone in understanding how genomic "dark matter" originates. This "dark matter" – called non-coding RNA – does not contain the blueprint for making proteins and yet it comprises more than 95 percent of the human genome. The researchers have discovered that essentially all coding and non-coding RNA originates at the same types of locations along the human genome. The team's findings eventually may help to pinpoint exactly where complex-disease traits reside, since the genetic origins of many diseases reside outside of the coding region of the genome.

A duo of scientists at Penn State University has

achieved a major milestone in understanding

genomic "dark matter" -- called non-coding RNA.

This "dark matter" is difficult to detect and no

one knows exactly what it is doing or why it is

there in our genome, but scientists suspect it may

be the source of inherited diseases. This research

achievement may help to pinpoint exactly where

complex-disease traits reside in the human

genome. This illustration shows, in the upper left

corner, a chromosome – a densely compressed

package containing one long, continuous strand

of DNA. The DNA is pervasively transcribed into

RNA, but only a very small fraction of the RNA

has the instructions (or codes) for making proteins.

The green circles in this illustration represent places

along the strand of DNA where transcription

originates. New research led by B. Franklin Pugh

of Penn State University shows that essentially all

RNA, whether or not it codes for proteins, originates

at the same types of locations along the strand

of DNA. The findings eventually may help to

pinpoint exactly where complex-disease traits

reside, since the genetic origins of many diseases

reside outside of the coding region of the genome.

Credit: National Institutes of Health and B.

Franklin Pugh, Penn State University. 

The research, which will be published as an Advance Online Publication in the journal Nature, was performed by B. Franklin Pugh, holder of the William chair in Molecular Biology at Penn State, and postdoctoral scholar Bryan Venters, who now holds a faculty position at Vanderbilt University.

In their research, Pugh and Venters set out to identify the precise location of the beginnings of transcription – the first step in the expression of genes into proteins.

"During transcription, DNA is copied into RNA – the single-stranded genetic material that is thought to have preceded the appearance of DNA on Earth – by an enzyme called RNA polymerase and, after several more steps, genes are encoded and proteins eventually are produced," Pugh explained.

He added that, in their quest to learn just where transcription begins, other scientists had looked directly at RNA. However, Pugh and Venters instead determined where along human chromosomes the proteins that initiate transcription of the non-coding RNA were located.

"We took this approach because so many RNAs are rapidly destroyed soon after they are made, and this makes them hard to detect," Pugh said.

"So rather than look for the RNA product of transcription we looked for the 'initiation machine' that makes the RNA. This machine assembles RNA polymerase, which goes on to make RNA, which goes on to make a protein."

Pugh added that he and Venters were stunned to find 160,000 of these "initiation machines," because humans only have about 30,000 genes.

"This finding is even more remarkable, given that fewer than 10,000 of these machines actually were found right at the site of genes. Since most genes are turned off in cells, it is understandable why they are typically devoid of the initiation machinery."

The remaining 150,000 initiation machines – those Pugh and Venters did not find right at genes – remained somewhat mysterious.

"These initiation machines that were not associated with genes were clearly active since they were making RNA and aligned with fragments of RNA discovered by other scientists," Pugh said.

"In the early days, these fragments of RNA were generally dismissed as irrelevant since they did not code for proteins."

Pugh added that it was easy to dismiss these fragments because they lacked a feature called polyadenylation – a long string of genetic material, adenosine bases – that protect the RNA from being destroyed. Pugh and Venters further validated their surprising findings by determining that these non-coding initiation machines recognized the same DNA sequences as the ones at coding genes, indicating that they have a specific origin and that their production is regulated, just like it is at coding genes.

"These non-coding RNAs have been called the 'dark matter' of the genome because, just like the dark matter of the universe, they are massive in terms of coverage – making up over 95 percent of the human genome. However, they are difficult to detect and no one knows exactly what they all are doing or why they are there," Pugh said.

"Now at least we know that they are real, and not just 'noise' or 'junk.' Of course, the next step is to answer the question, 'what, in fact, do they do?'"

