Manuel Ares - US grants

The relationships of the snRNA structural elements to their functions in spliceosome assembly and splicing are only generally understood. A persistent mystery in splicing is how the many RNA helicase family members achieve snRNA rearrangements during assembly of the spliceosome, catalysis, and disassembly. A second major question concerns how splicing regulators influence the basic process of spliceosome assembly. Many proteins that regulate splicing are known, but exactly how they do it remains quite opaque. A third and emerging set of questions concerns the role of splicing and other small RNA-mediated processes in genome function and evolution. We will address each of these major questions with a specific aim. (1) We will dissect functional interactions between the DEAD-box RNA helicase family member PrpSp and components of the U2 snRNP in particular Cus2p and U2 snRNA during spliceosome assembly and splicing in vitro and in vivo. Cus2p and PrpSp influence U2 snRNA structure and each other to control the delivery of U2 snRNA to the branchpoint region of the pre-mRNA in an ATP-dependent step-prespliceosome assembly. (2) We will explore the mechanism of Mer1p-activated splicing and its importance for the meiotic gene expression program in yeast. Since Merlp binds U1 snRNPs, we will focus on the composition and activities of the U1 snRNP with and without Merlp. A second part of this aim is to understand the roles of Merlp and the four known genes whose splicing depends on Mer1p in the meiotic gene expression program using splicing sensitive microarrays. (3) We will study the integration of splicing with other steps in the gene expression pathway and will begin to determine how the loss of introns containing conserved sequence affects cellular function. A large microarray study completed in the last funding period presents several specific hypotheses for how splicing and small RNAs are connected to other steps in gene expression. The areas of inquiry represented by these aims encompass the major issues in the splicing field: How do snRNPs work? How is splicing regulation achieved? And how is the process of splicing integrated into genome function and evolution? The combined strengths of genetics, biochemistry, and genomics available in yeast and the conserved properties of the splicing machinery indicate that fundamental knowledge uncovered by these efforts will translate directly to the understanding of human gene expression.

9318111 Ares Nucleic acid-protein interactions are critical to the correct expression and retrieval of genetic information. Selective transcription of genes requires selective interactions between protein and DNA. Construction of a messenger RNA containing the protein coding information of eukaryotic genes requires the recognition and removal of introns and RNA splicing, a process dependent on an elaborate set of RNA-RNA and RNA-protein interactions. The translation of messenger RNA into protein is carried out by a machinery made of RNA and protein as well, requiring another elaborate set of RNA-RNA and RNA-protein interactions. Control of the level of mRNA is a means by which the expression of genes is regulated, often governed by RNA structural elements in the mRNA. Detailed knowledge of how and when these DNA-protein, RNA-protein, and RNA-RNA interactions are established, maintained and disolved will be essential for understanding the expression of genetic information. A set of powerful "footprinting" or "structure probing" techniques for measuring the environments of nucleotides along a nucleic acid chain is well developed and is in general use among students of nucleic acid structure and function. These approaches rely on detecting differences in accessibility of the nucleic acid under study to some probe (a chemical or nuclease), capable of reacting with nucleic acid. The positions of reaction are mapped on the chain using a variety of techniques, all of which require radioactive labeling of the chain either directly or indirectly. The radioactive products are separated by size on a gel and the sites of reaction are calculated from the size of radioactive products. The amount of reaction at a particular site can be determined by the amout of radioactive material present in products of a particular size. For example, a protein that binds to a specific site on a nucleic acid chain will protect the chain from attack by the probe. After separation and com parison of bound andunbound nucleic acid, gel bands representing protected sites will contain less label. The technical limitations of this general approach are two fold. First, it is often difficult to isolate sufficient amounts of functionally relevant protein-nucleic acid complexes to obtain detectable signals, and second, some interactions are sufficiently subtle that protection from probes, though meaningful, may be only partial, and require careful quantitation of signal. Both of these limitations arise from a single cause: the requirement for exposure of radioactive gels to photographic film for detection and quantitation of radioactive signal. These limitations can be skirted by the use of a new technology, storage phosphor imaging, rather than autoradiography. Phosphor imaging involves exposure of the footprinting gel to a special storage phosphor screen, on which a compound sensitive to radiation is bound. The compound is converted by absorption of a radioactive particle. The screen is then inserted into a laser scanning device that determines the amount of converted compound present at every coordinate on the screen. This data is used to build a computer file that expresses the amount of radiactive material at each position as an image using a grey scale or color. The investigator can inspect the image on a monitor and with the image analysis software, analyze the data quantitatively. This technique is orders of magnitude more sensitive than autoradiography allowing better detection of signal, and unlike photographic film the response of the phosphor to radiation is linear, allowing accurate quantitation. This proposal aims for a material extension of our understanding of the function of nucleic acid-protein complexes and RNA-RNA interactions through the analysis of available footprinting data by the purchase and use of phosphor imaging technology. The Noller and Puglisi groups will use the device in their studies of ribosome structure and func tion in the process of translation. The Ares group will use it in their studies of the role of small nuclear RNAs in spliceosome assembly and pre-messenger RNA splicing. The Peck group will require this technology for understanding the assembly and function of RNA polymerase 111 transcription complexes, and the Silverthorne group will use it to study the role of mRNA structure in the regulation of mRNA stability.

