Non-coding RNAs in Oncogenesis
Posted February 03, 2011
Small RNAs do not code for proteins, but they nonetheless have an important role to play in the regulation of the development and execution of many cellular processes crucial to overall physiological well-being. Scientists are increasingly aware of these molecules' part in controlling disease pathogenesis and of their potential as targets for therapeutic intervention. At a November 16, 2010 symposium called Non-coding RNAs in Oncogenesis researchers gathered at The New York Academy of Sciences to explore the role of non-coding RNAs in oncogenesis, focusing on how they affect methylation patterns or interact with messenger RNAs to regulate gene expression, the role they play in cellular differentiation, and how they bind proteins to modulate protein function.
Greg Hannon of Cold Spring Harbor Laboratory began with an exploration of DNA methylation in the germ line, examining patterns in Drosophila and mammals. Drawing on some key differences between these two groups, Hannon explained that Piwi proteins and their associated piRNAs (or Piwi-interacting RNAs) suppress the mutagenic effects of transposons during fetal gonad development. Moreover, mammals repress transposable elements in a way that is correlated with the acquisition of stable DNA methylation markers, a finding that prompted Hannon's group to investigate the role of small RNAs in setting methylation patterns. In particular the group was eager to uncover how much methylation is default (with active protection to prevent methylation) versus how much is directed (with particular regions actively methylated). The group's research led them to conclude that multiple, interacting mechanisms direct DNA methylation in germ cells, and, importantly, that different mechanisms drive repeated methylation as transposons are uncovered and re-silenced during germ cell and zygotic development.
Pier Paolo Pandolfi from Harvard Medical School then articulated what he sees as a problem for cancer research. Although a vast number of mutations occur in human cancers, only a few of those occur in coding regions of the genome. Many of the rearrangements to non-coding regions, some of which recur in specific locations, fall in transcribed space. Pandolfi suggests that focusing on the familiar "cancer genes" without studying transcribed non-coding regions will greatly limit our understanding of fundamental cancer biology.
Pandolfi's talk then delved into the complex interactions of three subgroups of ncRNA: pseudogene transcripts, which are dysfunctional ancestral relatives of protein-coding genes, long non-coding RNAs (lncRNAs), and micro-RNAs (miRNAs). His group's work "flipped the logic regarding the way ... the miRNA-RNA network operates," as he put it. Whereas people generally focus on the ways miRNAs negatively regulate their messenger RNAs, Pandolfi chose to examine the oft-overlooked way those messenger RNAs recognize miRNAs. Messenger RNAs provide the targets for a particular set of miRNAs with miRNA Recognition Elements (MREs) located on the 3′ UTRs of the messengers.
In this way two RNAs with corresponding MREs can compete for a miRNA pool, and thereby “communicate” with one another. In Pandolfi’s example one ceRNA (competing endogenous RNA) in equilibrium with another could break the equilibrium by being over expressed, either physiologically or pathologically. Over expression of the first ceRNA would reduce the number of miRNAs available to bind to and negatively regulate the second ceRNA and would consequently increase the expression of that second ceRNA. After validating this hypothesis with PTEN (an important tumor suppressor gene) and its pseudogene, both of which are under miRNA regulation, Pandolfi was able to theorize that any RNA can have biological activity that is independent from or even counter to the protein it encodes simply because of the way it participates in the “target rivalry network” of MREs. Outlining the implications of this novel role for ncRNAs, Pandolfi explained that the results should motivate the cancer research community to re-study the mRNAs of all known coding genes, to examine introns for important MREs, to investigate pseudogenes as novel oncogenes or tumor suppressors, and to consider the effects of translocations on MREs.
David Spector from Cold Spring Harbor Laboratory explained that, as a way to study the behavior and to understand the possible structural and organizational functions of long, non-coding RNAs (lncRNAs), his group looked for lncRNAs that were misregulated in a developmental paradigm. Spector described long non-coding RNAs transcribed from the MEN1 locus, which is also home to the MEN1 gene that encodes the Menin protein. Of particular interest to Spector's group are two lncRNAs, called MENβ and MENε , and their involvement in the differentiation of myoblasts into myotubes. Spector's group determined that MENβ and MENε , upregulated upon myoblast differentiation, are not just localized to the cells' nuclei, but are actually further localized to a nuclear domain called the "paraspeckle." The presence of these RNAs and of certain RNA binding proteins in the paraspeckle (a discrete ribonucleoprotein) has led scientists to believe that this domain might play a role in retaining RNA transcripts needed during a rapid response, when the whole transcription process would be too time-consuming.
