Adrian R. Krainer - US grants

DESCRIPTION (provided by applicant): The central goal of this project is to understand fundamental mechanisms of human pre-mRNA splicing, a required step in the expression of most eukaryotic genes, as well as the regulation of this process. Specific mechanisms through which exons and introns are correctly identified by the spliceosome will continue to be investigated. These studies will focus on intronic sequences proximal to the splice sites, and on the RNA-binding proteins that recognize them. This project will also investigate an aspect of pre-mRNA splicing fidelity, namely the mechanisms underlying suppression of cryptic splice sites that are only used in the context of mutations elsewhere in a gene. In addition, the network of protein-protein interactions of several splicing-regulatory factors will be characterized. Finaly, the mechanisms underlying the interplay between pre- mRNA splicing and nonsense-mediated mRNA decay, an RNA quality-control process, will continue to be studied. This project will rely on integrative approaches, including biochemical, molecular, proteomics, and bioinformatics techniques, as well as both cell-based and in vitro assays. In addition to obtaining new insights into basic mechanisms of gene expression, these studies will improve the understanding of numerous mutations associated with various genetic diseases, as well as facilitate correct genetic diagnosis and therapeutics development for such diseases.

The RNA Processing Meeting provides an annual forum for researchers to share recent findings about RNA splicing and other RNA processing reactions, RNA catalysis, structure-function relationships in RNA and RNA-protein complexes, regulation at the level of alternative RNA processing and RNA editing and modification. The meetings have attracted and extraordinarily broad spectrum of scientists totalling approximately 400 from the U.S. and abroad. This total includes biochemists, molecular biologists, chemists, geneticists and cell biologists. A continuations of these meetings is proposed for 1991-1993. The organizer will select session chairmen and organize the meeting into 7 to 8 scientific session plus 3 poster sessions. Speakers will be chosen from the abstracts submitted. Poster sessions will provide all conferees with an opportunity to present their work, and will not be limited. Past meetings have shown that face-to-face contact engendered by this format enhances the quality and rate of progress in this field, promotes collabroative interactions, and ensures that the most up-to-date methods for analysis of RNA are communicated properly. This is the only meeting dedicated to this rapidly moving area of research that is held on an annual basis. The problems to be clarified by the Cold Spring Harbor RNA Processing Meeting include the pathway of assembly of the large complexes in which nuclear mRNA splicing takes place; the role of individual small nuclear mRNPs and proteins in splicing and other processing reactions; the mechanisms of trans-splicing; the role of RNA catalysis in a variety of steps in the maturation of tRNA, rRNA, and mRNA; and the mechanisms of RNA editing and modification.

The RNA Processing Meeting provides an annual forum for researchers to share recent findings about RNA splicing and other RNA processing reactions, RNA catalysis, structure-function relationships in RNA and RNA-protein complexes, regulation at the level of alternative RNA processing and RNA editing and modification. The meetings have attracted an extraordinarily broad spectrum of scientists, including biochemists, molecular biologists, chemists, geneticists and cell biologists. A continuation of these meetings is proposed for 1991-1993. The organizer will select session chairmen and organize the meeting into 7 or 8 scientific sessions Plus 3 poster sessions. Speakers will be chosen from the abstracts submitted. Poster sessions will provide all conferees with an opportunity to present their work, and will not be limited. Past meetings have shown that the face-to-face contact engendered by this format enhances the quality and rate of progress in this field, promotes collaborative interactions, and ensures that the most up-to-date methods for analysis of RNA are communicated promptly. This is the only meeting dedicated to this rapidly-moving area of research that is held on an annual basis. The problems to be clarified by the Cold Spring Harbor RNA Processing Meeting include the pathway of assembly of the large complexes in which nuclear mRNA splicing takes place; the roles of individual small nuclear RNPs and proteins in splicing and other processing reactions; the mechanisms of trans-splicing; the role of RNA catalysis in a variety of steps in the maturation of tRNA, rRNA and mRNA; and the mechanisms of RNA editing and modification.

