Model organism Caenorhabditis elegans.
Global Regulator of mRNA Editing Found
Protein controls editing, expanding the information content of DNA
An international team of researchers, led by scientists from the University of California, San Diego School of Medicine and Indiana University, have identified a protein that broadly regulates how genetic information transcribed from DNA to messenger RNA (mRNA) is processed and ultimately translated into the myriad of proteins necessary for life.
The findings, published today in the journal Cell Reports, help explain how a relatively limited number of genes can provide versatile instructions for making thousands of different messenger RNAs and proteins used by cells in species ranging from sea anemones to humans. In clinical terms, the research might also help researchers parse the underlying genetic mechanisms of diverse diseases, perhaps revealing new therapeutic targets.
“Problems with RNA editing show up in many human diseases, including those of neurodegeneration, cancer and blood disorders,” said Gene Yeo, PhD, assistant professor in the Department of Cellular and Molecular Medicine at UC San Diego. “This is the first time that a single protein has been identified that broadly regulates RNA editing. There are probably hundreds more. Our approach provides a method to screen for them and opens up new ways to study human biology and disease.”
“To be properly expressed, all genes must be carefully converted from DNA to messenger RNA, which can then be translated into working proteins,” said Heather Hundley, PhD, assistant professor of biochemistry and molecular biology at Indiana University and co-senior author of the study. RNA editing alters nucleotides (the building blocks of DNA and RNA) within the mRNA to allow a single gene to create multiple mRNAs that are subject to different modes of regulation. How exactly this process can be modulated, however, has never been clear.
Using the nematode Caenorhabditis elegans as their model organism and a novel computational framework, Hundley, Yeo and colleagues identified more than 400 new mRNA editing sites – the majority regulated by a single protein called ADR-1, which does not directly edit mRNA but rather regulated how editing occurred by binding to the messenger RNAs subject to editing.
“Cells process their genetic code in a way analogous to how the programming language Java compiles modern software. Both systems use an intermediate representation that is modified depending on its environment” said co-first author Boyko Kakaradov, a bioinformatics PhD student in the Yeo lab. “We’re now finding how and why the mRNA code is being changed en route to the place of execution.”
The scientists noted that a protein similar to ADR-1 is expressed by humans, and that many of the same mRNA targets exist in people too. “So it is likely that a similar mechanism exists to regulate editing in humans,” said Hundley, adding that she and colleagues will now turn to teasing out the specifics of how proteins like ADR-1 regulate editing and how they might be exploited “to modulate editing for the treatment of human diseases.”
A colored scanning electron micrograph of a human T lymphocyte. Image courtesy of the National Institute of Allergy and Infectious Disease
Using microRNA Fit to a T (cell)
Researchers show B cells can deliver potentially therapeutic bits of modified RNA
Researchers at the University of California, San Diego School of Medicine have successfully targeted T lymphocytes – which play a central role in the body’s immune response – with another type of white blood cell engineered to synthesize and deliver bits of non-coding RNA or microRNA (miRNA).
The achievement in mice studies, published in this week’s online early edition of the Proceedings of the National Academy of Sciences, may be the first step toward using genetically modified miRNA for therapeutic purposes, perhaps most notably in vaccines and cancer treatments, said principal investigator Maurizio Zanetti, MD, professor in the Department of Medicine and director of the Laboratory of Immunology at UC San Diego Moores Cancer Center.
“From a practical standpoint, short non-coding RNA can be used for replacement therapy to introduce miRNA or miRNA mimetics into tissues to restore normal levels that have been reduced by a disease process or to inhibit other miRNA to increase levels of therapeutic proteins,” said Zanetti.
“However, the explosive rate at which science has discovered miRNAs to be involved in regulating biological processes has not been matched by progress in the translational arena,” Zanetti added. “Very few clinical trials have been launched to date. Part of the problem is that we have not yet identified practical and effective methods to deliver chemically synthesized short non-coding RNA in safe and economically feasible ways.”
Zanetti and colleagues transfected primary B lymphocytes, a notably abundant type of white blood cell (about 15 percent of circulating blood) with engineered plasmid DNA (a kind of replicating but non-viral DNA), then showed that the altered B cells targeted T cells in mice when activated by an antigen – a substance that provokes an immune system response.
“This is a level-one demonstration for this new system,” said Zanetti. “The next goal will be to address more complex questions, such as regulation of the class of T cells that can be induced during vaccination to maximize their protective value against pathogens or cancer.
Single RNA generation of human iPS cells. Image by Peter Allen, UC Santa Barbara.
UC San Diego Researchers Develop Efficient Model for Generating Human iPSCs
Approach has potential to simplify generation of iPSCs for use in human stem cell therapies
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.
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 newborn 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.
Human breast cancer cell. Image courtesy of the National Cancer Institute.