Not only are these MEN RNAs localized to the paraspeckle, but Spector's research using an anti-sense approach to knock down MEN transcripts indicates that they are critical elements for the maintenance of the nuclear domain. In fact, Spector was able to determine that the paraspeckle domain forms adjacent to the MEN gene locus but not adjacent to the genes encoding any other RNAs that also happen to reside in the domain. Spector continued his talk with a closer examination of the MENβ and MENε RNAs and the features of their lncRNA family. His group's findings establish a model whereby ncRNAs can serve as a platform to recruit proteins to assemble a nuclear body.
John Rinn of Harvard Medical School also considered the role of ncRNAs in cell differentiation. In particular he postulated that large intergenic non-coding RNAs (lincRNAs) help give rise to cellular diversity, including regional specialization of the same "kinds" of cells. To investigate this role, however, Rinn first needed to establish a method for making testable, predictive hypotheses about the function of lincRNAs. For this he turned to a "guilt by association" model, wherein the function of lincRNAs could be predicted from the protein-coding pathways to which they correlate.
Clustering lincRNAs by common functionality allowed Rinn's group to zero in on lincRNAs with very strong correlations to known oncogenes or tumor suppressor genes. Rinn discussed the results of his group's work on TP53 (a known tumor suppressor, which codes for the p53 protein) and the lincRNA-p21 (or p21 for short), which is highly correlated with the TP53 gene's expression. The group was able to establish that p21 serves as a global repressor in the p53 pathway by binding a protein (hnRNP-k) and helping that protein to repress transcription at specific loci on the genome.
Since hundreds of lincRNAs bind to Polycomb and other complexes in a similar fashion, the behavior of lincRNA-p21 points to key regulatory roles for lincRNAs more generally. Rinn suggested that these ncRNAs might bridge proteins together to form diverse complexes within the cell, or as he put it, they carry out a "traffic control" function by telling proteins where to go at what time. Rinn finished his talk by explaining several other roles of lincRNAs that his group has uncovered: lncRNAs as modulators of cell fate, pluripotency, and adipogenesis. In general, lincRNAs can be seen as "molecular scaffolds, imparting localization and cell identity," concluded Rinn.
Ramin Shiekhattar from The Wistar Institute concluded the symposium with a presentation on defining and characterizing long non-coding RNAs (lncRNAs). Although, as this symposium demonstrated, lncRNAs provide an exciting domain for meaningful research, it is actually very difficult to describe them as a group and therefore to decide the bounds of that research area. By comparing global (genome-wide) data for protein coding genes with the data for lncRNAs, Shiekhattar's group was able to establish some general features of lncRNAs. Among other features, Shiekattar noted that the genes for lncRNAs, unlike protein-coding genes, generally have one transcript and no other isoforms. In addition, Shiekhattar analyzed the tissue-specific expression levels of lncRNAs and the chromatin signatures of lncRNA genes compared to protein-coding genes to characterize these RNA molecules further.
Shiekhattar finished his talk by taking up questions central to many of the symposium's presentations: what do these lncRNAs do and how do we begin to investigate their function? Echoing Pandolfi's others' hypotheses about the role of ncRNA in species and cellular diversity, Shiekhattar explained his research into the increased expression of many lncRNAs (over 70% of them) during the differentiation of adult human stem cells. For each gene locus that changes after differentiation, Shiekhattar warned one still must confirm that that locus is not simply part of the protein-coding genes it flanks. His own confirmation that the lncRNA TAL1 (a hematopoiesis regulator) and others were separate from adjacent protein-coding genes revealed an enhancer-like function for these lncRNAs. Ultimately, his results demonstrated an unanticipated role for a class of long ncRNAs in the activation of gene expression.