The long term objective of this work is to understand the mechanism of nuclear pre-mRNA splicing, a fundamental process that takes place in all eukaryotic cells and is a required step for the expression of most cellular and viral protein-coding genes. To this end, biochemical and molecular biological approaches will be employed for a detailed analysis of the structure and function of selected human protein factors required for splicing. Biochemical methods will also be used to identify, isolate, and characterize novel protein factors required for splicing. Biochemical complementation of selectively inactivated splicing extracts will be used as an assay for purifying these factors in their active states. Recombinant human splicing factor SF2/ASF and human immunodeficiency virus (HIV) or growth hormone pre-mRNAs will be used to study sequence-specific protein-RNA interactions between splicing factors and exonic splicing enhancer elements. The goal is to understand how these interactions can modulate the specificity and efficiency of pre-mRNA splicing. SF2/ASF mutants and related human proteins will also be analyzed to understand the molecular basis of these interactions. Selection and amplification cycling methods will be used to identify the RNA-binding specificity of each domain in these proteins. Physical studies of the structures of these proteins and domains in the presence and absence of target RNA will be initiated. Proteins related to human SF2/ASF will be sought in yeast to allow genetic dissection of the function of this class of proteins and comparative analysis between yeast and metazoan splicing factors. Errors in splicing specificity caused by mutations in intron-containing genes are often the cause of many human genetic diseases. Because the same mutations also affect splicing specificity in vitro, the molecular basis of the mechanisms of splicing specificity is amenable to biochemical analysis. Recent in vitro experiments made use of antisense modified oligonucleotides complementary to cryptic splice sites to correct aberrant splicing associated with beta-thalassemia mutations. In addition, genetic defects in the expression or structure of cellular splicing factors might be associated with inherited diseases or cancer. Inefficient use of splice sites, another aspect of splicing specificity, is an important feature of the life cycle of retroviruses, including HIV. The spliceosome, a multienzyme complex, and its individual components, remain to be explored as potential targets for novel therapeutic agents. In the long term it may be possible to identify or design drugs that change the concentration or biochemical properties of specific spliceosome constituents in such a way as to compensate for decreased or aberrant splicing in mutant genes. Thus, these studies, which are aimed at understanding basic cellular mechanisms of gene expression, may provide the basis for the development of new diagnostic tools and therapeutic agents.

9722797 Krainer A conference on "Eukaryotic mRNA Processing" will convene scientists studying various aspects of mRNA splicing and 3' -end processing mechanisms and regulation in metazoans and in yeast. Major advances have recently been made in these areas, and the conference will be a timely event for discussing the latest unpublished results and exchanging ideas, thereby fostering new developments in this rapidly moving field. The conference is a new international meeting to be held every other year at Cold Spring Harbor Laboratory. The first meeting will be held on August 20 - 24, 1997, and is expected to attract 300-400 scientists who are actively investigating various aspects of messenger RNA maturation in eukaryotic cells, using genetic, biochemical, molecular, and cell biological approaches. The major focus of the first meeting will be on nuclear events in mRNA maturation, particularly mRNA splicing and polyadenylation. It will include eight plenary sessions and two poster sessions. Speakers in the plenary sessions will be selected on the basis of the submitted abstracts, which will encourage active participation by junior scientists. The main topics covered will include catalytic mechanisms and specificity of mRNA splicing and 3' -end processing; developmental and cell-type specific regulation of gene expression by alternative splicing and polyadenylation; structure and function of snRNP particles and of hnRNPs, SR proteins, PRPs, and other yeast and metazoan protein factors involved in splicing or polyadenylation; spliceosome assembly; and localization and dynamics of processing reactions, factors, substrates, and products, in relation to nuclear architecture and to nuclear-cytoplasmic transport. The proper regulation of gene expression is essential to all life. One fundamental mode of regulation is the processing of RNA transcripts of genes. At this international meeting, between 300 and 400 scientists will gather to share, discuss, and analyze the most recent results and i deas in this field of mRNA processing in eukaryotes. ***

