The Cellular Functions of RNA Nucleases

The Cellular Functions of RNA Nucleases

Wednesday, November 2, 2011

The New York Academy of Sciences

Presented By

 

The ability to cleave nucleic acids is an essential activity for a wide array of cellular processes. Whether it is maintaining quality control of cellular mRNA or the generation of non coding RNAs, nucleases are fundamental components central to proper cell function. In this one-day forum, speakers will discuss the diverse roles that cellular nucleases perform in the biogenesis and/or quality control of rRNAs, mRNAs, and miRNAs. The forum will also focus on unique and common structural features shared between different nucleases and the diseases associated with nuclease malfunction. The overall objective of this event is to explore the current understanding of nucleases as they relate to a diverse array of cellular functions.

Networking Reception to Follow.

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Agenda

* Presentation times are subject to change


Wednesday, November 2, 2011

8:00 am

Registration and Breakfast

9:00 am

Opening Remarks
Jennifer Henry, PhD, The New York Academy of Sciences
Benjamin R. tenOever, PhD, Mount Sinai School of Medicine

Introduction to Nucleases

9:15 am

Conserved Function and Expression Profiles of RNA Nucleases
Stefanie Gerstberger, The Rockefeller University

Session I: Nucleases in rRNA Biogenesis

9:30 am

Structure and Mechanism of RNA 3’ Cyclase and the 18S rRNA Processosome Component Rcl1
Stewart Shuman, MD, PhD, Memorial Sloan Kettering Cancer Center

10:00 am

Utp24 in the Biogenesis of 18s rRNA
Susan J. Baserga, MD, PhD, Yale University

10:30 am

Coffee Break

Session II: Nucleases in miRNA Biogenesis

11:00 am

Non-canonical miRNA Biogenesis
Eric Lai, PhD, Memorial Sloan Kettering Cancer Center

11:30 am

Diverse Endonucleolytic Cleavage Sites in the Mammalian Transcriptome Depend Upon microRNAs, Drosha, and Additional Nucleases
Fedor V. Karginov, PhD, Cold Spring Harbor Laboratory

12:00 pm

A Role for Non-canonical microRNAs as Revealed by Rhenotypic Differences Between Dgcr8 and Dicer Knockouts
Robert H. Blelloch, MD, PhD, University of California, San Francisco

12:30 pm

Cytoplasmic Processing of Primary Viral microRNAs
Benjamin R. tenOever, PhD, Mount Sinai School of Medicine

1:00 pm

Lunch

Session III: Nuclease Structures

2:00 pm

Structures of Human Exonuclease-I DNA Complexes Reveal a Unified Understanding of 5′ Structure Specific Repair Nucleases
Lorena S. Beese, PhD, Duke University School of Medicine

2:30 pm

RNA Decay Activities of the Eukaryotic RNA Exosome
Christopher D. Lima, PhD, Memorial Sloan Kettering Cancer Center

3:00 pm

Coffee Break

3:30 pm

Structure and Nuclease Activity of C3PO
Dinshaw J. Patel, PhD, Memorial Sloan Kettering Cancer Center

4:00 pm

5’-3’ Exoribonucleases, RNA Degradation and Quality Control
Liang Tong, PhD, Columbia University

Session IV: Nucleases in Disease

4:30 pm

The Role of Dicer in Age-related Macular Degeneration
Jayakrishna Ambati, MD, University of Kentucky

1-hour reception to follow

Speakers

Organizers

Eric Lai, PhD

Memorial Sloan Kettering Cancer Center

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 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, Dr. 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. They have had particular interest in studying how these regulatory mechanisms direct the intricate patterning of Drosophila nervous system, and more recently, the self-renewal and differentiation of mammalian neural stem cells.