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Dike S, Balija VS, Nascimento LU, et al. The mouse genome: experimental examination of gene predictions and transcriptional start sites. Genome Res. 2004; 14(12):2424-2429.
Smith AD, Chung W, Hodges E, et al. Updates to the RMAP short-read mapping software. Bioinformatics 2009; 25(21):2841-2842.
Pier Paolo Pandolfi
Epping MT, Meijer LAT, Krijgsman O, et al. TSPYL5 suppresses p53 levels and function by physical interaction with USP7. Nat. Cell Biol. 2011; 13(1):102-108.
Filipowicz W, Bhattacharyya SN, Sonenberg N. Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat. Rev. Genet. 2008; 9(2):102-114.
Grosshans H, Filipowicz W. Proteomics joins the search for microRNA targets. Cell 2008; 134(4):560-562.
Poliseno L, Salmena L, Zhang J, et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 2010; 465(7301):1033-1038.
Shabalina SA, Spiridonov NA. The mammalian transcriptome and the function of non-coding DNA sequences. Genome Biol. 2004; 5(4):105.
Shabalina SA, Ogurtsov AY, Lipman DJ, Kondrashov AS. Patterns in interspecies similarity correlate with nucleotide composition in mammalian 3′ UTRs. Nucleic Acids Res. 2003; 31(18):5433-5439.
Shabalina SA, Ogurtsov AY, Rogozin IB, Koonin EV, Lipman DJ. Comparative analysis of orthologous eukaryotic mRNAs: potential hidden functional signals. Nucleic Acids Res. 2004; 32(5):1774-1782.
Stephens PJ, McBride DJ, Lin M, et al. Complex landscapes of somatic rearrangement in human breast cancer genomes. Nature 2009; 462(7276):1005-1010.
Guttman M, Amit I, Garber M, et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 2009; 458(7235):223-227.
Huarte M, Guttman M, Feldser D, et al. A large intergenic noncoding RNA induced by p53 mediates global gene repression in the p53 response. Cell 2010; 142(3):409-419.
Kirsch DG, Santiago PM, di Tomaso E, et al. p53 controls radiation-induced gastrointestinal syndrome in mice independent of apoptosis. Science 2010; 327(5965):593-596.
Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3):R25.
Trapnell C, Salzberg SL. How to map billions of short reads onto genomes. Nat. Biotechnol. 2009; 27(5):455-457.
Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 2009; 25(9):1105-1111.
Trapnell C, Williams BA, Pertea G, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 2010; 28(5):511-515.
Amaral PP, Mattick JS. Noncoding RNA in development. Mamm. Genome 2008; 19(7-8):454-492.
Amaral PP, Dinger ME, Mercer TR, Mattick JS. The eukaryotic genome as an RNA machine. Science 2008; 319(5871):1787-1789.
Barski A, Cuddapah S, Cui K, et al. High-resolution profiling of histone methylations in the human genome. Cell 2007; 129(4):823-837.
Clamp M, Fry B, Kamal M, et al. Distinguishing protein-coding and noncoding genes in the human genome. Proc. Natl. Acad. Sci. USA 2007; 104(49):19428-19433.
Fortschegger K, de Graaf P, Outchkourov NS, et al. PHF8 targets histone methylation and RNA polymerase II to activate transcription. Mol. Cell. Biol. 2010; 30(13):3286-3298.
Gingeras TR. Origin of phenotypes: genes and transcripts. Genome Res. 2007; 17(6):682-690.
Kapranov P, Cheng J, Dike S, et al. RNA maps reveal new RNA classes and a possible function for pervasive transcription. Science 2007; 316(5830):1484-1488.
Kapranov P, St Laurent G, Raz T, et al. The majority of total nuclear-encoded non-ribosomal RNA in a human cell is 'dark matter' un-annotated RNA. BMC Biol. 2010; 8(1):149.
Ørom UA, Derrien T, Beringer M, et al. Long noncoding RNAs with enhancer-like function in human cells. Cell 2010; 143(1):46-58.
Wang ET, Sandberg R, Luo S, et al. Alternative isoform regulation in human tissue transcriptomes. Nature 2008; 456(7221):470-476.
