The Cellular Functions of RNA Nucleases

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.