Benjamin R. tenOever, PhD

Mount Sinai School of Medicine

Dr. tenOever completed his postdoctoral formation in Molecular Biology from Harvard University in 2007 after receiving his PhD in Virology from McGill University in 2004. In August of 2007, Dr. tenOever joined Mount Sinai School of Medicine and is presently an Associate Professor of Microbiology. His work focuses on the molecular interactions between viruses and their host. 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. Dr. tenOever is both a Pew scholar and a Burroughs Wellcome investigator and 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

Dr. Tuschl received his PhD in chemistry from the University of Regensburg, in Germany, in 1995. 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. Dr. Tuschl was a junior investigator at the Max Planck Institute for Biophysical Chemistry before coming to Rockefeller in 2003 as associate professor. He was named professor in 2009. Dr. 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. In 2003 he received the Wiley Prize in Biomedical Sciences, the New York City Mayor’s Award for Excellence in Science and Technology and the Newcomb Cleveland Prize from the American Association for the Advancement of Science. Dr. Tuschl was the recipient of the European Molecular Biology Organization Young Investigator Award in 2001 and the Biofuture Award from the German government in 1999. He is also a Howard Hughes Medical Institute investigator.

Jennifer Henry, PhD

The New York Academy of Sciences

Marta Murcia, PhD

The New York Academy of Sciences

Speakers

Jayakrishna Ambati, MD

University of Kentucky

Jayakrishna Ambati, MD 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

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. Susan Baserga received a BS in Biology from Yale College and an MD and PhD (Human Genetics) from Yale in 1988. The focus of Susan's research is on the function of ribonucleoproteins in pre-rRNA processing and pre-ribosome assembly. Her laboratory website is http://info.med.yale.edu/mbb/baserga/.

Lorena Beese, PhD

Duke University School of Medicine

Lorena S. Beese, PhD, is a James B. Duke Professor in the Department of Biochemistry, at Duke University Medical Center. She earned a B.A. degree in mathematics and biology from Oberlin College and a Ph.D. in Biophysics from Brandeis University. Dr. Beese completed postdoctoral training at Yale University in the Department of Molecular Biophysics and Biochemistry under the direction of Dr. 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. Dr. 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. Dr. 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 Blelloch, MD, PhD

University of California, San Francisco

Dr. 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. He 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

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

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.

Christopher D. Lima, PhD

Memorial Sloan Kettering Cancer Center

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 Dr. Alfonso Mondragon. After completing his postdoctoral studies as a Helen Hay Whitney Fellow at Columbia University under the supervision of Dr. 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. Dr. 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, Dr. 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

Stewart Shuman, MD, PhD

Memorial Sloan Kettering Cancer Center

Liang Tong, PhD

Columbia University

Liang Tong received his B. Sc. degree in chemistry from Peking University in China, and his PhD in protein crystallography from the University of California at Berkeley, working in the laboratory of Sung-Hou Kim. He was a post-doctoral fellow in the laboratory of Michael Rossmann at Purdue University in West Lafayette, IN. He then established a structure-based drug design laboratory at Boehringer Ingelheim Pharmaceuticals, Inc. in Ridgefield, CT. 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.

Abstracts

Introduction to Nucleases

Conserved Function and Expression Profiles of RNA Nucleases
Stefanie Gerstberger, The Rockefeller University

Specialized pathways involving nucleases, helicases, RNA-binding proteins and other protein cofactors, regulate maturation and turnover of coding and non-coding RNAs. In many instances, the constituents conferring specificity to these nucleolytic pathways remain unknown or poorly characterized. I will give a short introduction to eukaryotic RNA nucleases, which are the key enzymes in these processes, with emphasis on their conservation and tissue specific expression patterns, as these often provide insights into function and target specificity of different nucleases.