Bernard D, Prasanth KV, Tripathi V, et al. A long nuclear-retained non-coding RNA regulates synaptogenesis by modulating gene expression. EMBO J. 2010; 29(18):3082-3093.
Mao YS, Sunwoo H, Zhang B, Spector DL. Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs. Nat. Cell Biol. 2011; 13(1):95-101.
Prasanth KV, Camiolo M, Chan G, et al. Nuclear organization and dynamics of 7SK RNA in regulating gene expression. Mol. Biol. Cell 2010; 21(23):4184-4196.
Spector DL, Lamond AI. Nuclear speckles. Cold Spring Harb Perspect Biol. 2010.
Wilusz JE, Spector DL. An unexpected ending: noncanonical 3′ end processing mechanisms. RNA 2010; 16(2):259-266.
Senthil K. Muthuswamy, PhD
Senthil Muthuswamy received his PhD with William Muller in Biology from McMaster University in Hamilton, Canada and did his postdoctoral fellowship with Joan Brugge at Harvard Medical School. He began his independent faculty position at Cold Spring Harbor Laboratory, New York and now at Ontario Cancer Institute and Campbell Family Institute for Breast Cancer Research as the Margaret Lau Chair in Breast Cancer Research. He is a recipient of Rita Allen Scholar award, V Foundation scholar award, the US Army Era of Hope Scholar Award, and CSBMCB young investigator award (formerly Merck–Frost Prize). Muthuswamy's research goal is to understand the how cell polarity pathways regulate epithelial cell morphogenesis, differentiation and tumorigenesis. Research from his laboratory has demonstrated that oncogenes interact with polarity proteins to disrupt apical–basal polarity and to transform polarized breast epithelial structures. The observations made in his lab provide direct evidence for a role for polarity pathways during tumorigenesis and identify them as novel targets that can be exploited for diagnosis and treatment of carcinoma.
Pier Paolo Pandolfi, MD, PhD
Pandolfi received his MD in 1989, and his PhD in 1995, from the University of Perugia, Italy, and he studied philosophy at the University of Rome, Italy. He received post-graduate training at the National Institute for Medical Research and the University of London in the UK. Pandolfi became an Assistant Member in 1994 and then a full Member of the Molecular Biology Program and the Department of Human Genetics at Memorial Sloan–Kettering Cancer Center. He was also a professor of Molecular Biology and Human Genetics at the Weill Graduate School of Medical Sciences at Cornell University, a professor, Molecular Biology in Pathology and Laboratory Medicine at Weill Cornell Medical College, and Head of the Molecular and Developmental Biology Laboratories at MSKCC. Pandolfi presently holds the Reisman Endowed Chair of Medicine, and is a professor of Pathology at Harvard Medical School. He serves as the director of research, Beth Israel Deaconess Cancer Center, director of the Cancer Genetics Program, and chief of the Division of Genetics in the Department of Medicine, Beth Israel Deaconess Medical Center. Among many other awards, Pandolfi has received the NIH MERIT Award for superior competence and outstanding productivity in research. And, in 2006, he was elected as a member of the American Society for Clinical Investigation (ASCI) and the American Association of Physicians (AAP), and in 2007 as Member of the European Molecular Biology Organization (EMBO).
Jennifer Henry, PhD
The New York Academy of Sciences
Jennifer Henry received her PhD in plant molecular biology from the University of Melbourne, Australia, with Paul Taylor at the University of Melbourne and Phil Larkin at CSIRO Plant Industry in Canberra, specializing in the genetic engineering of transgenic crops. She was then appointed as Associate Editor, then Editor, of Functional Plant Biology at CSIRO Publishing. She moved to New York for her appointment as a Publishing Manager in the Academic Journals division at Nature Publishing Group, where she was responsible for the publication of biomedical journals in nephrology, clinical pharmacology, hypertension, dermatology, and oncology.
Jennifer joined the Academy in 2009 as Director of Life Sciences and organizes 35–40 seminars each year. She is responsible for developing scientific content in coordination with the various life sciences Discussion Group steering committees, under the auspices of the Academy's Frontiers of Science program. She also generates alliances with outside organizations interested in the programmatic content.
