Donald C. Rio - US grants

The P. element transposon of Drosophila melanogaster is of importance for transformation of the fruitfly but is also of intrinsic interest as a biological phenomenon. It has been described at the molecular and genetic level by several laboratories. The transposon moves only in somatic cells although it is present in germ line cells. The answer to that mystery was found to lie in the processing of the transcript. The transcript contains three introns and in somatic cells, all three are processed out to form a mature mRNA. In germ line cells, one of the introns is not processed out and the mature message is not completed. The present study is directed towards understanding the genetic control of P. element transposition using an integrated approach utilizing genetics, biochemistry and molecular biology. An attempt will be made to develop an in vitro system in order to study the biochemical mechanism of transposition as well as the factors involved with differential splicing events. This study should result in greater insights into gene regulation at the cellular and genetic levels.

DESCRIPTION: The overall objective of the research proposed in this application is to elucidate molecular mechanisms used to regulate pre-mRNA splicing during development of higher metazoan organism using a combination of molecular biology and biochemistry. The specific aims are as follows: 1. Refine biochemical reconstitution assays to study the somatic repression of P element IVS3 pre-mRNA splicing in vitro. 2. Identify and characterize the 65 kD and 40 kD RNA-binding proteins that are part of the P element IVS3 5' exon inhibitory complex. 3. Characterize the biochemical properties of the recombinant PSI (97 kD) and hrp48 proteins. 4. Analyze the expression patterns of PSI and isolate P element insertion mutations in the PSI gene; characterize mutations in the hrp48 gene.

The overall objective of the research proposed in this grant is to elucidate the biochemical and regulatory mechanisms of a eukaryotic transposable element in a higher metazoan using an integrated approach that combined biochemistry, genetics and molecular biology. Our efforts will be focused on understanding the biochemical mechanism of P element transposition and the mechanisms by which these eukaryotic DNA rearrangement reactions are regulated in the fruit fly, Drosophila melanogaster. DNA rearrangements play a variety of roles in the normal development and genomic flux of eukaryotic cells. These studies will also provide necessary information for the development of P elements as genetic tools in other organisms. In order to accomplish our overall objective, we will: 1.Express the Drosophila P element 87kD transposase and 66kD repressor proteins and shorter peptides in mammalian or E. coli host-vector systems. 2.Develop rapid immunoaffinity or affinity chromatography methods to facilitate the biochemical analysis of the P element proteins. 3.Analyze the biochemical activities and properties of the P element proteins as they relate to their biological role in transposition and its regulation. 4.Define the domain structures of transposase and the 66kD proteins; determine the location of the DNA and nucleotide ligand binding sites. 5.Analyze the biochemical and genetic properties of the DNA binding region(s), the nucleotide binding domain and leucine zipper regions of the P element proteins. 6.Develop an in vitro assay for P element transposition and analyze the mechanism and regulation of transposition. 7.Identify other proteins that interact with the P element proteins using genetic and biochemical methods and explore their role in transposition. 8.Develop P element-mediated transposition as a genetic tool in other organisms.

The goal of this proposal is to understand the mechanisms involved in the maternal control (cytotype) of P element transposition in the fruit fly, Drosophila melanogaster. P element transposition is the cause of a syndrome of genetic traits known as hybrid dysgenesis. These traits include high rates of sterility and mutation, as well as chromosomal rearrangements and abnormal germline development. P element transposition can be controlled genetically in crosses where P element-bearing females (P strains) are mated to P strain males. Normally hybrid dysgenesis only occurs when P strain males are mated to females lacking P elements (M strains). P strain females produce eggs that are said to possess "P cytotype" or the ability to repress P element transposition, whereas eggs from M strain females possess the permissive "M cytotype." P cytotype shows an unusual pattern of inheritance through the female germline. The molecular basis of the maternal effect and inheritance of P cytotype will be investigated using P element-mediated transformation, genetic assays for P element repression and P element mRNA and protein localization. Germline transformation will be used to generate Drosophila strains carrying modified P element transposons engineered to express the 66kD P element repressor protein during oogenesis and to localize the 66kD protein mRNA to the posterior germline precursor (pole) cells. Genetic assays will be used to monitor P cytotype repressor activity of these transformants relative to normal P strains. RNA in situ hybridization and antibody staining will be used to correlate expression of the 66kD mRNA and protein during oogenesis with the maternal effect and inheritance of P cytotype repression. The relative levels of the 66kD and transposase mRNAs in purified germline precursor (pole) cells will be determined using the polymerase chain reaction (PCR).