The Monoclonal Antibody Shared Resources provides a central site for the production of monoclonal antibodies by Cold Spring Harbor Cancer Center personnel. The purpose of the facility is to provide the resources and expertise to generate these reagents in a highly efficient and cost- effective manner. CSHL Cancer Center scientists provide specific antigens, and the Shared Resource personnel handle all stages of immunization, fusion, primary screening of hybridomas, cell culture, single-cell cloning, freezing and stage of hybridoma lines, and large-scale production of monoclonal antibodies. Immunization and test bleeds are coordinated with the personnel with the personnel of the Animal Shared Resource. Specialized antibody screens are coordinated with individual scientists. The Shared Resources promotes scientific and technical interactions among Cancer Center scientists and stimulates a great deal of cancer research that is heavily dependent on monoclonal antibody reagents.

The proposed conference on "Eukaryotic mRNA Processing": will convene scientists studying various aspects of mRNA splicing and 3'-end processing mechanisms and regulation in higher eucaryote and in yeast. Major advances have recently been made in these areas, and the proposed conference will be a timely event for discussing the latest unpublished results and exchanging idea, therapy fostering new developments in this rapidly moving field. The proposed conference will be held on August 25-29, 199 and is the second meeting of a conference that is held every other year at Cold Spring Harbor Laboratory. The first meeting was held on August 20-24, 1997, and attracted 272 scientists internationally, who are actively investigating various aspects of messenger RNA maturation, particularly mRNA splicing and polyadenylation, but the scope will now be slight expanding to encompass nuclear mRNA editing. The meeting format will consist of seven plenary sessions and three poster sessions. Speakers in the plenary sessions will be will be selected on the basis of the submitted abstracts which will encourage active participation by junior scientists. The main topics covered will include: catalytic mechanisms and specificity of mRNA splicing and 3'-end processing: developmental and cell-type specific regulation of gene expression by alterative splicing and polyadenylation; structure and function of snRNP particles and of hnRNPs, SR proteins, PRPs, and other yeast, metazoan, and/or plant protein factors involved in splicing of polyadenylation; spliceosome assembly; nuclear mRNA editing; mRNA turnover; and localization and dynamics of processing reactions, factors, substrates, and products, in relation to nuclear architecture and to nuclear- cytoplasmic transport.

The control of gene expression at the post-transcriptional level is a fundamental problem in biology, with relevance to cancer. The mechanisms for the regulation of alternative splicing of cellular and viral genes will be investigated, focusing on global mechanisms that affect the expression of large sets of pre -mRNAs in different tissues, developmental stages, and/or in response to external signals. Three families of human alternative splicing factors will be investigated: (I) the SF7 factors, exemplified by the recently identified SF7A protein; (ii) the hnRNP A/B proteins, of which the best characterized is hnRNP A1; and (iii) the SR proteins, whose prototype is SF2/ASF. The SR proteins function also as constitutive splicing factors, but this project will focus on their ability to modulate alternative splicing in vivo and in vitro in a concentration- dependent manner. This activity is antagonized by hnRNP A/B proteins to modulate alternative 5' splice site selection, and by SF7 proteins to determine alternative 3' splice site selection. The molecular mechanisms by which individual members of these families of RNA-binding proteins modulate alternative splice site selection, and how they achieve substrate specificity, will continue to be studies using biochemical, molecular, and reverse genetic approaches. In addition, the hypothesis that specific pairwise combinations of these antagonistic factors are used to regulate alternative splicing of specific sets of pre-mRNAs will be tested, and experiments are proposed to identify natural, specific pre-mRNA targets for regulations by each of these proteins in vivo. These studies are directly relevant to the overall goals of the program project. As global regulators of alternative pre-mRNA splicing, the SR, hnRNP A/B, and SF7 proteins are excellent candidates to account for the observed aberrant patterns of mRNA expression of numerous genes in transformed cells. Among potential targets of these regulators are several critical genes involved in the establishment or maintenance of the transformed phenotype, or in progression of malignancy. Regulation of alternative splicing is responsible for generating oncongenic and non-oncognic forms of many cellular and viral oncogenes. Therefore, a abetter understanding of the basic mechanisms of alternative splicing regulation, and of the specificity of this process, may lead, in the long term, to the identification of drugs that specifically affect the synthesis of particular protein isoforms that play critical roles in tumorigenesis.