Completion of the DNA sequence of the yeast genome has made accessible a large number of questions about the organization and expression of eukaryotic genomes. Important among these questions is defining a complete minimum protein set necessary for eukaryotic cell growth and regulation, key to understanding human cancer. A hallmark of the eukaryotes is the abundant presence of introns, internal gene sequences not found in the mature messenger RNAs (mRNAs) that specify the protein coding capacity of the genome. The presence of introns clouds our ability to see open reading frames in the genomic sequence. To understand the complete coding capacity of the yeast genome, and of other eukaryotic genomes, we must first be able to recognize introns in the genomic sequence. With the complete sequence of the yeast genome in hand, we have the opportunity to map the positions of all the nuclear pre-mRNA introns in the yeast genome, and thus reveal its protein coding capacity. At this writing 220 yeast introns are known or predicted, but these have been identified in a biased, ad hoc fashion. We have developed a powerful molecular approach to the direct detection of introns in a manner not biased by the contents of the gene in which it is embedded. Oligonucleotides complementary to the unique lariat sequence formed during splicing ("branchmers") specifically prime reverse transcription of lariat intron RNA. Mutations that inactivate the lariat debranching enzyme cause dramatic accumulation of intron RNA in yeast. Thus branchmer oligonucleotides will be used to generate expressed intron probes. Our aims are (1) to create and screen libraries of "expressed intron tag" clones derived from strains of yeast that accumulate large-amounts of intron RNA. These clones will be sequenced to generate a database of expressed intron sequences, (2) to identify genomic sequences similar to known introns using informatic approaches and test these for splicing potential in vivo, and (3) to refine repeated applications of each approach until a complete set of confirmed introns is mapped to the sequence of the genome. Finding all the introns will be essential to the complete understanding of the coding capacity of the genome.

DESCRIPTION: The essential RNT1 (ribonuclease three) gene encodes a double strand-specific endoribonuclease from the yeast Saccharomyces cerevisiae. The RNT1 protein appears similar to bacterial RNase III in structure and function and is required for pre-ribosomal RNA processing events in yeast. Eukaryotic pre-rRNA processing is also dependent on small nucleolar RNAs, and at least one processing event is dependent on both yeast RNase III and U3 snoRNA. In bacteria, RNase III is involved in a wide variety of RNA processing events; thus the identification of the enzyme in yeast offers an unusual opportunity to explore the role of this fundamental enzyme in eukaryotic gene expression and RNA biogenesis, where it may influence mRNA stability or the replication of RNA viruses. An overarching hypothesis to be tested is that yeast RNase III is required for the synthesis or degradation of several unstable nonribosomal RNAs necessary for cell growth. In addition, the mechanism of snoRNA facilitation of RNase III activity deserves investigation. Three aims are proposed: 1) To characterize the structure, function and substrate specificity of the yeast RNase III enzyme using reverse genetics and biochemistry; 2) to identify and characterize substrates of RNase III whose processing is key to cell growth; and 3) To develop systems to study the mechanism by which snoRNAs function in concert with RNase III in pre-rRNA processing and ribosome assembly. The first specific aim will be addressed by studying the function of RNT1 protein and its mutants to determine which parts of the protein are required for substrate binding, cleavage, metal binding and multimerization. In the second specific aim genetic screens for suppressors and enhancers (synthetic lethal mutations) for two available temperature sensitive mutations in the RNT1 gene will be used to identify key substrates whose cleavage by RNase III is essential for cell growth. The third specific aim will require an effort to reproduce the observed in vivo requirement for both RNase III and the U3 snRNA in the A0 processing event in vitro. Together, the proposed experiments should provide an initial characterization of a conserved and fundamental eukaryotic gene product about which little is known.