The overall objective of the research proposed in this grant is to elucidate the biochemical and regulatory mechanisms of a eukaryotic transposable element in a higher metazoan using an integrated approach that combines biochemistry, genetics and molecular biology. Our efforts will focus on understanding the biochemical mechanism of P element transposition and the mechanisms by which these eukaryotic DNA rearrangement reactions are regulated in the fruit fly, Drosophila melanogaster. We will also investigate the role of the Drosophila Ku and DNA-dependent protein kinase subunits in DNA repair and P element transposition. These studies will provide necessary information for the development of P elements as genetic tools in other organisms as well as provide insights into metazoan DNA repair mechanisms. In order to accomplish our overall objective, we will: 1. Analyze the phenotypes of mutant P element transposase proteins using an in vivo excision assay. 2. Express and purify wild type and mutant transposase proteins using mammalian vaccinia virus vectors. 3. Analyze the biochemical properties of purified recombinant transposase protein; further develop biochemical assays for its activities. 4. Analyze the properties of the 66kD and KP repressor proteins in vitro using purified recombinant E. coli proteins. 5. Identify other proteins that interact with the 66kD P element repressor protein and explore their role in P cytotype repression. 6. Analyze the role of the Drosophila proteins IRBP(Dmku70), DmKu80 and DmDNA-PK(p450) in P element transposition. 7. Develop P element-mediated transposition as a genetic tool in other organisms.

