This proposal describes experiments to investigate processing of pre-messenger RNA in higher eucaryotes. In the last several years, our understanding of the mechanics of pre-messenger RNA processing has increased considerably, in no small measure because of the development of in vitro systems competent to catalyze splicing and polyadenylation of simple precursor RNAs. Upon addition to a splicing extract, an exogenous precursor RNA rapidly assembles into a large ribonucleoprotein complex (the spliceosome) containing hnRNP and snRNP factors known to be required for the cleavage and ligation steps of splicing. The next few years promise to be exciting ones for the splicing field during which the rules of spliceosome assembly and participation in the splicing reaction will be determined. We plan to investigate both the assembly and activity of the spliceosome using in vitro processing extracts. Seven experimental approaches currently in use in the laboratory will be used to address basic questions about spliceosome structure and function, including: 1. Examination of snRNP protein phosphorylation and its relationship to assembly and activity of the spliceosome; 2. Development of two-dimensional gels for the examination of spliceosomes; 3. Fractionation of splicing extracts using size-based approaches; 4. Use of RNP gel electrophoresis to deduce the pathway of spliceosome assembly and disassembly; 5. Examination of the involvement of matrix proteins in in vitro splicing; 6. Examination of snRNP assembly and sub-populations by RNP gel analysis of complexes assembled on isolated snRNAs, and 7. Examination of the relationship between splicing and polyadenylation through the use of precursor RNAs containing introns and poly(A) sites.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Cytology Study Section (CTY)
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Baylor College of Medicine
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Auboeuf, Didier; Dowhan, Dennis H; Dutertre, Martin et al. (2005) A subset of nuclear receptor coregulators act as coupling proteins during synthesis and maturation of RNA transcripts. Mol Cell Biol 25:5307-16
Dowhan, Dennis H; Hong, Eugene P; Auboeuf, Didier et al. (2005) Steroid hormone receptor coactivation and alternative RNA splicing by U2AF65-related proteins CAPERalpha and CAPERbeta. Mol Cell 17:429-39
Auboeuf, Didier; Dowhan, Dennis H; Li, Xiaotao et al. (2004) CoAA, a nuclear receptor coactivator protein at the interface of transcriptional coactivation and RNA splicing. Mol Cell Biol 24:442-53
Auboeuf, Didier; Dowhan, Dennis H; Kang, Yun Kyoung et al. (2004) Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes. Proc Natl Acad Sci U S A 101:2270-4
Auboeuf, Didier; Honig, Arnd; Berget, Susan M et al. (2002) Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science 298:416-9
Honig, Arnd; Auboeuf, Didier; Parker, Marjorie M et al. (2002) Regulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72. Mol Cell Biol 22:5698-707
Stickeler, E; Fraser, S D; Honig, A et al. (2001) The RNA binding protein YB-1 binds A/C-rich exon enhancers and stimulates splicing of the CD44 alternative exon v4. EMBO J 20:3821-30
Grossie Jr, V B (2001) Influence of the ward colon tumor on the innate and endotoxin-induced inflammatory response of the rat. Cancer Invest 19:698-705
Zeng, C; Berget, S M (2000) Participation of the C-terminal domain of RNA polymerase II in exon definition during pre-mRNA splicing. Mol Cell Biol 20:8290-301
McCullough, A J; Berget, S M (2000) An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites. Mol Cell Biol 20:9225-35

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