Krainer
On August 25 - 29, 1999, the second biannual "Conference on Eukaryotic mRNA Processing" will be held at the Cold Spring Harbor Laboratory in Cold Spring Harbor, New York. The conference focuses on the mechanisms and regulation of splicing and 3'-end processing of mRNA in yeast and higher eukaryotes. Major advances have recently been made in these areas, and the conference will enable the community to discuss the latest unpublished results and exchange ideas, thereby fostering new developments in this rapidly moving field. The first meeting was held on August 20 - 24, 1997, and attracted an international group of 272 scientists who are actively investigating various aspects of mRNA maturation using genetic, biochemical, molecular, and cellular approaches. As in the previous meeting, the major focus will be on nuclear events in mRNA maturation, particularly mRNA splicing and polyadenylation; the scope, however, will be slightly expanded to encompass nuclear mRNA editing. The meeting format will consist of seven plenary sessions and three poster sessions. Speakers in the plenary sessions will be selected on the basis of the submitted abstracts, a system that accommodates active participation by more junior scientists. The main topics covered will include catalytic mechanisms and specificity of mRNA splicing and 3'-end processing; developmental and cell-type specific regulation of gene expression by alternative splicing and polyadenylation; structure and function of snRNP particles and of hnRNPs, SR proteins, PRPs, and other yeast, metazoan, and/or plant protein factors involved in splicing or polyadenylation; spliceosome assembly; nuclear mRNA editing; mRNA turnover; and localization and dynamics of processing reactions, factors, substrates, and products, in relation to nuclear architecture and to nuclear-cytoplasmic transport.

Messenger RNA is a class of molecules absolutely essential to all living organisms. The processing of mRNA in eukaryotic cells is a highly complex phenomenon involving a multitude of molecular players requiring intricate and precise regulation. Understanding it is essential to helping human society through biology. This conference will enable the international research community studying the many aspects of this phenomenon to exchange their information, insights and ideas.

DESCRIPTION (provided by applicant): The objective of this project is to develop a new approach that can be applied, in the long term, to therapeutic intervention in spinal muscular atrophy (SMA). SMA is an autosomal-recessive, pediatric neuromuscular disorder, characterized by the degeneration of spinal motor neurons. The protein encoded by the SMA-determining gene SMN1 is necessary for the survival of motor neurons. This gene is defective or absent in SMA patients, and its mouse homolog is essential for embryonic development. In humans, a second, nearly identical gene, SMN2, allows affected individuals to survive, but in most patients it cannot express sufficient amounts of active protein to fully compensate for the absence of SMN1. Splicing of the SMN2 pre-mRNA is predominantly via skipping of exon 7, whereas this exon is constitutively included in spliced SMN1 mRNA. Only the small fraction of correctly spliced SMN2 mRNA encodes functional SMN protein. A single-nucleotide difference between the two genes at position 6 of exon 7 - in synonymous codons - is responsible for the difference in splicing patterns. Increasing the extent of exon inclusion in the SMN2 transcripts should generate higher levels of functional protein, and is therefore expected to have therapeutic value. An SF2/ASF-dependent exonic splicing enhancer in exon 7 appears to be defective in SMN2, and recognition of this exon is very sensitive to subtle perturbations. Novel, small antisense molecules will be developed to promote exon 7 inclusion during SMN2 pre-mRNA splicing. The rational design of these molecules is based on recent advances in studies of pre-mRNA splicing factors and signals involved in exon definition. The new compounds will be tested and optimized using in vitro splicing of SMN2 pre-Mrna, and delivered into cultured cells. The best compounds will be tested for therapeutic effects, and will be further optimized, by delivering them systemically and locally into mice. These experiments will make use of the recently-developed mouse Smn knockout strain, rescued by a human SMN2 transgene, which is a useful animal model of SMA.