Acquisition of DNA Analysis Instrumentation for Molecular Studies Our goal is to establish a common core facility for DNA sequence analysis. We propose to purchase: an Automated DNA Sequencer; a molecular biology CATALYST Labstation; an Oligo Synthesizer; a PhosphorImager workstation; a Fluorimager; a Computer System; a Biomek II robot; a Thermocycler: an Automated Film Developer: a Vacuum Centrifuge: a Photodocumentation Station; and two Ultra Cold Freezers (-80 and -30). The participating faculty address a wide range of research related to biodiversity and biotechnology. Their efforts share a common theme of DNA sequence analysis and fall into two major areas: 1) Molecular genetic approaches to study gene expression, RNA processing, and molecular phylogeny, and 2) Molecular approaches in the study of Population Genetics, Ecology, and Evolution. In the area of Molecular genetic approaches to study gene expression. RNA processing. and molecular phylogeny, faculty research and research training include: Whalen's molecular genetic analyses of interactions between bacterial pathogens and plants; Marquez-Magana's investigations of the molecular mechanisms that regulate flagellin gene expression in Bacillus subtilis; and Davis's functional significance and regulation of spliced leader RNA trans-splicing in metazoa, the evolution of parasitism in flatworms, and the molecular phylogeny of flatworms and early metazoa. The focus of Ramirez' s research is to clone the RECI gene and further characterize the RPD3 (a.k.a. REC3) genes of Saccharomyces cerevisiae which are implicated in the repair of damaged DNA and in mitotic recombination. Goldman focuses on a structural unit of the eukaryotic chromosome, the chromatin domain or loop, and its potential role as a functional unit in the regulation of gene expression in mammalian X- chromosome inactivation and genomic imprinting. Perara investigates molecular approaches to the problem of protein transport across cell ular membranes, with the primary focus on a systematic comparison of the molecular mechanisms of secretory protein transport across the endoplasmic reticulum (ER) membrane and the bacterial periplasmic membrane. In the area of Molecular approaches in the study of Population Genetics. Ecology and Evolution, faculty research and research training include: Parker/Cullings' studies of plant community and evolutionary ecology, dynamics of plant recruitment, life history evolution and community turnover rates; Smith/Bayliss' investigations of evolutionary ecology and conservation biology with research focus on the role of transition zones between rain forest and savannah in speciation and their role as related to conservation of rain forest species; Arp's studies of physiological adaptation to environment, sulfide tolerance and detoxification in mudflat invertebrates, ecology and physiology of hydrothermal vent organisms; Randall's investigations of evolution of communication and social organization in desert rodents; and Rmutman' s use of modem molecular genetics to examine population structure, biogeography and intraspecific phylogeny, and the genetic architecture of complex phenotypes. Larson's research is in population biology of nearshore teleost fishes, including their behavior, feeding ecology, life histories, and population structure. Desjardin focuses on taxonomy and phylogeny of fleshy fungi, primarily Agaricales, using traditional and modern aspects of systematics, including cladistic and morphometric analysis, utilizing morphological, ecological, physiological, genetic and molecular data. Hollibaugh addresses physiological ecology of bacterial communities in geochemical cycles and foodwebs. Orrego employs the techniques of molecular genetics for the study of genetic variation in natural populations as displayed by sequences obtained via long distance PCR methods. Our proposed instrumentation will increase the amount and efficiency of our research an d training efforts and enable us to train a greater number of students. The requested equipment will provide the highest resolution available for the analysis of DNA/RNA using PCR amplification, DNA sequencing, Denaturing Gradient Gel Electrophoresis (DGGE), Single Strand Conformation Polymorphisms (SSCP), and a variety of other DNA/RNA detection and analytical methods. With the increased throughput capacity for large numbers of samples that can be processed rapidly and efficiently with longer sequence reading (>700 bp), we will be able to do new high resolution analysis on much greater numbers of samples more rapidly than in the past. For example, the ABI 377 Sequencer provides new longer gel formats that allow one to read 700 bases/run and 36 lanes/run that can be completed in 8 hour (or overnight) runs and 300-500 bp runs in 2.25 hours, allowing 3 runs in an eight hour period. Several faculty (Davis, Hollibaugh, and Parker) will collectively sequence in excess of 10,000 samples/year which represents one run of 36 samples each day for a year. While this alone clearly justifies our need, the acquisition of the proposed instrumentation would also allow us the capacity to triple this number. We will also use the equipment for faculty enhancement short courses; since 1991 we have presented 10 such courses-to over 240 faculty from 35 states.

The goal of this proposal is to understand the mechanisms involved in the function of the conserved arginine-serine (R/S) domain containing pre-mRNA splicing factor U2 snRNP auxiliary factor (U2AF) in the fruit fly, Drosophila melanogaster. This proposal capitalizes on the strengths of Drosophila as an experimental organism and allows a multidisciplinary approach to biological problems. Using a combination of genetics, molecular biology, and biochemistry, we will elucidate the mechanisms by which R/S domains function in pre-mRNA splicing. R/S domains have been found in many mammalian and Drosophila general and regulatory splicing factors but in vivo experiments analyzing their function at physiological levels have not been performed. We have isolated the genes encoding both Drosophila U2AF homologs, have shown that recombinant dU2AF50 has all of the RNA binding and splicing activities of hU2AF65 and have expressed the dU2AF38 in protein in E. coli. Cytogenetic and germline transformation experiments carried out in our lab have shown that previously identified lethal mutations at position 14C1-2 on the X chromosome in an uncharacterized complementation group called 9-21 correspond to the large U2AF subunit, dU2AF50. We have also shown that the dU2AF38 small subunit gene is located on the second chromosome and have identified lethal dU2AF38 mutations. Cytogenetic experiments have localized the dU2AF38 gene to position 21B-C on the second chromosome and we have now isolated lethal mutations in the dU2AF38 small subunit gene demonstrating the gene is essential. These observations provide the basis assaying the function of mutant forms of an essential pre-mRNA splicing factor in a transgenic metazoan organism. There already exist a number of biochemical assays for U2AF function and thus any in vivo phenotype of mutant U2AF derivatives can be directly correlated with altered biochemical activities in vitro. During this funding period in order to accomplish the goals of this proposal, we will 1) Carry out genetic analysis of wild type and mutant U2AF large and small subunit derivatives in vivo; 2) Define the U2AF heterodimer interaction domains in vivo and in vitro; 3) Use Schizosaccharomyces pombe and sequence information for mutant prp-2 alleles to create temperature-sensitive dU2AF50 mutants and test their function in Drosophila; 4) Assay wild type and mutant U2AF heterodimers for splicing activity and splicing complex assembly in vitro; and 5) Structure studies on the RNA binding domain of Sex-lethal and dU2AF50 and the interaction domain of the dU2AF heterodimer. It is clear that many human oncogenes are expressed differentially by alternative pre-mRNA splicing. These oncogene protein isoforms may have different functions in different tissues. Because many human diseases, such as beta-thalassemia, results from defects in pre-mRNA splicing, it is likely that alternative splicing defects may lead to oncogenic transformation of certain somatic cell types.

