The long-term goal of this project, which remains unchanged from the previous granting periods, is the study of pre-mRNA splicing in human cells.The main focus of this proposal will be the study of sequence elements in pre-mRNA that play a role in the selection of splice sites in a multiintron precursor. The mechanisms that determine splice site selection in constitutive and alternative splicing are not well understood and present one of the major unresolved questions in pre-mRNA splicing. This proposal will attempt to elucidate these mechanisms by the following experimental approaches: 1) Transcripts with a short internal exon and two introns, a model for a multiintron pre-mRNA, will be spliced in vitro. Preliminary results show that in such model transcripts the internal exon is either skipped or included in the spliced product depending on the sequence of the splice sites and the length of the internal exon. The details of the mechanism of splice site selection as exemplified by exon skipping/inclusion will be further elucidated. In particular, a hypothesis will be tested that the selection of splice sites depends on the interplay between the """"""""strength"""""""" of the splice sites flanking the short internal exon and the length of this exon. Additional mechanisms of splice site selection such as """"""""exon definition"""""""" and splice site competition win also be investigated. 2) The interactions of splicing factors with model pre-mRNAs mentioned above will be investigated. Formation of splicing complexes will be analyzed by native gel electrophoresis. The kinetics and nature of the interactions of splicing factors with splice sites will be determined by nuclease protection assays. The model transcripts will also be used to study the role of splicing factors in alternative splicing. A hypothesis will be tested that the relative concentrations of """"""""housekeeping"""""""" splicing factors are responsible for alternative exon skipping or exon inclusion. We will also test whether factors that appear to be specific for certain alternatively spliced pre-mRNAs can induce alternative splicing in our model systems. 3) The patterns of splicing of model pre-mRNAs in vitro, in cell-free extracts, and in vivo in transiently and stably transfected cells will be compared. The goal of these experiments is to determine if coupling of transcription and splicing affects the patterns of splice site selection and if there are any additional mechanisms that control splice site selection in vivo.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Biology Study Section (MBY)
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University of North Carolina Chapel Hill
Schools of Medicine
Chapel Hill
United States
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Tian, H; Kole, R (2001) Strong RNA splicing enhancers identified by a modified method of cycled selection interact with SR protein. J Biol Chem 276:33833-9
Sierakowska, H; Sambade, M J; Schumperli, D et al. (1999) Sensitivity of splice sites to antisense oligonucleotides in vivo. RNA 5:369-77
Dominski, Z; Kole, R (1996) Effects of exon sequences on splicing of model pre-mRNA substrates in vitro. Acta Biochim Pol 43:161-73
Dominski, Z; Ferree, P; Kole, R (1996) Antisense 2'-O-methyloligoribonucleotides hybridized to RNA block a nuclear, ATP-dependent 3'-5' exonuclease. Antisense Nucleic Acid Drug Dev 6:37-45
Tian, H; Kole, R (1995) Selection of novel exon recognition elements from a pool of random sequences. Mol Cell Biol 15:6291-8
Dominski, Z; Kole, R (1994) Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing. Mol Cell Biol 14:7445-54
Dominski, Z; Kole, R (1994) Identification of exon sequences involved in splice site selection. J Biol Chem 269:23590-6
Dominski, Z; Kole, R (1992) Cooperation of pre-mRNA sequence elements in splice site selection. Mol Cell Biol 12:2108-14
Shukla, R R; Dominski, Z; Zwierzynski, T et al. (1990) Inactivation of splicing factors in HeLa cells subjected to heat shock. J Biol Chem 265:20377-83
Sierakowska, H; Shukla, R R; Dominski, Z et al. (1989) Inhibition of pre-mRNA splicing by 5-fluoro-, 5-chloro-, and 5-bromouridine. J Biol Chem 264:19185-91

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