eBriefing

The Cellular Functions of RNA Nucleases

The Cellular Functions of RNA Nucleases
Reported by
Kristen Delevich

Posted March 26, 2012

Presented By

Overview

Nucleic acid cleavage is an essential reaction for a wide array of cellular processes. Whether maintaining quality control of cellular mRNA or generating non-coding RNAs, nucleases are central to proper cell function. Speakers attending The Cellular Functions of RNA Nucleases symposium, presented by the Non-coding RNA Discussion Group on November 2, 2011, discussed the diverse roles that cellular nucleases perform in the biogenesis or quality control of rRNAs, mRNAs, and miRNAs. The forum focused on unique and common structural features among different nucleases and the diseases associated with the nucleases' malfunction.

Stefanie Gerstberger, a graduate student at the Rockefeller University, started the meeting with an introduction to the conserved function and expression profiles of RNA nucleases. RNA nucleases are enzymes that cleave the phosphodiester bonds that join nucleotides along the backbones of RNA polymer chains. The simplest distinction to draw among the RNA nucleases is where along a polynucleotide chain they cleave: exonucleases work from either the 3′ or 5′ ends, cleaving one nucleotide at a time, while endonucleases cleave within the polynucleotide chain.

Under most conditions the phosphorous-oxygen (P-O) bond is highly stable, but nucleases utilize a variety of nucleophiles to attack one of the two bridging scissile phosphates and to initiate a bimolecular nucleophilic substitution reaction (SN2, for short). The most commonly used nucleophile is a deprotonated water molecule (OH-) that mediates nucleophilic attack and direct hydrolysis of the P-O bond. Although the fundamental chemistry of RNA cleavage is the same, there is a remarkable diversity and complexity in RNA nuclease structure and catalytic mechanisms.

Gerstberger delineated catalytic mechanisms of RNA nucleases along the lines of their dependence on metal ions. Nucleases that use external nucleophiles (like deprotonated water) are metal-dependent because metal ions activate a nucleophile and stabilize the transition state of the ensuing bimolecular nucleophilic substitution reaction. On the other hand, metal-independent RNA nucleases use the 2′-hydroxyl (OH-) of ribose internal to the RNA structure itself as a nucleophile. Metal-independent RNA nucleases invariably generate a 2′,3′ cyclic phosphate intermediate. Generally speaking, metal-independent nucleases exhibit less substrate specificity than their metal-dependent counterparts and require greater regulation. This means that metal-independent nucleases are more often spatially restricted and kept in check by specific inhibitors. All known exonucleases are metal-dependent, while both metal-dependent and metal-independent catalytic mechanisms are found in the endonuclease family.

Gerstberger discussed how the activity of RNA nucleases is required for a diverse set of cellular processes, including maturation and processing of RNA in the nucleus, RNA turnover, degradation of foreign RNA, and regulation of RNA species during stress. The ability of nucleases to specifically recognize their RNA targets is essential to the integrity of these processes and, therefore, to cell survival. It isn't difficult to imagine the disastrous effects of a nuclease run amok, carrying out unwanted or uncontrolled degradation of RNA.

Use the tabs above to find a meeting report and multimedia from this event.

 

Presentations available from:
Jayakrishna Ambati, MD (University of Kentucky)
Susan J. Baserga, MD, PhD (Yale University)
Lorena S. Beese, PhD (Duke University School of Medicine)
Robert Blelloch, MD, PhD (University of California, San Francisco)
Stefanie Gerstberger (The Rockefeller University)
Fedor V. Karginov, PhD (Cold Spring Harbor Laboratory)
Eric Lai, PhD (Memorial Sloan Kettering Cancer Center)
Christopher D. Lima, PhD (Memorial Sloan Kettering Cancer Center)
Benjamin R. tenOever, PhD (Mount Sinai School of Medicine)
Liang Tong, PhD (Columbia University)

Journal Articles

Jayakrishna Ambati

Kaneko H, Dridi S, Tarallo V et al. DICER1 deficit induces Alu RNA toxicity in age-related macular degeneration. Nature 2011 Mar 17;471(7338):325-30.