The overall objective of the research proposed in this grant is to elucidate the biochemical and regulatory mechanisms of a eukaryotic transposable element in a higher metazoan using an integrated approach that combines biochemistry, genetics and molecular biology. Our efforts will focus on understanding the biochemical mechanism of P element transposition and how these eukaryotic transposable DNA elements tie into the DNA repair pathways of the fruit fly, Drosophila melanogaster. The experiments in this proposal will provide necessary information for the development of P elements as genetic tools in other organisms. In order to accomplish our overall objective, we will: 1. Analyze the phenotypes of mutant P element transposase proteins using an in vivo excision assay. 2. Analyze the biochemical properties of wild type and mutant transposase proteins; further develop biochemical assays for their activities. 3. Analyze the biochemical effects of phosphorylation of the transposase protein; identify the kinases involved in these phosphorylation events. 4. Determine what role GTP binding and/or hydrolysis play in P element transposition. 5. Analyze the role of the Drosophila proteins IRBP, DmKup70, DmKup80 and DmDNA-PKcs (p470) in P element transposition. 6. Facilitate the development of P element-mediated transposition as a genetic tool in other organisms.

DESCRIPTION (provided by applicant): A request is made for a shared instrumentation grant to NCRR for a Micromass hybrid quadrupole time of flight (Q ToF) mass spectrometer. The instrument will be housed in the Center for Integrated Genomics (CIG), a new research unit on the Berkeley campus. The instrument will be used for protein identification following one- or two-dimensional gel electrophoresis, using MS-MS sequencing of proteolytic peptides. Projects supported by this instrument will involve human and Tetrahymena telomerase, Drosophila, Ciona and Artmia transcription factors, Drosophila P element transposase and DNA repair proteins, Drosophila RNA binding / RNA splicing factors and Xenopus embryonic transcription and signal transduction factors. The instrument will also be used to analyze protein phosphorylation of transcription factors, DNA recombination enzymes, telomerase and RNA splicing factors using nano-LC MS-MS and precursor ion scanning in the positive or negative mode. An instrument of this type, using nano-elctrospray ionization in conjunction with a hybrid quadrupole-time-of-flight mass analyzer is not available on the U.C., Berkeley campus.