Enhancer RNAs Alter Gene Expression
New class of molecules may be key emerging “enhancer therapy”
In a pair of distinct but complementary papers, researchers at the University of California, San Diego School of Medicine and colleagues illuminate the functional importance of a relatively new class of RNA molecules. The work, published online this week in the journal Nature, suggests modulation of “enhancer-directed RNAs” or “eRNAs” could provide a new way to alter gene expression in living cells, perhaps affecting the development or pathology of many diseases.
Enhancers are sequences in the genome that act to boost or “enhance” the activity or expression of nearby genes. They “often behave in a cell-specific manner and play an important role in establishing a cell’s identity and functional potential,” said Christopher Glass, MD, PhD, a professor in the department of Medicine and Cellular and Molecular Medicine at UC San Diego and principal investigator of one of the papers.
Although enhancers have been recognized for more than 25 years, scientists have labored to fully flesh out the breadth and complexity of what enhancers do and how they do it. In 2010, it was discovered that enhancers directed expression of RNA on a broad scale in neurons and macrophages, a type of immune system cell. Dubbed eRNAs, they were different from other classes of nuclear non-coding RNAs, and raised new questions about their potential roles in the functions of enhancers. The two Nature papers attempt to answer some of these questions.
In the first, principal investigator Glass and colleagues investigated a pair of related transcriptional repressors called Rev-Erb-alpha and Rev-Erb-beta (proteins with important roles in regulating the circadian rhythm in many cell types) in mouse macrophages. Using genome-wide approaches, they found that the Rev-Erb proteins repressed gene expression in macrophages primarily by binding to enhancers. Collaboration with researchers at the Salk Institute for Biological Studies revealed that the repressive function of Rev-Erbs was highly correlated with their ability to repress the production of eRNAs.
In the second paper, principal investigator Michael G. Rosenfeld, MD, a professor in the UC San Diego Department of Medicine and Howard Hughes Medical Institute investigator, and colleagues looked at estrogen receptor binding in human breast cancer cells – and its impact on enhancer transcription. In contrast to the repressive functions of Rev-Erbs, estrogen receptors (ERs) activate gene expression; but, like Rev-Erbs, they primarily function by also binding to enhancers. ER binding was shown to be associated with increases in enhancer-directed eRNAs in the vicinity of estrogen-induced genes, and to exert roles on activation of coding target genes.
Both papers offer new evidence that eRNAs significantly contribute to enhancer activity, and therefore to expression of nearby genes. “Because many broadly expressed genes that play key roles in essential cellular functions are under the control of cell-specific enhancers, the ability to affect enhancer function by knocking down eRNAs could potentially provide a new strategy for altering gene expression in vivo in a cell-specific manner,” said Glass, noting that in his research, anti-sense oligonucleotides were developed in conjunction with Isis Pharmaceuticals, which suppressed enhancer activity and reduced expression in nearby genes.
Three UC San Diego Scientists Garner ENCODE Grants
Recently, the ENCyclopedia Of DNA Elements, otherwise known as ENCODE, made national news with the single-day publication in multiple journals of dozens of related papers intended to more fully flesh out the functional components of the human genome.
The findings were a big step, but the blueprint of human biology remains incomplete. This week, the National Human Genome Research Institute, part of the National Institutes of Health, announced new grants worth $30.3 million this year alone to expand and deepen the effort.
Three scientists at UC San Diego were among the recipients. Bing Ren, PhD, head of the Laboratory of Gene Regulation at the Ludwig Institute for Cancer Research at UCSD, and colleagues have been awarded $11.4 million over four years (roughly $2.86 million per year) to continue their work developing a working catalog of the mouse genome.
“The goal is to enhance use of this model organism in studying a wide range of tissues not readily accessible in the human (genome), and to tap into the power of comparative genomic analysis to increase understanding of the function of the human genome,” said an NHGRI official.
Earlier this year, Ren and colleagues published a paper in Nature that described mapping for the first time a significant portion of the functional sequences of the mouse genome. Specifically, they looked at genome regions containing cis-regulatory elements, key stretches of DNA that appear to regulation the transcription of genes. Misregulation of genes can result in diseases like cancer.
In addition to Ren’s grant, Gene Yeo, PhD, assistant professor of cellular and molecular medicine, and Xiang-Dong Fu, PhD, professor of cellular and molecular medicine (both, along with Ren, are members of the Institute of Genomic Medicine) are part of a team headed by Brenton Graveley, PhD, of the University of Connecticut Health Center that was awarded a four-year, $9.3 million grant to analyze human RNA transcripts to identify protein-binding sites and investigate their function. Proteins that bind to RNA can directly regulate protein production from RNA molecules, as well as affect protein production by regulating degradation of RNA molecules. The project is ENCODE’s first production scale effort to map protein-binding sites in RNA.