 

Session I: Nucleases in rRNA Biogenesis

Rcl1 in rRNA Processing
Stewart Shuman, MD, PhD, Memorial Sloan Kettering Cancer Center

RNA 3′-phosphate cyclase (Rtc) enzymes are a widely distributed family that synthesize RNA 2′,3′ cyclic phosphate ends via three steps: reaction with ATP to form a covalent Rtc–AMP intermediate; transfer of adenylate to an RNA 3′-phosphate to form RNA(3′)pp(5′)A; and attack of the vicinal O2′ on the 3′-phosphorus to form a 2′,3′ cyclic phosphate and release AMP. We have determined the crystal structures of E. coli Rtc at multiple steps along the reaction pathway, including: Rtc•ATP and Rtc•ATP•Mn2+ substrate complexes, the covalent Rtc-(histidinyl-Ne)-AMP intermediate, and an Rtc•AMP product complex. They illuminate the basis for ATP substrate specificity, the mechanism of nucleotidyl transfer, the stereochemical transitions at the AMP phosphate, and the critical role of the metal in orienting the PPi leaving group of ATP during the RtcA adenylylation step. We show that the subsequent steps in the cyclization pathway are metal-independent.
 
Eukaryal Rcl1 is an essential nucleolar protein required for U3 snoRNA-guided pre-rRNA processing at sites flanking the 18S rRNA sequence. A potential catalytic role for Rcl1 during pre-RNA cleavage has been suggested based on its primary structure similarity to RNA 3′ cyclase. We've determined the 2.6 Å crystal structure of a biologically active yeast Rcl1, which illuminates its modular 4-domain architecture and overall homology to RNA cyclases, while revealing numerous local differences that account for why Rtcs possess metal-dependent adenylyltransferase activity and Rcls do not. A conserved oxyanion binding site in Rcl1 was highlighted for possible catalytic or RNA binding functions. However, the benign effects of mutations in and around the anion site on Rcl1 activity in vivo militate against such a role.
 

Utp24 in the Biogenesis of 18s rRNA
Susan J. Baserga, MD, PhD, Yale University

Ribosome biogenesis is a complex process that requires over 150 trans-acting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and novel components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results are consistent with the idea that Utp24 might be the long-sought endonuclease that cleaves the pre-rRNA at sites A1 and/or A2.

 

Session II: Nucleases in miRNA Biogenesis

Ago2-mediated miRNA Biogenesis
Eric Lai, PhD, Memorial Sloan Kettering Cancer Center

One of the largest surprises of the past decade has been the elucidation of diverse post-transcriptional regulatory pathways mediated by non-coding RNAs <30 nucleotides in length, which guide Argonaute (AGO) complexes to target genes. Three major classes of small RNAs include miRNAs, siRNAs and piRNAs. Collectively, these mediate conserved strategies of gene regulation critical for normal development, physiology, and germline integrity. Moreover, their study has provided powerful experimental tools, insights into disease, and new opportunities for therapy. My laboratory has studied canonical and atypical biogenesis mechanisms for all three classes of small RNAs, and I will discuss recent progress on Drosha-independent and Dicer-independent mechanisms of miRNA biogenesis.
 

Diverse Endonucleolytic Cleavage Sites in the Mammalian Transcriptome Depend Upon microRNAs, Drosha, and Additional Nucleases
Fedor V. Karginov, PhD, Cold Spring Harbor Laboratory

The functional lifespan of a mammalian miRNA is determined by a variety of mechanisms. Among these, animal microRNAs in complex with Argonaute proteins bind to many mRNA targets, generally with imperfect complementarity, leading to destruction of the mRNA through the normal cellular decay machinery. The ancestral "slicer" endonuclease activity of Argonaute2, which requires more extensive complementarity with the target RNA, is not used in this pathway. Only two examples of microRNA-guided slicing of mRNA targets have been reported, neither of which can fully account for the deep conservation of Ago2 catalytic activity or the requirement for an intact Ago2 nuclease domain for mouse viability. Here, we assess the endonucleolytic function of Ago2 and other nucleases by identifying cleavage products retaining 5′-phosphate groups in mouse ES cells on a transcriptome-wide scale. We detect a significant signature of Ago2-dependent cleavage events and validate several targets. Unexpectedly, a broader class of Ago2-independent cleavage sites is also observed, indicating participation of additional nucleases in site-specific mRNA cleavage. Within this class, we identify a cohort of Drosha-dependent mRNA cleavage events that functionally regulate mRNA levels in mES cells, including one in the Dgcr8 mRNA. Together, these results highlight the underappreciated role of endonucleolytic cleavage in controlling mRNA fates in mammals.
 