[unreadable] DESCRIPTION (provided by applicant): Alternative pre-mRNA splicing is a common mechanism for regulating gene expression in metazoans. Indeed, RNA processing is a conduit through which genomic information is transferred to proteomic information. In fact, it is now recognized that 30-40% of the known human and mouse disease gene mutations affect the splicing process. Thus, understanding how introns are recognized and how patterns of alternative splicing are set up may allow therapeutic intervention. Cis-acting sequence elements in the pre- mRNA, known as silencers and enhancers, are known to modulate splice site use. MicroRNAs have been shown to control gene expression at the levels of translation and RNA stability and some microRNAs (and piRNAs or rasiRNAs) act in the nucleus as ribonucleoprotein (RNP) complexes with arognaute/piwi family proteins to alter chromatin structure/organization and control the activity/expression of mobile genetic elements. The overall objective of the research proposed in this grant is to ask if microRNAs can control alternative pre-mRNA splicing patterns in Drosophila using gene expression and splice junction microarrays. Our efforts will be focused on the use of alternative splice junction microarrays along with microRNA blocking or over-expression methods to ask how global splicing patterns change when the function of a single microRNA is perturbed. In order to accomplish our overall objectives, we will: 1. Analyze the genome-wide effects on alternative pre-mRNA splicing patterns using Drosophila splice junction microarrays after blocking microRNA function, blocking microRNA maturation or by using microRNA over-expression in cell culture. 2. Analyze the genome-wide effects on alternative pre-mRNA splicing patterns using Drosophila splice junction microarrays after blocking microRNA function or maturation in whole animals using dicer-1, dicer-2 and ago-2 mutants. PUBLIC HEALTH RELEVANCE Alternative pre-mRNA splicing is a common mechanism for regulating gene expression in metazoans. MicroRNAs have been shown to control gene expression at the levels of translation and RNA stability and some microRNAs (and piRNAs or rasiRNAs) act in the nucleus as ribonucleoprotein (RNP) complexes with arognaute/piwi family proteins to alter chromatin structure/organization and control the activity/expression of mobile genetic elements. The overall objective of the research proposed in this grant is to ask if microRNAs can control alternative pre-mRNA splicing patterns in Drosophila using gene expression and splice junction microarrays. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This proposal attempts to develop methods for the purification and characterization of individual nuclear pre-mRNA-protein complexes. Because binding of eukaryotic RNA binding proteins to nascent transcripts occurs in the nucleus during transcription, it is believed that the particular constellation of RNA binding proteins that a given transcript acquires to form a distinct ribonucleoprotein (RNP) complex will control its RNA processing, RNA export and RNA stability. These post-transcriptional pathways are critically important for gene expression. Moreover, because >95% of human genes generate multiple transcript isoforms it is also important to find out if differentially spliced mRNA isoforms have distinct patterns of RNA binding proteins. The hypothesis being tested is that different nuclear pre-mRNAs/RNPs have distinct protein compositions which contribute to their fate. If successful, this approach would have a major impact in our understanding of how nuclear RNP structure controls the RNA processing and fates of newly transcribed messenger RNA molecules. In order to approach this question, we will: 1. Develop technology to purify individual Drosophila nuclear pre-mRNPs using biotinylated anti-sense chimeric LNA-DNA oligonucleotides targeted to specific pre-mRNAs and analyze their protein composition by mass spectrometry. This proposal will outline one specific aim that builds on the expertise of my lab in evaluating genome-wide patterns of alternative pre-mRNA splicing and the distribution of RNA splicing factors on nuclear pre-mRNAs. PUBLIC HEALTH RELEVANCE: This proposal attempts to develop methods for the purification and characterization of individual nuclear pre-mRNA-protein complexes. Because binding of eukaryotic RNA binding proteins to nascent transcripts occurs in the nucleus during transcription, it is believed that the particular constellation of RNA binding proteins that a given transcript acquires to form a distinct ribonucleoprotein (RNP) complex will control its RNA processing, RNA export and RNA stability. These post-transcriptional pathways are critically important for gene expression and can be perturbed in disease states.