Pugh added that the implications of this research could represent one step towards solving the problem of "missing heritability" – a concept that describes how most traits, including many diseases, cannot be accounted for by individual genes and seem to have their origins in regions of the genome that do not code for proteins.

"It is difficult to pin down the source of a disease when the mutation maps to a region of the genome with no known function," Pugh said.

"However, if such regions produce RNA then we are one step closer to understanding that disease."

Source: Penn State 

Contact: Barbara K. Kennedy

Reference:

Genomic organization of human transcription initiation complexes

Bryan J. Venters & B. Franklin Pugh

Nature (2013), doi:10.1038/nature12535
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Synthetic mRNA Can Induce Self-repair and Regeneration of the Infarcted Heart

Synthetic mRNA Can Induce Self-repair and Regeneration of the Infarcted Heart

Sunday, 08 September 2013

A team of scientists at Karolinska Institute and Harvard University has taken a major step towards treatment for heart attack, by instructing the injured heart in mice to heal by expressing a factor that triggers cardiovascular regeneration driven by native heart stem cells. The study, published in Nature Biotechnology, also shows that there was an effect on driving the formation of a small number of new cardiac muscle cells.

Dr. Kenneth Chien was recently recruited as a

researcher to Karolinska Institutet and now

share his time between Sweden and the US.

Credit: Ulf Sirborn. 

"This is the beginning of using the heart as a factory to produce growth factors for specific families of cardiovascular stem cells, and suggests that it may be possible to generate new heart parts without delivering any new cells to the heart itself ", says Kenneth Chien, a Professor at the medical university Karolinska Institute in Sweden and Harvard University, US, who led the research team behind the new findings.

The study is based upon another recent discovery in the Chien lab, which was published in Cell Research. This study shows that VEGFA, a known growth factor for vascular endothelial cells in the adult heart, can also serve as a switch that converts heart stem cells away from becoming cardiac muscle and towards the formation of the coronary vessels in the fetal heart. To coax the heart to make the VEGFA, the investigators in the Nature Biotechnology study used new technology where synthetic messenger RNA (mRNA) that encodes VEGFA is injected into the muscle cell. Then, heart muscle produces a short pulse of VEGFA. The mRNA is synthetically modified so that it escapes the normal defense system of the body that is known to reject and degrade the non-modified mRNA as a viral invader.

The study, performed in mice, shows that only a single administration of a short pulse of expression of VEGFA is required, if it can be delivered to the exact region where the heart progenitors reside. The therapeutic effect is long term, as shown by markedly improved survival following myocardial infarction with a single administration of the synthetic mRNA when given within 48 hours after the heart attack. The long-term effect appears to be based on changing the fate of the native heart stem cells from contributing to cardiac fibrotic scar tissue and towards cardiovascular tissue.

"This moves us very close to clinical studies to regenerate cardiovascular tissue with a single chemical agent without the need for injecting any additional cells into the heart," says Professor Chien.

At the same time, he points out that these are still early days and there remains much to be done. In particular, it will become of interest to engineer new device technology to deliver the synthetic mRNA via conventional catheter technology. It also will be critical to move these studies, which are based in mouse models, to other animals, which is currently in progress.

Source: Karolinska Institute

Contact: Katarina Sternudd

Reference:

Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction

Lior Zangi, Kathy O Lui, Alexander von Gise, Qing Ma, Wataru Ebina, Leon M Ptaszek, Daniela Später, Huansheng Xu, Mohammadsharif Tabebordbar, Rostic Gorbatov, Brena Sena, Matthias Nahrendorf, David M Briscoe, Ronald A Li, Amy J Wagers, Derrick J Rossi, William T Pu & Kenneth R Chien

Nature Biotechnology, 8 September 2013, DOI: 10.1038/nbt.2682

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How 'Junk DNA' Can Control Cell Development

How 'Junk DNA' Can Control Cell Development

Saturday, 03 August 2013

Researchers from the Gene and Stem Cell Therapy Program at Sydney's Centenary Institute have confirmed that, far from being "junk", the 97 per cent of human DNA that does not encode instructions for making proteins can play a significant role in controlling cell development.

And in doing so, the researchers have unravelled a previously unknown mechanism for regulating the activity of genes, increasing our understanding of the way cells develop and opening the way to new possibilities for therapy.