DESCRIPTION (provided by applicant): The control of gene expression at the post-transcriptional level is a fundamental problem in biology, with relevance to cancer. The basic mechanisms for the regulation of alternative splicing of cellular and viral genes in different tissues, developmental stages, or in response to external signals, will be investigated. We will carry out detailed structural and functional studies of hnRNP A1 and its derivatives, focusing on its nucleic acid-binding properties' its activities in alternative 5' splice-site selection, splicing silencing and telomere-length regulation, and the regulation of its localization and activity in response to genotoxic stress. We will carry out genetic screens in cultured mammalian cells to identify protein components involved in the fidelity of pre-mRNA splicing. Finally, we will continue the development of new technology involving rational design of compounds that mimic one of the activities of SR proteins, the activation of splicing via exonic splicing enhancers. These compounds will be tested for their ability to correct specific splicing defects caused by mutations in cancer susceptibility genes, and to modulate the expression of alternatively spliced isoforms of an apoptosis gene. These studies are directly relevant to the overall goals of the Program Project. As global regulators of alternative pre-mRNA splicing, hnRNP A1 and its antagonists, the SR proteins, may be responsible for the observed aberrant patterns of mRNA expression of numerous genes in transformed cells. Among potential targets of these regulators are several critical genes involved in the establishment or maintenance of the transformed phenotype, or in progression of malignancy. Regulation of alternative splicing is responsible for generating oncongenic and non-oncogenic forms of many cellular and viral oncogenes. Therefore, a better understanding of the basic mechanisms of alternative splicing regulation, and of the fidelity of this process, may lead to the design of drugs to specifically affect the synthesis of particular protein isoforms that play critical roles in tumorigenesis.

The purpose of the Cold Spring Harbor Laboratory training program is to train highly qualified postdoctoral fellows with a broad expertise in basic cancer research. The program emphasizes a multi-disciplinary approach which encompasses concepts and state-of-the-art techniques for molecular biology, biochemistry, protein chemistry, protein chemistry, genetics and cell biology. Specific research training is offered in the analysis of the activities of viral and cellular oncogenes and their roles in controlling gene expression, DNA replication, and phenotype of the transformed cell. Each fellow will conduct an independent research program in collaboration with one of the thirty-one senior staff faculty. Trainees will have access to other faculty, who interact extensively for advice, technical help, and collaboration. Faculty members already have an active research program and represent a variety of expertise. The postdoctoral trainees will be encouraged to publish papers and to present data and scientific meetings. Trainees will participate in several in-house seminar series and discussion groups as well as have access to the Laboratory's extensive summer meetings/workshop/postgraduate advanced course program, which runs from March through December. This allows the trainees to communicate with speakers and participating attendees of the meetings and courses. Attendees come from around the world, and there is a free flow of information in an informal atmosphere. The trainees will be highly qualified candidates who have previously obtained a professional doctorate level degree. Postdoctoral candidates are chosen from among the large number of recent Ph.D.'s or M.D.s who apply or come to the attention of the staff. They are promising young scientists who are accepted into the laboratory of one of the senior staff scientists on the faculty. The qualification of prospective trainees must be approved by the Training Grant Faculty Committee. Each trainee has maximum freedom to select a project in conjunction with his or her faculty preceptor. We are seeking to renew our support for eight trainees.

PROJECT SUMMARY (See instructions): The central goal of Project 2 is to understand the role of alternative splicing in cancer. Cancer cells display extensive qualitative and quantitative dysregulation of splicing, and a subset of the isoforms that are inappropriately expressed can contribute to tumorigenesis. The mechanisms and pathways through which the splicing-factor oncoprotein SRSF1 and related members of the SR protein family transform cells will be investigated. Organotypic culture, as well as orthotopic and transgenic mouse models will be used to study transformation promoted by these splicing factors and their cooperation with other oncogenes in different cancer contexts. How these factors themselves are regulated in normal cells and upregulated in cancer will be addressed. High-throughput methods will be employed to systematically identify the splicing targets of SR proteins in human cells, and selected targets involved in tumorigenesis will be analyzed in detail. Splicing factors that contribute to the distinctive glycolytic metabolism of cancer cells will be identified and characterized, and alternative splicing of pyruvate kinase pre-mRNA will be investigated as a potential therapeutic target by specifically manipulating this process in tumors.