DESCRIPTION (provided by applicant): PROJECT SUMMARY / ABSTRACT Alternative pre-mRNA splicing in Drosophila. D. Rio - PI Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in metazoans. Indeed, RNA processing is a conduit through which genomic information is transferred to proteomic information. Because most eukaryotic genes are split and have the potential for alternative splicing, a dramatic increase in proteomic diversity among cells, tissues and organisms is a direct consequence of alternative splicing. In fact, it is now recognized that 30-40% of the known human and mouse disease gene mutations affect the splicing process. Thus, understanding how introns are recognized and how patterns of alternative splicing are set up may allow therapeutic intervention. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific splicing patterns in metazoans. Our previous work has focused on characterization of the tissue-specific Drosophila P element splicing silencer element. Recent work from our group has focused on tissue-specific regulation of alternative splicing and how the action of hnRNP proteins, PSI and the hnRNP A/B family proteins, hrp48, 40, 38 and 36 results in splicing silencer function. We wish to extend these studies to investigate the function of a Drosophila paralog of U2AF, called LS2, that functions as a splicing repressor. Building on previous R21 funding, we will continue investigations of the microRNA pathway protein, Ago-2, in alternative splicing pathways. Finally, using a variety of approaches, we want to identify and functionally characterize new cellular splicing silencers that are controlled by multiple splicing factors. One advantage this proposal has is the small genome size and well-annotated Drosophila genome that allows a comprehensive analysis of both alternative splicing patterns and the genome-wide distribution of RNA binding proteins. Because most eukaryotic genes are split and have the potential for alternative splicing, increased proteomic diversity among cells, tissues and organisms is a direct consequence of alternative splicing. Because the protein factors involved in controlling patterns of alternative splicing have homologs in humans and like humans Drosophila has extensive alternative splicing, results obtained in this system will have direct relevance and application to human health.

DESCRIPTION (provided by applicant): The human genome is composed of > 50 % transposable elements, which are an important source of human genetic variation. 3% of the human genome consists of DNA-based (cut-and-paste) transposons and 43 of the 47 genes related to transposable elements are derived from these mobile elements. Most of these genes remain uncharacterized with the exception of the lymphoid-specific RAG1/RAG2 V(D) recombinase and the SETMAR protein, a fusion between a mariner transposase and a histone methylase SET domain. One member of this collection of transposon-related genes is called THAP9, which is homologous along its entire length to the Drosophila P element transposase protein. THAP9 and eleven other human genes contain a THAP domain, a newly recognized C2CH zinc-coordinating site-specific DNA binding domain. There are > 300 THAP domain-containing proteins in the genomes of eukaryotes, but only THAP9 is homologous beyond the THAP domain. Comparative genomic analyses indicate that the THAP9 gene is present in 21 vertebrate genomes surveyed, but is absent from rodents and that there are P element-like transposons with terminal inverted repeats in the zebrafish genome. These observations suggest that the THAP9 genes were derived from ancient transposons, much like the scenario for evolution of the V(D)J recombination- RAG1/RAG2 recombinase system. We wish to characterize the human THAP9 gene, which our preliminary data indicate is an active DNA transposase. The long-term goal of this research is to understand how genes related to transposable elements function in the human genome. Because transposable elements cause mutations, genome rearrangements, lead to genetic variation and alter gene expression, they are important components of genome evolution and gene expression programs. The experiments outlined in this proposal will characterize the functional activities of the THAP9 protein, and will potentially identify a new family of human transposable elements. The central hypothesis of this proposal is that the human THAP9 gene encodes an active DNA transposase. We will test this hypothesis in several ways and attempt to find active transposable elements in the human genome that use the THAP9 protein as a transposase. The rationale behind this proposal is to discover an active DNA-based transposon system in humans. In order to address the functional role THAP9 plays in human cells, we will: 1) Investigate and characterize the activities of human THAP9 in cells; 2) Map human THAP9 genomic DNA binding sites using chromatin immunoprecipitation-HTP sequencing (ChIP-seq); 3) Proteomic and biochemical analysis of human THAP9 protein.