Using the latest gene sequencing techniques and sophisticated computer analysis, a research group led by Professor John Rasko and including Centenary's Head of Bioinformatics, Dr William Ritchie, has shown how particular white blood cells use non-coding DNA to regulate the activity of a group of genes that determines their shape and function. The work is published today in the scientific journal Cell.

"This discovery, involving what was previously referred to as "junk", opens up a new level of gene expression control that could also play a role in the development of many other tissue types," Rasko says.

"Our observations were quite surprising and they open entirely new avenues for potential treatments in diverse diseases including cancers and leukaemias."

The researchers reached their conclusions through studying introns — non-coding sequences which are located inside genes.

As part of the normal process of generating proteins from DNA, the code for constructing a particular protein is printed off as a strip of genetic material known as messenger RNA (mRNA). It is this strip of mRNA which carries the instructions for making the protein from the gene in the nucleus to the protein factories or ribosomes in the body of the cell.

But these mRNA strips need to be processed before they can be used as protein blueprints. Typically, any non-coding introns must be cut out to produce the final sequence for a functional protein. Many of the introns also include a short sequence — known as the stop codon — which, if left in, stops protein construction altogether. Retention of the intron can also stimulate a cellular mechanism which breaks up the mRNA containing it.

Dr Ritchie was able to develop a computer program to sort out mRNA strips retaining introns from those which did not. Using this technique the lead molecular biologist of the team, Dr Justin Wong, found that mRNA strips from many dozens of genes involved in white blood cell function were prone to intron retention and consequent break down. This was related to the levels of the enzymes needed to chop out the intron. Unless the intron is excised, functional protein products are never produced from these genes. Dr Jeff Holst in the team went a step further to show how this mechanism works in living bone marrow.

So the researchers propose intron retention as an efficient means of controlling the activity of many genes.

"In fact, it takes less energy to break up strips of mRNA, than to control gene activity in other ways," says Rasko.

"This may well be a previously-overlooked general mechanism for gene regulation with implications for disease causation and possible therapies in the future."

Source: Centenary Institute 

Contact: Tamzin Byrne

Reference:

Orchestrated Intron Retention Regulates Normal Granulocyte Differentiation 

Justin J.-L. Wong, William Ritchie, Olivia A. Ebner, Matthias Selbach, Jason W.H. Wong, Yizhou Huang, Dadi Gao, Natalia Pinello, Maria Gonzalez, Kinsha Baidya, Annora Thoeng, Teh-Liane Khoo, Charles G. Bailey, Jeff Holst, John E.J. Rasko

Cell, Volume 154, Issue 3, 583-595, 1 August 2013, 10.1016/j.cell.2013.06.052

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Researchers Develop Efficient Model for Generating Human iPSCs

Approach has potential to simplify generation of iPSCs for use in human stem cell therapies

Thursday, 01 August 2013

Researchers at the University of California, San Diego School of Medicine report a simple, easily reproducible RNA-based method of generating human induced pluripotent stem cells (iPSCs) in the August 1 edition of Cell Stem Cell. Their approach has broad applicability for the successful production of iPSCs for use in human stem cell studies and eventual cell therapies.

This graphic depicts single RNA generation

of human iPS cells. Credit: Peter Allen, UC

Santa Barbara. 

Partially funded by grants from the California Institute for Regenerative Medicine (CIRM) and the National Institutes of Health (NIH), the methods developed by the UC San Diego researchers dramatically improve upon existing DNA-based approaches – avoiding potential integration problems and providing what appears to be a safer and simpler method for future clinical applications.

The generation of human iPSCs has opened the potential for regenerative medicine therapies based on patient-specific, personalized stem cells. Pluripotent means that these cells have the ability to give rise to any of the body's cell types. The human iPSCs are typically artificially derived from a non-pluripotent adult cell, such as a skin cell. They retain the characteristics of the body's natural pluripotent stem cells, commonly known as embryonic stem cells. Because iPSCs are developed from a patient's own cells, it was first thought that treatment using them would avoid any immunogenic responses. However, depending on methods used to generate such iPSCs, they may pose significant risks that limit their use. For example, using viruses to alter the cell's genome could promote cancer in the host cell.