Meister G. Vision: Dicer leaps into view. Nature 2011 Mar 17;471(7339):308-9.

Susan Baserga

Bleichert F, Granneman S, Osheim YN et al. The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation. Proc. Natl. Acad. Sci. USA 2006 Jun 20;103(25):9464-9.

Dragon F, Gallagher JE, Compagnone-Post PA, Mitchell BM et al. A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis. Nature 2002 Jun 27;417(6892):967-70.

Osheim YN, French SL, Keck KM et al. Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae. Mol. Cell 2004 Dec 22; 16(6):943-954.

Lorena S. Beese

Ceska TA, Sayers JR. Structure-specific DNA cleavage by 5′ nucleases. Trends in Biochemical Sciences 1998 Sep 1; 23(9):331-336.

Orans J, McSweeney EA, Iyer RR et al. Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family. Cell 2011 Apr 15;145(2):212-23.

Wang W, Hellinga HW, Beese LS. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis. Proc. Natl. Acad. Sci. USA 2011 Oct 25;108(43):17644-8.

Robert H. Blelloch

Babiarz JE, Hsu R, Melton C et al. A role for noncanonical microRNAs in the mammalian brain revealed by phenotypic differences in Dgcr8 versus Dicer1 knockouts and small RNA sequencing. RNA 2011 Aug;17(8):1489-501.

SubramanyamD, Blelloch R. From microRNAs to targets: pathway discovery in cell fate transitions. Curr. Opin. Genet. Dev. 2011 Aug;21(4):498-503.

Schofield CM, Hsu R, Barker AJ et al. Monoallelic deletion of the microRNA biogenesis gene Dgcr8 produces deficits in the development of excitatory synaptic transmission in the prefrontal cortex. Neural Dev. 2011 Apr 5;6:11.

Fedor V. Karginov

Cheloufi S, Dos Santos CO, Chong MM et al. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature 2010 Jun 3;465(7298):584-9.

Karginov FV, Cheloufi S, Chong MM et al. Diverse endonucleolytic cleavage sites in the mammalian transcriptome depend upon microRNAs, Drosha, and additional nucleases. Mol. Cell 2010 Jun 25;38(6):781-8.

Liu J, Carmell MA, Rivas FV et al. Argonaute2 is the catalytic engine of mammalian RNAi. Science 2004 Sep 3;305(5689):1437-41.

Stephanie Gertsberger

Doma MK, Parker R. RNA quality control in eukaryotes. Cell 2007;131(4):660-668.

Houseley J, LaCava J, Tollervey D. RNA-quality control by the exosome. Nat. Rev. Mol. Cell Biol. 2006;7(7):529-539.

Nicholson A. ed, Ribonucleases, Vol 26. Springer Berlin Heidelberg 2011.

Yang W. Nucleases: diversity of structure, function and mechanism. Q. Rev. Biophys. 2011;44(1):1-93.

Zuo Y, Deutscher MP. Exoribonuclease superfamilies: structural analysis and phylogenetic distribution. Nucleic Acids Res. 2001;29(5):1017-1026.

Eric Lai

Flynt AS, Greimann JC, Chung WJ et al. MicroRNA biogenesis via splicing and exosome-mediated trimming in Drosophila. Mol. Cell 2010 Jun 25;38(6):900-7.

Westholm JO, Lai EC. Mirtrons: microRNA biogenesis via splicing. Biochime. 2011 Nov;93(11):1897-904.

Yang JS, Lai EC. Alternative miRNA biogenesis pathways and the interpretation of core miRNA pathway mutants. Mol. Cell 2011 Sep 16;43(6):892-903.

Christopher D. Lima

Januszyk K, Lima CD. Structural components and architectures of RNA exosomes. Adv. Exp. Med. Biol. 2010;702:9-28. Review.