? DESCRIPTION (provided by applicant): Mobile genetic elements or transposons are found in the genomes of all organisms. These elements can move via DNA or RNA intermediates. About 50% of the human genome is made up of transposable elements with ~ 2.7% corresponding to DNA-based transposons. Many of these putative transposons or transposase-related genes are uncharacterized. Our previous studies have focused on the P element family of DNA transposons in Drosophila. P element transposase functions as a tetramer, using GTP as a cofactor for transposition. N-terminal domain of the transposase corresponds to a C2CH THAP DNA binding domain, which is a member of a prevalent family of DNA binding domains found exclusively in animal genomes. One THAP gene, called THAP9, is homologous to the Drosophila P element transposase and is present in primates, Xenopus, zebrafish and Ciona, but is absent from rodents. Recent work from our lab has shown that the human and zebrafish THAP9 genes can mobilize the Drosophila and zebrafish P element transposons in human and Drosophila cells. This proposal is focused on understanding what role the human THAP9 gene may play in human embryonic stem cells and how the Drosophila P element transposase protein recognizes and assembles with the transposon ends, donor DNA, target DNA and GTP/Mg2+ to form an active protein-DNA complex. These studies are aimed at gaining mechanistic insights. Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in metazoans and is a conduit through which genomic sequence is transferred to proteomic information. Most eukaryotic genes are split and have the potential for alternative splicing, dramatically increasing proteomic diversity. Many human and mouse disease gene mutations affect the splicing process. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific alternative splicing patterns. Our previous work has focused on characterization of the tissue-specific Drosophila P element pre-mRNA exonic splicing silencer element. Recent work from our group has focused on how the action of the RNA binding proteins, PSI and hrp48. Using this information, we want to identify new Drosophila cellular splicing silencer elements that are controlled by these two splicing factors. The PSI protein also interacts with U1 snRNP and PSI mutant Drosophila strains that abolish this interaction exhibit male courtship behavior defects and altered pre-mRNA splicing of the Drosophila male-specific fruitless pre-mRNA isoforms. We want to investigate how the PSI protein controls fruitless pre-mRNA splicing and how it controls binding of U1 snRNP on the Drosophila transcriptome. U1 snRNP has distinct roles in U1 snRNP binding sites in PCPA (premature cleavage and polyadenylation), splicing at intron 5' splice sites and at potential new splicing silencers.

PROJECT SUMMARY / ABSTRACT Profiling the locations of U1 snRNP binding across the nuclear human and Drosophila transcriptomes. D. Rio ? P.I. Spliceosomal U1 snRNP functions in the nucleus to influence both pre-mRNA alternative splicing and polyadenylation site usage, which are two key gene expression mechanisms. The goal of this proposal is to develop a method to comprehensively map the locations of U1 snRNP across the nuclear transcriptomes of Drosophila and human cells and systematically categorize the function of the spliceosomal U1 snRNP at specific sites in modulating global pre-mRNA splicing and polyadenylation patterns. In order to address this question we will: 1) Develop a genome-wide mapping and profiling method for characterizing the binding sites of U1 snRNP across the nuclear transcriptomes of human and Drosophila cells. To do this, we will develop a novel and highly-specific two-step immunoaffinity selection strategy in combination with an RNase T1 nuclease protection assay to characterize the widespread targeting of U1 snRNP (a complex of 10 proteins and one non-coding, small RNA) to intron 5' splice sites, premature cleavage and polyadenylation (PCPA) sites and splicing silencer elements. We will develop novel computational methods to extensively and accurately profile significant U1 snRNP binding sites; 2) Categorize and define U1 snRNP binding sites as bone fide or cryptic 5' splice sites, telescripting sites or splicing silencer elements.  For this purpose we will use state-of-the-art cDNA sequencing after perturbation of U1 snRNP activity in human and Drosophila cells. We will use the accurate U1 snRNP profile maps generated in Aim 1 to correlate with altered pre-mRNA splicing patterns, polyadenylation events and splicing control elements transcriptome-wide. Mapping U1 snRNP binding sites to the nuclear transcriptome will link pre-mRNA splicing patterns, splicing silencer elements and PCPA sites to decode the function of U1 snRNP-mediated post-transcriptional regulation at specific binding sites. These new methods will be transformative by allowing predictions about where and how U1 snRNP binding to nuclear pre-mRNA affects constitutive splicing, alternative splicing, surveillance by premature transcript cleavage and polyadenylation (PCPA) and alternative polyadenylation, all of which are profoundly perturbed in many disease states. The proposed research will reveal for the first time a bona-fide transcriptome-wide map of U1 snRNP binding to nuclear pre-mRNAs and allow the definition of molecular function of the U1 snRNP- mediated post-transcriptional regulatory pathways in both RNA surveillance and RNA processing of the human and Drosophila transcriptomes. This information has the potential to allow the development of new therapeutic strategies to treat disease through investigation and manipulation of U1 snRNP function.