[unreadable] DESCRIPTION (provided by applicant): This proposal requests partial support for a Gordon Research Conference on The Biology of Post-Transcriptional Gene Regulation, to be held at Colby College in Waterville, Maine, from June 29 to July 4, 2008. This meeting is the third one of a Gordon Conference series held every other year. The broad and long-term goal of the conference is to improve our current understanding of fundamental mechanisms and regulation of RNA biogenesis in normal and disease states. The emphasis is on post- transcriptional transactions involving mRNA, but this extends to transcriptional coupling, and to factors involved in translation and its control, including tRNA and microRNAs. The specific aims of this meeting will be to convene approximately 45 speakers representing key areas of post-transcriptional gene regulation, with a total of 150 participants, for a five-day meeting. The program will consist of nine morning or evening sessions that will broadly address cutting-edge issues in: coupling between transcription and RNA processing; genomics and evolution of RNA-processing signals and factors; RNA splicing catalysis, fidelity, regulation, and involvement in disease; RNA turnover, including nonsense-mediated mRNA decay; RNA transport and localization; non-coding RNA; and translational control. In addition, small poster sessions on four late afternoons 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 Colby College will stimulate productive, informal discussions among established and junior principal investigators, postdoctoral trainees and graduate students from the U.S. and abroad. The significance of this application is that this GRC has a unique format and brings together sets of researchers who, despite having converging interests, have infrequent opportunities to meet as a group. The resulting discussions and cross- fertilization will help define critical areas to propel this field forward. In addition, elucidating the basic mechanisms of post-transcriptional gene regulation will be essential to gain a full understanding of the organization, function, and evolution of the human genome. The health relatedness of this application reflects the extensive involvement of RNA-processing dysfunction in numerous genetic diseases, neurological and neuromuscular disorders, and cancer. PUBLIC HEALTH RELEVANCE The relevance of this application to public health reflects the extensive involvement of defects in RNA metabolism associated with many human genetic diseases, neurological and neuromuscular disorders, and cancer. In addition, elucidating the basic mechanisms of the steps of gene regulation that are the focus of this conference, will be essential to gain a full understanding of the organization, function, and evolution of the human genome. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The successful completion of the human genome project and mapping of many disease genes, combined with advances in the molecular understanding of gene-expression pathways, provides unprecedented opportunities to design novel mechanism-based therapies for various genetic diseases. Nonsense mutations account for a large fraction of the causal mutations in nearly all genetic diseases. By definition, nonsense mutations introduce premature termination codons (PTCs), resulting in truncated proteins, and usually severe disease presentations. Translational readthrough drugs, such as ataluren, allow the synthesis of some full-length, functional protein from defective genes with nonsense mutations. However, nonsense-mediated mRNA decay (NMD)¿a ubiquitous mRNA quality-control pathway¿diminishes the effectiveness of readthrough drugs. We will selectively abrogate NMD of mRNAs harboring PTCs, so as to increase their availability for readthrough drugs. To explore the feasibility of our approach, we will initially focus on cell-culture experiments with several nonsense alleles of CFTR, MECP2, DMD, and HBB genes, which cause cystic fibrosis, Rett syndrome, Duchenne muscular dystrophy, and beta-thalassemia, respectively. We will systematically test the effect of inhibiting NMD on mRNA accumulation. Using reporter cell lines, we will then combine transcript-specific NMD inhibition with ataluren treatment, and determine whether there is increased synthesis of full-length protein, compared to ataluren treatment alone. The results of this exploratory study are expected to provide proof of principle for the effectiveness of targeted inhibition of NMD to enhance the efficacy of readthrough drugs. The proposed experiments could lead to a broadly applicable therapeutic approach that would be used in combination with readthrough drugs to treat a large number of severe genetic diseases. PUBLIC HEALTH RELEVANCE: Nonsense mutations result in truncated protein production and cause many severe genetic diseases. Drugs like ataluren can promote the synthesis of intact proteins from genes with nonsense mutations; however, cells have an intrinsic mechanism that diminishes the output of genes with nonsense mutations. We will develop a method to bypass this mechanism for individual mutant genes, so as to enhance the effectiveness of ataluren and similar drugs, and thereby provide more effective therapies for a variety of genetic diseases.