A Role for Non-canonical microRNAs as Revealed by Rhenotypic Differences Between Dgcr8 and Dicer Knockouts
Robert H. Blelloch, MD, PhD, University of California, San Francisco

The DGCR8/DROSHA complex is essential for processing pri-miRNAs to pre-miRNAs, while DICER processes pre-miRNAs to mature miRNA. However, Dicer also exists in species that do not have miRNAs, but instead produce Dicer-dependent endo-siRNAs. By deep sequencing of all small RNAs in wild-type, Dgcr8 null, and Dicer null mouse embryonic stem cells, we discovered the existence of at least 3 classes of DICER-dependent small RNAs—canonical miRNAs (require DGCR8/DROSHA), non-canonical miRNAs (bypass DGCR8/DROSHA), and endo-siRNAs (long double stranded RNAs that bypass DGCR8/DROSHA and are consecutively processed by Dicer). The biological roles of noncanonical miRNAs and endo-siRNA remain largely unknown in mammals. However, by comparing phenotypes of DGCR8 and DICER deletion in various tissues together with deep sequencing in those tissues, we have discovered tissue specific roles for non-canonical miRNAs and endo-siRNAs in the brain and oocytes respectively. These results together with ongoing studies will be discussed.
 

Cytoplasmic Processing of Primary Viral microRNAs
Benjamin R. tenOever, PhD, Mount Sinai School of Medicine

The discovery of microRNAs (miRNAs) revealed an unappreciated level of post-transcriptional control utilized by the cell to maintain optimal protein levels. While canonical synthesis of these miRNAs occurs in a stepwise fashion, initiated by the nuclear microprocessor Drosha and DGCR8, rare non-canonical processing mechanisms have also been identified. In an effort to determine whether RNA viruses could exploit both canonical and non-canonical miRNA biogenesis pathways, we engineered nuclear and cytoplasmic viruses to encode a primary miRNA transcript. Surprisingly, viruses originating from both subcellular compartments were capable of generating robust levels of miRNAs, irrespective of nuclear access. Furthermore, while nuclear viruses required the canonical host machinery for miRNA processing, cytoplasmic viruses, grafted with the same hairpin, generated the miRNA in the absence of DGCR8. Virus-derived miRNAs, regardless of origin, associated with the RNA induced silencing complex and conferred post transcriptional gene silencing both in vitro and in vivo. These studies illustrate the existence of an unappreciated cytoplasmic nuclease activity of unknown physiological function. These findings, the components involved, and the implications of this processing, will be discussed.

 

Session III: Nuclease Structures

Structures of Human Exonuclease-I DNA Complexes Reveal a Unified Understanding of 5′ Structure Specific Repair Nucleases
Lorena S. Beese, PhD, Duke University Medical School

DNA repair is essential for maintaining genome stability and defects in repair pathways underly a number of human diseases including cancer. Human exonuclease 1 (hExo1) is involved in several essential DNA repair and recombination processes. hExo1 is a member of the 5′ structure-specific nuclease family of exonucleases and endonucleases that includes FEN-1, XPG, and GEN1. Structures of hExo1 in complex with DNA substrates, followed by mutagenesis studies, lead to a proposal for a common mechanism by which this nuclease family recognizes and processes diverse DNA structures1. Frayed 5′ ends of nicked duplexes resemble flap junctions, unifying the mechanisms of endo- and exonucleolytic processing. The relative arrangement of substrate binding sites in these enzymes provides a solution to the complex geometrical puzzle of substrate recognition and processing. In DNA mismatch repair hExoI is activated MutS homologs and becomes the processive nuclease required for 5′ directed DNA mismatch repair. A model for hExo1 activation and its implications for human mismatch repair will be presented.
 