DESCRIPTION (provided by applicant): The correct secondary structures and the relationships of structural elements to the function of snRNAs in splicing have been appreciated for a number of years. Roles of snRNAs in recognition of each other and the premessenger RNA reactive sites that establish the structure of the active spliceosome have been demonstrated. A continuing mystery in the analysis of splicing is how the snRNA rearrangements are achieved and coordinated during assembly of the spliceosome, catalysis, and disassembly. A second major issue concerns how, at the biochemical level, the regulation of splicing impinges on the basic process of spliceosome assembly. A third and emerging set of questions concerns the role of splicing in genome function and evolution. Experiments addressing each of these major questions are proposed. By characterizing the functional interactions between U2 snRNA, the DExD/H ATPase Prp5p and Cus2p, the enforcer of ATP-dependence at the Prp5p step it will be possible to understand the mechanism of prespliceosome assembly. Critical to this process, the yeast SF3a (Prp9p, Prp11p, Prp21p), and SF3b (Cus1p, Hsh49p, Hsh155p, Rse1p) protein complexes are present near the catalytic core of the spliceosome. The hypothesis is that protein factors that interact with U2 snRNA play essential roles in the regulation, progression and fidelity of splicing reactions. A unique opportunity to study positive regulation of splicing is by exploring the functional interactions between Mer1p and the U1 snRNP during splicing activation. The hypothesis is that Mer1p accelerates a key step in spliceosome assembly or splicing. Finally a modified microarray technology capable of monitoring all introns in the yeast genome in parallel will be applied to questions concerning the global regulation of splicing. The results should reveal general features of presliceosome assembly in terms of the basal machinery at the key step of prespliceosome formation, and in instances of splicing regulation. Genome-wide views of splicing have never been developed, and the new technology will allow this important process to be understood in terms of regulatory networks. These studies will be important foundations for investigations into systems with more complex splicing, such as human cells.

DESCRIPTION (provided by applicant): Alternative splicing is a key process in the control of mammalian gene expression and a major source of protein diversity. Errors in splicing regulation are implicated in many disease processes, including cancer and inherited disorders of the neuromuscular systems. However, the cellular circuits that control splicing regulation are mostly unknown. New methods that measure splicing changes on a genome-wide scale make possible the discovery of coordinately regulated networks of alternative splicing. The elucidation of the regulatory events underlying this coordinate control will be essential for understanding how groups of exons are controlled during development and disease. This project will support the continued development and dispersal of parallel technologies for measuring alternative splicing initiated by the Black, Fu and Ares labs through prior R24 funding. In the initial project period, several different approaches were developed. Most notably, two splicing- sensitive microarrays, one for mouse and one for human cells, each measuring splicing of about 1300 alternative splicing events in about 1000 genes, were successfully designed, printed and used to capture and analyze data. These arrays were applied to a diverse set of experiments and were successful in uncovering several systems of coordinate splicing control important in cellular differentiation and homeostasis. We propose to continue this productive collaboration with the following aims: (1) We will continue to apply the arrays and analysis methods produced during the previous funding period to questions of splicing regulation, and we will expand their use to additional laboratories studying splicing; (2) we will improve the design and analysis of splicing-sensitive arrays to make them more comprehensive, and reliable, as well as more widely available; and (3) we will develop a promising new approach to genome-wide splicing analysis using high density sequencing methods. This project will broaden the study of splicing regulation to the level of the whole genome, allowing the integration of specific splicing regulatory pathways into our understanding of gene regulation and genome function. PUBLIC HEALTH RELEVANCE Many human diseases, including both cancer and inherited diseases of the neuromuscular systems, are caused by alterations in gene function through a process called alternative pre-mRNA splicing. Although individual changes in splicing have been linked to particular disorders, it is not well understood how programs of splicing affect the larger biology of the cell, and hence how abnormalities in these programs lead to disease. This project will extend our work on methods for examining splicing regulation on a genome wide scale that will allow elucidation of these larger programs of genetic change in disease.