The central goal of Project 2 is to understand the role of alternative splicing in cancer. Cancer cells display extensive qualitative and quantitative dysregulation of splicing, and a subset of the isoforms that are inappropriately expressed can contribute to tumorigenesis. The mechanisms and pathways through which the splicing-factor oncoprotein SRSF1 and related members of the SR protein family transform cells will be investigated. Organotypic culture, as well as orthotopic and transgenic mouse models will be used to study transformation promoted by these splicing factors and their cooperation with other oncogenes in different cancer contexts. How these factors themselves are regulated in normal cells and upregulated in cancer will be addressed. High-throughput methods will be employed to systematically identify the splicing targets of SR proteins in human cells, and selected targets involved in tumorigenesis will be analyzed in detail. Splicing factors that contribute to the distinctive glycolytic metabolism of cancer cells will be identified and characterized, and alternative splicing of pyruvate kinase pre-mRNA will be investigated as a potential therapeutic target by specifically manipulating this process in tumors.

Antibody and Phage Display Shared Resource - Project Summary/Abstract The Antibody Shared Resource provides a central site for the production of monoclonal and polyclonal antibodies. The purpose of this Shared Resource is to provide the resources and expertise to generate these valuable reagents in a highly efficient and cost-effective manner. In the case of monoclonal antibodies, CSHL Cancer Center scientists provide specific antigens and the Shared Resource personnel handle all stages of immunization, fusion, primary screening of hybridomas, cell culture, single-cell cloning, freezing and storage of hybridoma lines, and large-scale production of monoclonal antibodies in cell culture or in vivo. Immunization, test bleeds, and production of ascites fluids are coordinated with the personnel of the Animal Shared Resource. Specialized monoclonal antibody screens are closely coordinated with individual scientists. Polyclonal antibody production is outsourced, but the Shared Resource personnel are in charge of processing and distributing of samples, thus reducing the costs and maximizing efficiency. The Shared Resource also tests and implements new techniques, reagents, and assay improvements related to the different stages of antibody production. After extensive discussions among the Leadership of the Cancer Center, followed by consultation with the Cancer Center External Advisory Board, it was decided that the inclusion of phage- display technology would ensure that this important Shared Resource remains at the cutting edge. This technology provides the capability for rapid production and optimization of high-affinity synthetic antibodies in a manner to complement existing expertise and services of this Shared Resource. It is anticipated that this step forward will not only furnish Cancer Center members with essential reagents, but also may open up new therapeutic opportunities.

PROJECT SUMMARY - PROJECT 2 The central goal of Project 2 is to understand the various roles of alternative splicing in cancer, and to exploit cancer-specific features of this process to develop targeted-therapeutic approaches. Cancer cells display extensive qualitative and quantitative dysregulation of splicing, and a subset of the numerous isoforms that are inappropriately expressed contribute to tumorigenesis or altered cell metabolism. The mechanisms and pathways through which the splicing-factor oncoproteins SRSF2 and SRSF1 transform cells will continue to be investigated. Cell-culture models, as well as orthotopic and genetic mouse models will be used to study tumorigenesis promoted by these splicing factors upon mutation or overexpression in different cancer contexts, with an emphasis on recurrent mutations in myeloid dysplasias. High-throughput RNA-sequencing and computational analysis will be employed to identify and compare the splicing targets of these SR proteins in different cancer contexts, and selected targets will be characterized and manipulated to evaluate their contributions to tumorigenesis and potential as therapeutic targets or biomarkers. One key event, alternative splicing of pyruvate kinase pre-mRNA, which controls the distinctive glycolytic metabolism of cancer cells, will be thoroughly investigated as a potential therapeutic target, by specifically manipulating this process in vivo, using antisense technology and mouse models of glioma and hepatocellular carcinoma.