1. Orans, J., McSweeney. E.A., Iyer, R.R., Hast, M.A., Hellinga, H.W., Modrich, P, & Beese, L.S. (2011) Structures of human exonuclease 1 DNA complexes suggest a unified mechanism for nuclease family. Cell 145(2): 212-23.
 

RNA Decay Activities of the Eukaryotic RNA Exosome
Christopher D. Lima, PhD, Memorial Sloan Kettering Cancer Center

RNA lifetime and abundance is regulated by controlling the balance between RNA transcription and RNA degradation. Two principle RNA decay pathways exist in eukaryotes, one catalyzes degradation 5′ to 3′ while the other degrades RNA in the 3′ to 5′ direction. The 3′ to 5′ decay pathway requires the activities of the RNA exosome, a large multi-subunit protein complex that contains a non-catalytic core of nine subunits and two additional subunits that catalyze processive and distributive 3′ to 5′ RNA exoribonuclease activities, Rrp44 and Rrp6. In budding yeast, ten of the eleven genes are essential for growth, suggesting the importance of the RNA exosome and its activities in cellular function. While recent efforts illuminated fundamental aspects of eukaryotic exosome structure and function, many questions remain with respect to the individual and collective activities for exosome subunits in RNA processing and decay. We are focused on characterizing the activities for human counterparts to yeast Rrp44 and Rrp6 and on defining how the exosome 9 subunit core contributes to substrate selection and RNase activities.
 

Structure and Nuclease Activity of C3PO
Dinshaw J. Patel, PhD, Memorial Sloan Kettering Cancer Center

Abstract forthcoming.
 

5′–3′ Exoribonucleases, RNA Degradation and Quality Control
Liang Tong, PhD, Columbia University

The 5′–3′ exoribonucleases (XRNs) belong to a family of highly conserved enzymes in eukaryotes and have important functions in RNA metabolism and RNA interference. XRN1 (175 kD) is primarily cytosolic and is involved in degradation of decapped mRNAs, nonsense mediated decay, microRNA decay, and is essential for proper development. XRN2 (115 kD, Rat1 in yeast) is primarily nuclear and has important roles in RNA trafficking, transcription termination by RNA polymerase II (Pol II), rRNA and snoRNA processing, and other processes.
 
The crystal structures of Rat1 (1) and Xrn1 (2) reveal a unique active site landscape for these enzymes and explain their exclusive exonuclease activity. Xrn1 contains four additional domains that are important for nuclease activity and may also be a platform for interacting with other proteins. Unexpectedly, we discovered that the activating protein partner of Rat1, Rai1, is a new eukaryotic enzyme with RNA 5′ pyrophosphohydrolase (PPH) activity (1) as well as decapping activity towards unmethylated 5′ cap (3). We have shown that Rai1 is part of a novel RNA quality surveillance pathway in yeast, promoting the degradation of mRNAs with 5′ capping defects (3).
 
The presentation will also cover results from our latest research on these important enzymes.
 
Supported in part by a grant from the NIH.

 

Session IV: Nucleases in Disease

The Role of Dicer in Macular Degeneration
Jayakrishna Ambati, MD, University of Kentucky

Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell degeneration. We have identified a dramatic reduction in the expression of the microRNA (miRNA)-processing enzyme DICER1 in the RPE of humans with GA. We found that conditional ablation of Dicer1, but not multiple other miRNA-processing enzymes, induces RPE degeneration in mice. We determined that DICER1 knockdown induces accumulation of Alu repetitive RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. DICER1 knockdown also induced RPE degeneration in non-human primates. Alu RNA is also increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. We found that Alu RNA is a substrate for DICER1, and that DICER1-digested Alu RNA cannot induce RPE degeneration in mice. In addition, Alu RNA can travel from one RPE cell to another, which is compatible with the hypothesis that the centrifugal spread of GA is due to poisoning of neighboring RPE cells by unhealthy RPE cells. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.
 

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