Januszyk K, Liu Q, Lima CD. Activities of human RRP6 and structure of the human RRP6 catalytic domain. RNA 2011 Aug 17;(8):1566-77.

Liu Q, Greimann JC, Lima CD. Reconstitution, activities, and structure of the eukaryotic RNA exosome. Cell 2006 Dec 15;127(6):1223-37. Erratum in: Cell 2007 Oct. 5;131(1):188-9.

Dinshaw J. Patel

Liu Y, Ye X, Jiang F et al. C3PO, an endoribonuclease that promotes RNAi by facilitating RISC activation. Science 2009 Aug 7;325(5941):750-3.

Tian Y, Simanshu DK, Ascano M et al. Multimeric assembly and biochemical characterization of the Trax–translin endonuclease complex. Nat. Struct. Mol. Biol. 2011. May 8;(18):658–664.

Ye X, Huang N, Liu Y et al. Structure of C3PO and mechanism of human RISC activation. Nat. Struct. Mol. Biol. 2011 Jun;18(6):650-7.

Stewart Shuman

Chakravarty AK, Shuman S. RNA 3′-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5′-monophosphate ends. J. Biol. Chem. 2011 Feb 11;286(6):4117-22.

Horn DM, Mason SL, Karbstein K. Rcl1 protein, a novel nuclease for 18 S ribosomal RNA production. J. Biol. Chem. 2011 Sep 30;286(39):34082-7.

Tanaka N, Smith P, Shuman S. Crystal structure of Rcl1, an essential component of the eukaryal pre-rRNAprocessome implicated in 18S rRNA biogenesis. RNA 2011 Apr;17(4):595-602.

Benjamin R. tenOever

Christov CP, Gardiner TJ, Szüts D et al. Functional requirement of noncoding Y RNAs for human chromosomal DNA replication. Mol. Cell Biol. 2006 Sep;26(18):6993-7004.

Langlois RA, Shapiro JS, Pham AM et al. In vivo delivery of cytoplasmic RNA virus-derived miRNAs. Mol. Ther. 2011 Nov 15.

Varble A, Chua MA, Perez JT et al. Engineered RNA viral synthesis of microRNAs. Proc. Natl. Acad. Sci. USA 2010 Jun 22;107(25):11519-24.

Liang Tong

Chang JH, Xiang S, Xiang K et al. Structural and biochemical studies of the 5′→3′ exoribonuclease Xrn1. Nat. Struct. Mol. Biol. 2011 Mar;18(3):270-6.

Jiao X, Xiang S, Oh C et al. Identification of a quality-control mechanism for mRNA 5′-end capping. Nature 2010 Sep 30;467(7315):608-11.

Xiang S, Cooper-Morgan A, Jiao X et al. Structure and function of the 5′→3′ exoribonuclease Rat1 and its activating partner Rai1. Nature 2009 Apr 9;458(7239):784-8.

Organizers

Eric Lai, PhD

Memorial Sloan Kettering Cancer Center
e-mail | website | publications

Eric Lai's interest in developmental biology began at Harvard, where he studied the C. elegans homeoprotein Ceh-20 for his BA thesis with Gary Ruvkun. He did his PhD with James Posakony at UC San Diego, where he characterized a new family of Notch pathway components in Drosophila and characterized the repression of Notch target genes by novel 3′ UTR sequence motifs. He continued to study the mechanism of Notch signaling as a postdoc with Gerald Rubin at UC Berkeley, but shifted his focus to small RNAs upon realizing that the post-transcriptional regulatory motifs he studied earlier were in fact microRNA binding sites. In 2005 Lai joined the Developmental Biology faculty at Sloan Kettering Institute in New York City. Eric Lai's lab currently studies two general topics: (1) the biogenesis and biological activities of small regulatory RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and piwi-interacting RNAs (piRNAs), and (2) the determination of cell fates via cell–cell signaling mediated by the Notch pathway. His group combines biochemical, genetic, and computational strategies towards understanding gene regulation at transcriptional and post-transcriptional levels.