Methods previously developed to generate integration-free iPSCs were not easily and efficiently reproducible. Therefore, the UC San Diego researchers focused their approach on developing a self-replicating, RNA-based method (one that doesn't integrate into the DNA) with the ability to be retained and degraded in a controlled fashion, and that would only need to be introduced once into the cell.

Using a Venezuelan equine virus (VEE) with structural proteins deleted, but non-structural proteins still present, the scientists added four reprogramming factors (OCT4, KLF4, SOX2 with either c-MYC or GLIS1). They made a single transfection of the VEE replicative form (RF) RNA into new-born or adult human fibroblasts, connective tissue cells that provide a structural framework for many other tissues.

"This resulted in efficient generation of iPSCs with all the hallmarks of stem cells," said principal investigator Steven Dowdy, PhD, professor in the UC San Diego Department of Cellular & Molecular Medicine.

"The method is highly reproducible, efficient, non-integrative – and it works."

Dowdy added that it worked on both young and old human cells. He explained that this is important since – in order to be used therapeutically in fighting disease or to create disease models for research – iPSCs will need to be derived from the cells of middle-aged to old adults who are more prone to the diseases scientists are attempting to treat. In addition, reprogramming factors can be easily changed.

Source: University of California - San Diego 

Contact: Debra Kain

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Scientists Analyse Genetic Makeup of Human and Mouse Embryos in Unprecedented Detail

Single-cell RNA sequencing could lead to improved diagnoses of genetic diseases

Wednesday, 31 July 2013

UCLA scientists, in collaboration with teams in China, have used the powerful technology of single-cell RNA sequencing to track the genetic development of a human and a mouse embryo at an unprecedented level of accuracy. 

The technique could lead to earlier and more accurate diagnoses of genetic diseases, even when the embryo consists of only eight cells. 

The study was led by Guoping Fan, professor of human genetics and molecular biology and member of both the Jonsson Comprehensive Cancer Center and the Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research. The findings were published in the online edition of the journal Nature and will appear later in the print edition.

Single-cell RNA sequencing allows researchers to determine the precise nature of the total gene transcripts, or all of the genes that are actively expressed in a particular cell.

"The advantages of this technique are twofold," Fan said.

"It is a much more comprehensive analysis than was achievable before and the technique requires a very minimal amount of sample material — just one cell."

Besides its implications for genetic diagnoses — such as improving scientists' ability to identify genetic mutations like BRCA1 and BRCA2, which predispose women to breast cancer and ovarian cancer, or genetic diseases that derive from protein dysfunction, such as sickle cell disease — the technology may also have important uses in reproductive medicine. 

The technique marks a major development in genetic diagnoses, which previously could not be conducted this early in embryonic development and required much larger amounts of biological material.

"Previous to this paper we did not know this much about early human development," said Kevin Huang, the study's co-first author and a postdoctoral scholar in Fan's laboratory.

"Now we can define what 'normal' looks like, so in the future we will have a baseline from which to compare possible genetic problems. This is our first comprehensive glance at what is normal."

With single-cell RNA sequencing, much more gene transcription was detected than before.

"The question we asked is, 'How does the gene network drive early development from one cell to two cells, two cells to four cells, and so on?'" Fan said.

"Using the genome data analysis methods developed by co-author Steve Horvath at UCLA, we have uncovered crucial gene networks and we can now predict possible future genetic disorders at the eight-cell stage."

Source: University of California - Los Angeles

Contact: Shaun Mason 

Reference:

Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing

Zhigang Xue, Kevin Huang, Chaochao Cai, Lingbo Cai, Chun-yan Jiang, Yun Feng, Zhenshan Liu, Qiao Zeng, Liming Cheng, Yi E. Sun, Jia-yin Liu, Steve Horvath & Guoping Fan

Nature (2013), doi:10.1038/nature12364

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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at

http://cellnews-blog.blogspot.com/

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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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ZenMaster

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For more on stem cells and cloning, go to CellNEWS at

http://cellnews-blog.blogspot.com/

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