The Gordon Research Conference (GRC) on "The Biology of Post-Transcriptional Gene Regulation" will be held at Salve Regina University in Newport, Rhode Island, from July 18th to July 23rd, 2010. This meeting is the fourth in a GRC series that has been held every other year since 2004. The goal of this series is the elucidation of the fundamental mechanisms and regulation of RNA biogenesis. The emphasis is on post-transcriptional transactions involving the generation and metabolism of mRNA, but this extends to transcriptional coupling, and to factors involved in translation and its control, including tRNA and non-coding (e.g., micro) RNAs. Representing key areas of post-transcriptional gene regulation, the speakers will include a number of internationally known experts in this field, along with more junior speakers chosen on the basis of the content of their submitted abstracts. Approximately 150 participants are expected for the five-day meeting. The program will consist of nine morning or evening sessions that broadly address cutting-edge issues in the areas of coupling between transcription and RNA processing; genomics and evolution of RNA-processing signals and factors; RNA splicing catalysis, fidelity, and regulation; RNA turnover, including nonsense-mediated mRNA decay; RNA transport and localization; non-coding RNA; translational control; and RNA-protein interactions. In addition, four small poster sessions will enable all participants to contribute to, and learn about, these topics. Free early-afternoon and late-evening periods in the relatively isolated summer setting of Salve Regina University should stimulate productive, informal discussions among established and more junior investigators, postdoctoral trainees, and graduate students from the U.S. and abroad.

This Gordon Research Conference has a unique format that brings together sets of researchers - graduate students, post-doctoral fellows, principal investigators at all career stages, grant administrators, scientists from the pharmaceutical and publishing industries who, despite converging interests, have only infrequent opportunities to meet as a group. The resulting intellectual cross-fertilization will help define critical areas and thus help propel this field forward. Understanding the basic mechanisms of post-transcriptional gene regulation is essential for a full understanding of the organization, function, and evolution of genomes. Speakers for this conference include 17 females and five underrepresented minorities. The selection of attendees from the pool of applicants will employ the principles of affirmative action with respect to minorities, women, and junior scientists. Funds from the National Science Foundation will support the attendance of young investigators working in U.S. laboratories, including graduate students, post-doctoral fellows, and beginning principal investigators.

PROJECT DESCRIPTION The central role of splicing in the eukaryotic gene expression pathway is well established, and yet our understanding of how spliceosomal snRNPs recognize introns and assemble into an active spliceosome remains in shadow. The recent explosion of cryo-EM structure models has exposed the spliceosome?s complex journey from one state to the next, detailing its elaborate changes in composition and conformation. A surprise has been just how big some of the conformational changes are ? so big that we have no understanding of how they might occur. The challenge now is to understand the molecular basis by which these amazing transitions take place. The veil has yet to be lifted on the structural details of how U2 selects the branchpoint, a process of keen interest that is affected by recurrent splicing factor mutations in many cancers. How do the RNA elements and splicing proteins required for branchpoint recognition select the appropriate site? With the recent structures, we can infer a kaleidoscope of changing U2 interactions with itself, with other RNAs and with proteins. Years ago, we showed that a U2 stem IIa to stem IIc RNA rearrangement promotes the first catalytic step of splicing, and the structures now suggest that this rearrangement happens as the 3? half of the U2 snRNP swings 150Å and the U2- branchpoint helix moves 50Å into the catalytic center. How are these movements triggered and what ensures they are completed properly? Recently Karla Neugebauer developed single molecule methods to determine both the position of RNA polymerase on the template and the splicing status of the pre-mRNA. We will use her method with reporters in which we have engineered changes in the gene that delay RNA polymerase and affect splicing outcomes. How does the timing of polymerase transit affect RNA processing? To address these exciting questions in a comprehensive way, we propose the following specific aims. We will (1) determine how early interactions between the U2 snRNP and the intron lead to the establishment of the extended U2-intron pairing upon ATP hydrolysis, (2) use the recent cryo-EM structures as a guide to characterize factors controlling the dynamics of U2 snRNA during splicing, and (3) determine mechanisms by which the dynamics of transcript elongation impact splicing decisions. The overarching hypothesis of this application is that combined structure and function analysis of the core components of the spliceosome will provide the mechanistic and structural basis for understanding the regulation of this central step in eukaryotic gene expression. The recent structures of the yeast spliceosome, the power of yeast genetics and biochemistry, and the fundamental conservation of the splicing machinery promise to translate directly into new understanding of the mechanisms of gene regulation in eukaryotes, including humans, where defects in splicing are increasingly recognized as contributors to disease, and interventions that address splicing are increasingly recognized as pathways to treatment and cures.