Benjamin R. tenOever, PhD

Mount Sinai School of Medicine
e-mail | website | publications

Benjamin tenOever completed his postdoctoral training in Molecular Biology at Harvard University in 2007 after receiving his PhD in Virology from McGill University in 2004. In August of 2007, tenOever joined Mount Sinai School of Medicine where he is presently an Associate Professor of Microbiology. His work focuses on the molecular interactions between viruses and their hosts. More specifically, the lab studies the host transcriptional response to infection and the means by which the virus circumvents these activities to propagate the infection. This research encompasses the study of cellular antiviral proteins and small RNAs, of both cellular and virus origin, which contribute to the outcome of infection. The overall objective of this lab is to gain an understanding of the molecular basis of virus pathogenicity in an effort to generate improved therapeutics. tenOever is both a Pew scholar and a Burroughs Wellcome investigator and is the recipient of a number of prestigious honors including young investigator awards from the American Society of Microbiology, the National Academy of Sciences, and the White House.

Thomas Tuschl, PhD

The Rockefeller University
e-mail | website | publications

Thomas Tuschl received his PhD in chemistry from the University of Regensburg, in Germany, in 1995. From there, he went to the Max Planck Institute for Experimental Medicine in Göttingen, Germany, pursuing research with Fritz Eckstein. He next joined the biology department at the Massachusetts Institute of Technology and the Whitehead Institute for Biomedical Research, where he worked with Phillip A. Sharp and David P. Bartel. Tuschl was a junior investigator at the Max Planck Institute for Biophysical Chemistry before coming to Rockefeller in 2003 as an associate professor. He was named Professor in 2009. Tuschl's most recent honors include the Ernst Jung Prize for Medicine in 2008 and the Max Delbrück Medal and the Karl Heinz Beckurtz Award in 2007. In 2006 he received the Molecular Bioanalytics Prize from Roche Diagnostics. In 2005 he was named a fellow of the New York Academy of Sciences and received the Meyenburg Prize, the Irma T. Hirschl Trust Career Scientist Award and the Ernst Schering Award. He is also a Howard Hughes Medical Institute investigator.

Jennifer Henry, PhD

The New York Academy of Sciences
e-mail

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. Henry 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.

Marta Murcia, PhD

The New York Academy of Sciences

Until December 2011, Marta Murcia was Senior Program Manager, Life Sciences at the New York Academy of Sciences. She has a multidisciplinary background with a PhD in Organic-Medicinal Chemistry (Universidad Complutense, Madrid) and 7 years of postdoctoral experience in the fields of the Structure-based Drug Design and Computational Biology at Weill Cornell Medical College and Mount Sinai School of Medicine, New York. Murcia joined the Academy's Life Sciences team in September 2008, where she organized major scientific conferences in a broad range of topics such as cancer medicine, stroke, epigenetics, pain, and many others. In addition, Murcia managed the Academy's Non-coding RNA Biology Discussion Group, which meets periodically to provide a discussion forum for scientists engaged in research on non-coding RNA.


Speakers

Jayakrishna Ambati, MD

University of Kentucky
e-mail | website | publications

Jayakrishna Ambati is Professor of Physiology and Professor & Vice-Chair of Ophthalmology and Visual Sciences at the University of Kentucky. He holds the Dr. E. Vernon Smith & Eloise C. Smith Endowed Chair in Macular Degeneration Research. His laboratory has revealed novel mechanisms of age-related macular degeneration and angiogenesis. He is the 2010 ARVO Cogan Awardee and the winner of the 2010 Roger H. Johnson Memorial Award for Macular Degeneration Research. He is the first ophthalmologist to win the Doris Duke Distinguished Clinical Scientist Award and the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. Research to Prevent Blindness has awarded him its Senior Scientific Investigator Award, Lew R. Wasserman Merit Award, and Physician-Scientist Award. He was elected to The American Society for Clinical Investigation and was the first ophthalmologist to be elected to The Association of American Physicians. He serves on the Editorial Board of Investigative Ophthalmology & Visual Science and is an Associate Editor of Ophthalmology.