Parsing a process of life
Transcription is the first step in gene expression, the process by which information contained in a gene is used to make functional products, such as proteins. It’s fundamental to life and, not surprisingly, extraordinarily complicated.
In the July 22, 2012 issue of Nature Structural & Molecular Biology, Dong Wang, PhD, assistant professor in the Skaggs School of Pharmacy and Pharmaceutical Science, and colleagues further elucidate how transcription is altered by some forms of cytosine.
Cytosine, of course, is one of the four main bases that comprise DNA and RNA (along with adenine, guanine and thymine; uracil replacing thymine in RNA). There are at least five forms of cytosine in human DNA. Wang and colleagues have discovered that two recently identified forms of cytosine, known as 5fC and 5caC, significantly reduce the transcription rate in vitro.
The finding, said Wang, suggests that some forms of cytosine (and perhaps other players yet-to-be-identified) may provide another layer of regulation and fine-tuning to the transcription process. By slowing the activity of RNA polymerase II, a major transcriptional enzyme, 5fC and 5caC may make it easier for other enzymes, proteins and factors to play their parts in the larger act of gene expression.
Photo: Structure of RNA Polymerase II, a key enzyme in mammalian cells that catalyzes the transcription of DNA into messenger RNA, the molecule that in turn dictates the order of amino acids in proteins. Courtesy of National Institute of General Medical Sciences.
A photomicrograph of superficial keratinocytes or skin cells. Image courtesy of Thomas Deerinck, National Center for Microscopy and Imaging Research, UC San Diego.
What Happens When We Sunburn
Researchers describe inflammatory mechanism for first time
The biological mechanism of sunburn – the reddish, painful, protective immune response from ultraviolet (UV) radiation – is a consequence of RNA damage to skin cells, report researchers at the University of California, San Diego School of Medicine and elsewhere in the July 8, 2012 Advance Online Publication of Nature Medicine.
The findings open the way to perhaps eventually blocking the inflammatory process, the scientists said, and have implications for a range of medical conditions and treatments.
“For example, diseases like psoriasis are treated by UV light, but a big side effect is that this treatment increases the risk of skin cancer,” said principal investigator Richard L. Gallo, MD, PhD, professor of medicine at UC San Diego School of Medicine and Veterans Affairs San Diego Healthcare System. “Our discovery suggests a way to get the beneficial effects of UV therapy without actually exposing our patients to the harmful UV light. Also, some people have excess sensitivity to UV light, patients with lupus, for example. We are exploring if we can help them by blocking the pathway we discovered.”
Using both human skin cells and a mouse model, Gallo, first author Jamie J. Bernard, a post-doctoral researcher, and colleagues found that UVB radiation fractures and tangles elements of non-coding micro-RNA – a special type of RNA inside the cell that does not directly make proteins. Irradiated cells release this altered RNA, provoking healthy, neighboring cells to start a process that results in an inflammatory response intended to remove sun-damaged cells.
We see and feel the process as sunburn.
The art depicts RNAs wound in an undecipherable knot bound by hnRNP proteins, describing the intractable problem of RNA regulation as a “Gordian Knot.” As Alexander the Great discovered, the best way to unravel such a knot is a brute force approach, and thus Huelga et al. attack this puzzle through multiple, genome-wide swords (splicing arrays, CLIP-seq, and RNA-seq).
Understanding how RNA binding proteins control the genetic splicing code is fundamental to human biology and disease – much like editing film can change a movie scene. Abnormal variations in splicing are often implicated in cancer and genetic neurodegenerative disorders.
In a step toward deciphering the “splicing code” of the human genome, researchers at the University of California, San Diego School of Medicine have comprehensively analyzed six of the more highly expressed RNA binding proteins collectively known as heterogeneous nuclear ribonucleoparticle (hnRNP) proteins.
This study, published online Feb 16 in Cell Press’ new open-access journal Cell Reports, describes how multiple RNA binding proteins cooperatively control the diversity of proteins in human cells by regulating the alternative splicing of thousands of genes.
In the splicing process, fragments that do not typically code for protein, called introns, are removed from gene transcripts, and the remaining sequences, called exons, are reconnected. The proteins that bind to RNA are important for the control of the splicing process, and the location where they bind dictates which pieces of the RNA are included or excluded in the final gene transcript — in much the same fashion that removing and inserting scenes, or splicing, can alter the plot of a movie.
“By integrating vast amounts of information about these key binding proteins, and making this data widely available, we hope to provide a foundation for building predictive models for splicing and future studies in other cell types such as embryonic stem cells,” said principal investigator Gene Yeo, PhD, assistant professor in the Department of Cellular and Molecular Medicine and the Institute for Genomic Medicine at UC San Diego, and a visiting professor at the Molecular Engineering Laboratory in Singapore. “If we can understand how these proteins work together and affect one another to regulate alternative splicing, it may offer important clues for rational drug design.”