Abstract

The experiments described in this proposal are designed to gain insights into the mechanism regulation of pre-RNA splicing in vertebrate cells. The work focuses on members of a family of conserved splicing factors known as SR proteins, but other proteins that participate in splicing and/or its regulation will also be analyzed. Studies examining the roles of snRNAs in splicing catalysis will be continued. The following specific aims are proposed. 1. Novel functions of ASF/SF2. The recent discovery that ASF/SF2 is necessary for the maintenance of genome stability by preventing mutagenic co-transcriptional R loops will be pursued in detail. In vitro assays to investigate the mechanism and specificity of RNP formation will be developed, with the ultimate goal of recreating R-loop formation and the subsequent DMA double strand breaks (DSBs) that occur in the absence of proper RNP formation and/or splicing. The mechanism of DMA DSB formation will also be investigated. A genetic screen to identify suppressors of ASF/SF2-induced cell death will be continued. 2. Properties of SRp38. The discovery that SRp38 can function as a sequence-specific activator of splicing will be pursued and the identity of an SRp38-specific co-activator determined. Analysis of the signaling pathway that leads to SRp38-dephosphorylation will be investigated. The mechanism by which Hsp27 functions to prevent SRp38 dephosphorylation and induce splicing thermo tolerance will be pursued. Analysis of a recently created SRp38 knockout mouse will be continued. 3. Other splicing regulators. The mechanism by which hnRNP A1 contributes to SMN2 exon 7 exclusion will be analyzed. The possibility that creation of hnRNP A1-dependent exonic splicing silencers by mutation, for example in BRCA1 and BRCA2 transcripts, occurs relatively frequently will be investigated. The identity of an RNAPII CTD-specific splicing co-activator will be established and its mechanism of action analyzed. The basis for the requirement of a PPP family phosphatase for the second catalytic step of splicing will be determined. 4. snRNAs and splicing catalysis. Characterization of RNA-Y, a novel product formed with purified segments of U2 and U6 RNAs in a reaction very similar to the first step of splicing, will be completed. Collaborative efforts to determine the crystal structure of the active U2/U6 complex will be continued. Experiments to identify protein cofactors that facilitate U2/U6- mediated catalysis and/or U2/U6 complex formation will be pursued. Select residues in U2/U6 will be subjected to mutagenesis to elucidate how they function in catalysis. ? ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048259-22
Application #
7455010
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1992-07-01
Project End
2010-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
22
Fiscal Year
2008
Total Cost
$500,956
Indirect Cost

  Institution

Name
Columbia University (N.Y.)
Department
Biology
Type
Other Domestic Higher Education
DUNS #
049179401
City
New York
State
NY
Country
United States
Zip Code
10027

  Publications

Shkreta, Lulzim; Toutant, Johanne; Durand, Mathieu et al. (2016) SRSF10 Connects DNA Damage to the Alternative Splicing of Transcripts Encoding Apoptosis, Cell-Cycle Control, and DNA Repair Factors. Cell Rep 17:1990-2003
Conlon, Erin G; Lu, Lei; Sharma, Aarti et al. (2016) The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains. Elife 5:
Zhang, Jian; Lieu, Yen K; Ali, Abdullah M et al. (2015) Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities. Proc Natl Acad Sci U S A 112:E4726-34
Coady, Tristan H; Manley, James L (2015) ALS mutations in TLS/FUS disrupt target gene expression. Genes Dev 29:1696-706
Zhou, Xuexia; Wu, Wenwu; Li, Huang et al. (2014) Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation. Nucleic Acids Res 42:4019-30
Li, Huang; Wang, Zhijia; Zhou, Xuexia et al. (2013) Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression. Proc Natl Acad Sci U S A 110:E2687-95
Manley, James L (2013) SELEX to identify protein-binding sites on RNA. Cold Spring Harb Protoc 2013:156-63
Zhang, Jian; Manley, James L (2013) Misregulation of pre-mRNA alternative splicing in cancer. Cancer Discov 3:1228-37
Kanehiro, Yuichi; Todo, Kagefumi; Negishi, Misaki et al. (2012) Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1. Proc Natl Acad Sci U S A 109:1216-21
Tan, Adelene Y; Riley, Todd R; Coady, Tristan et al. (2012) TLS/FUS (translocated in liposarcoma/fused in sarcoma) regulates target gene transcription via single-stranded DNA response elements. Proc Natl Acad Sci U S A 109:6030-5

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