Susan J. Baserga, MD, PhD

Yale University
e-mail | website | publications

Susan J. Baserga is a Professor at Yale University with a primary appointment in Molecular Biophysics & Biochemistry and joint appointments in the Departments of Genetics and Therapeutic Radiology. Baserga received a BS in Biology from Yale College and an MD and PhD (Human Genetics) from Yale in 1988. The focus of her research is on the function of ribonucleoproteins in pre-rRNA processing and pre-ribosome assembly.

Lorena S. Beese, PhD

Duke University School of Medicine
e-mail | website | publications

Lorena S. Beese is a James B. Duke Professor in the Department of Biochemistry at Duke University Medical Center. She earned a BA degree in mathematics and biology from Oberlin College and a PhD in Biophysics from Brandeis University. Beese completed postdoctoral training at Yale University in the Department of Molecular Biophysics and Biochemistry under the direction of Thomas A. Steitz. She joined the Duke faculty in 1992 and has served as Co-director for the Structural and Chemical Biology Program in the Duke Comprehensive Cancer Center, Director of Graduate Studies for the Structural Biology and Biophysics Program, and Director of the Center for Structural Biology. Beese's research focuses on understanding the molecular mechanisms that underlie DNA replication and human mismatch repair. She has carried out pioneering work that elucidated the structure and mechanism of protein prenyltransferases, enzymes that catalyze essential post-translational modifications to cell-signaling molecules. Additionally, she has contributed to development of therapeutics for treatment of cancer and infectious disease. Beese was elected to the National Academy of Sciences in 2009. Other honors include a Searle Scholar Award, a MERIT Award from the National Institutes of Health.

Robert H. Blelloch, MD, PhD

University of California, San Francisco
e-mail | website | publications

Robert Blelloch is an Associate Professor at University of California–San Francisco. He is a member of the Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, the Center of Reproductive Sciences, and the Diller Cancer Center. He holds appointments in the Departments of Urology, Obstetrics/Gynecology, and Pathology. He is clinically trained in Clinical Pathology and Transfusion Medicine. His research interests are in the epigenetic regulation of stem cells and cancer. His major focus in the past few years has been on the role of non-coding small RNAs, microRNAs, and endo-siRNAs in regulating embryonic stem cell self-renewal and differentiation. Blelloch is currently expanding these efforts into somatic stem cells and cancer. He has expertise in genetics, genomics, epigenetics, small RNAs, stem cell biology, embryology, and cancer.

Stefanie Gerstberger

The Rockefeller University
e-mail | website | publications

Stefanie Gerstberger is a graduate student within the Tri-Institutional Chemical Biology program of The Rockefeller University, Memorial Sloan-Kettering Cancer Research Center and Weill Cornell Medical School, and a member of the Tuschl laboratory. She studied Biochemistry and Chemistry at Oxford University and received her MChem degree in 2009.

Fedor V. Karginov, PhD

Cold Spring Harbor Laboratory
e-mail | website | publications

Fedor Karginov received a BS degree in chemistry from University of Virginia, Charlottesville, VA. He underwent graduate studies to earn a PhD in chemistry and biochemistry at the University of Colorado in Boulder, CO, in the laboratory of Olke Uhlenbeck, where he studied the modular structure and enzymology of RNA helicases. Fedor went on to a postdoctoral position at Cold Spring Harbor Laboratory in the group of Gregory Hannon. His research focus and interests include mammalian microRNA biology, roles and mechanisms of Argonaute proteins, and identification of microRNA targets.