Greg Hannon, PhD
Greg Hannon is a Professor in the Watson School of Biological Sciences at Cold Spring Harbor Laboratory. He received a BA in biochemistry and a PhD in molecular biology from Case Western Reserve University. From 1992 to 1995, he was a postdoctoral fellow of the Damon Runyon–Walter Winchell Cancer Research Fund, where he explored cell cycle regulation in mammalian cells. After becoming an Assistant Professor at Cold Spring Harbor Laboratory in 1996 and a Pew Scholar in 1997, in 2000, he began to make seminal observations in the emerging field of RNA interference. In 2002 Hannon accepted a position as Professor at CSHL where he continued to reveal that endogenous non-coding RNAs, then known as small temporal RNAs and now as microRNAs, enter the RNAi pathway through Dicer and direct RISC to regulate the expression of endogenous protein coding genes. He assumed his current position in 2005 as a Howard Hughes Medical Institute Professor and continues to explore the mechanisms and regulation of RNA interference as well as its applications to cancer research.
John L. Rinn, PhD
After studying biochemistry and earning his BS at the University of Minnesota, Duluth, John Rinn moved to Yale University to do his graduate work. In 2004 he earned his PhD in Molecular Biophysics & Biochemistry under the direction of his thesis advisor Michael Snyder. In his graduate research, Rinn was one of the first to discover an abundance of RNA molecules emanating from non-coding, often referred to as 'junk regions' of the human genome. He continued to pursue these mysterious RNA molecules for his postdoctoral work leading to the discovery of a novel type of non-coding RNA, termed HOTAIR, encoded on one chromosome that silences a large region on a different chromosome. This work also revealed a genetic code of large non-coding RNAs and HOX genes that determine the localization of an adult human skin cell in the body. Rinn is a now senior-associate member of the Broad Institute and assistant professor of Stem Cell and Regenerative Biology at Harvard University. Inspired by HOTAIR the Rinn lab developed a novel approach to hone in on functional RNA molecules. This led to a recent discovery of a class of large intergenic non-coding RNAs (lincRNAs), involved in numerous key biological processes.
Ramin Shiekhattar, PhD
Ramin Shiekhattar is a Professor of Gene Expression and Regulation program at Wistar Institute of the University of Pennsylvania, and he holds the Herbert Kean Professorship. He received a BS in chemistry and a PhD in biochemistry from University of Kansas. He was a postdoctoral fellow (1993–1996) under a National Research Service Award (NRSA) to investigate the mechanism of eukaryotic transcription in human cells. Shiekhattar joined the faculty of the Wistar Institute in 1997 as an assistant professor and began his seminal contributions to the field of epigenetics through biochemical characterization of chromatin-modifying as well as RNA-processing complexes. These include the identification of BRCA1-associated complexes, novel histone demethylase complexes as well as the discovery of the RNA processing machineries such as the Microprocessor and Integrator complexes. In 2007 the Shiekhattar lab extended their analysis of the role of RNA in epigenetic regulation by assessing the role of long non-coding RNAs. This work has revealed that a class of long non-coding RNAs in human cells behave similar to classically defined enhancer elements and consequently begins a new chapter in the biological scope of non-coding RNAs in mammalian development.
David Spector, PhD
David L. Spector is Director of Research at Cold Spring Harbor Laboratory, where he has been a member of the faculty since 1985. He received his PhD from Rutgers University in 1980. Spector's research centers on understanding the organization and regulation of gene expression in living cells. His laboratory's work is focused on implementing innovative approaches to elucidate the spatial and temporal aspects of gene expression and in identifying and characterizing the function of nuclear retained long non-coding RNAs. An expert in microscopy, Spector also directs the Microscopy Shared Resource at CSHL. He has edited numerous microscopy techniques manuals that are used in laboratories throughout the world and he serves on the editorial boards of the Journal of Cell Science, Epigenetics & Chromatin, and Current Opinion in Cell Biology. He has also been elected to the Council of the American Society of Cell Biology. In 2006 he received the Winship Herr Award for Excellence in Teaching from the Watson School of Biological Sciences.