NIH R35 GM118121; DNA transposons and alternative pre-mRNA splicing. D. Rio ? PI. PROJECT SUMMARY / ABSTRACT DNA transposons and alternative pre-mRNA splicing. D. Rio ? PI Mobile genetic elements or transposons are found in the genomes of all organisms. These elements can move via DNA or RNA intermediates. About 50% of the human genome is made up of transposable elements with ~ 2.7% corresponding to DNA-based transposons. Many of these putative transposons or transposase-related genes are uncharacterized. Our previous studies have focused on the P element family of DNA transposons in Drosophila. P element transposase functions as a tetramer, using GTP as a cofactor for transposition. N-terminal domain of the transposase corresponds to a C2CH THAP DNA binding domain, which is a member of a prevalent family of DNA binding domains found exclusively in animal genomes. One THAP gene, called THAP9, is homologous to the Drosophila P element transposase and is present in primates, Xenopus, zebrafish and Ciona, but is absent from rodents. Recent work from our lab has shown that the human and zebrafish THAP9 genes can mobilize the Drosophila and zebrafish P element transposons in human and Drosophila cells. We have also used cryo-EM to solve the structure of the P element transposase strand transfer complex. This proposal is focused on understanding what role the human THAP9 gene may play in human embryonic stem cells and the reaction pathway that the Drosophila P element transposase protein uses to recognize and assemble with the transposon ends, donor DNA, target DNA and GTP/Mg2+ to form an active protein-DNA complex. These studies are aimed at gaining mechanistic insights. Alternative pre-mRNA splicing is an important mechanism for regulating gene expression in metazoans and is a conduit through which genomic sequence is transferred to proteomic information. Most eukaryotic genes are split and have the potential for alternative splicing, dramatically increasing proteomic diversity. Many human and mouse disease gene mutations affect the splicing process. in fact, somatic mutations in splicing factor and spliceosomal genes have been linked to human diseases, such as cancer and the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Our previous work has focused on characterization of the tissue-specific Drosophila P element pre- mRNA exonic splicing silencer element. Recent work from our group has focused on how the action of the RNA binding proteins, PSI and hrp48 and the human RNA binding splicing factors hnRNPA1 and DDX5. We are using this information to identify new Drosophila cellular splicing silencer elements that are controlled by PSI and hrp48. We are also analyzing mutant forms of hnRNPA1 that are linked to ALS to find splicing pattern defects that could be used as biomarkers for the disease or provide clues to have neurons are dying in the disease. Splicing silencers are a major type of RNA control element generating tissue- or cell type-specific alternative splicing patterns. The PSI protein also interacts with U1 snRNP and PSI mutant Drosophila strains that abolish this interaction exhibit male courtship behavior defects and altered pre-mRNA splicing of the Drosophila male-specific fruitless pre-mRNA isoforms. We want to investigate how the PSI protein controls fruitless pre-mRNA splicing and how it controls binding of U1 snRNP on the Drosophila transcriptome. U1 snRNP has distinct roles in U1 snRNP binding sites in PCPA (premature cleavage and polyadenylation), splicing at intron 5' splice sites, at cryptic 5' splice sites and at splicing silencers (from our work).