Eric Lai, PhD

Memorial Sloan Kettering Cancer Center
e-mail | website | publications

Christopher D. Lima, PhD

Memorial Sloan Kettering Cancer Center
e-mail | website | publications

Christopher D. Lima is a Professor and Member in the Structural Biology Program at the Memorial Sloan-Kettering Cancer Center in New York City. He received his PhD in 1994 from Northwestern University for his work on E. coli topoisomerase I under the supervision of Alfonso Mondragon. After completing his postdoctoral studies as a Helen Hay Whitney Fellow at Columbia University under the supervision of Wayne A. Hendrickson he joined the faculty at the Weill Medical College of Cornell University in 1998. He moved his laboratory to the Sloan-Kettering Institute in 2003. Lima received the Louise and Allston Boyer Young Investigator Award, the Mayor's Award for Excellence in Science and Technology, the Beckman Young Investigator Award, and the Rita Allen Scholar Award. Since starting his lab, Lima's research has focused on pathways that contribute to RNA processing, 5′ cap formation, and RNA decay and on mechanisms that underlie post-translational protein modification by the ubiquitin-like modifier SUMO.

Dinshaw J. Patel, PhD

Memorial Sloan Kettering Cancer Center
e-mail | website | publications

Dinshaw Patel received his BSc in Chemistry from the University of Bombay, India before pursuing an MS in Chemistry at the California Institute of Technology. After his MS, he east ventured to New York University where he received his PhD in 1968. Patel completed postdoctoral training in an academic setting at New York University Medical School and in industry at AT&T Bell Laboratories. From 1984 to 1992 Patel was a Professor of Biochemistry and Molecular Biophysics at Columbia University. Since 1992 he has been a member of the Structural Biology Program at Memorial Sloan-Kettering Cancer Center, and since 1994, a professor at Weill Cornell Medical College. His research group applies crystallographic and solution NMR techniques to investigate macromolecular-mediated recognition, regulation, and catalysis. The major thrust of his laboratory's work is currently in the structural biology of RNA silencing and the histone/epigenetics code.

Stewart Shuman, MD, PhD

Memorial Sloan Kettering Cancer Center
e-mail | website | publications

Stewart Shuman is a Member of the Molecular Biology Program at Memorial Sloan-Kettering Cancer Center. He is also a professor of the Tri-Institutional Training Program in Chemical Biology, with a focus on Biological Chemistry and Biochemistry. His lab focuses on nucleic acid metabolism and specifically on 4 problems in nucleic acid metabolism: (i) the coordination of eukaryotic mRNA synthesis and processing, (ii) the structure and function of eukaryotic type I DNA topoisomerases, (iii) DNA damage recognition and repair by DNA ligases, and (iv) mechanisms and biological roles of RNA repair enzymes. Shuman has received numerous awards during his career and in 1990 he was named a Pew Scholar.

Benjamin R. tenOever, PhD

Mount Sinai School of Medicine
e-mail | website | publications

Liang Tong, PhD

Columbia University
e-mail | website | publications

Liang Tong received his BSc degree in chemistry from Peking University in China and his PhD in protein crystallography from the University of California, Berkeley, working in the laboratory of Sung-Hou Kim. He was a post-doctoral fellow in the laboratory of Michael Rossmann at Purdue University. He then established a structure-based drug design laboratory at Boehringer Ingelheim Pharmaceuticals, Inc. He joined the faculty at Columbia University in 1997 and is now Professor of Biological Sciences. His current research interests include enzymes involved in RNA degradation and quality control, proteins involved in pre-mRNA 3′-end processing, as well as enzymes involved in fatty acid metabolism.


Kristen Delevich

Kristen Delevich is a graduate student in the Watson School of Biological Sciences at Cold Spring Harbor Laboratory. Her work in Bo Li's laboratory focuses on the role of a schizophrenia susceptibility gene on synaptic physiology in the maturing rodent cortex. She earned her BS as a double-major in Neuroscience and Philosophy at the University of Pittsburgh. Her previous research has spanned from human to monkey to mouse brains. When she's not at the rig patch-clamping, you can find her at her home-base in Brooklyn.