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The major goals of our research is to identify and characterize the components of the yeast spliceosome involved in the precise recognition of the 3' splice site region of nuclear pre-mRNA introns.
We have recently identified novel interactions of two factors with the 3' splice sites of yeast introns. One of these factors appears to bind to the 3' splice site in a sequence specific manner at a very early stage of splicing complex assembly and may be a homolog of the mammalian splicing factor SAP155. Using a powerful 3' splice site cross-linking assay that we developed, we are currently examining the role of this and other factors in the crucial events of initial recognition of the 3' splice site. The U2, U5, and U6 small nuclear RNA components of the spliceosome appear to form the active site for the second catalytic step of splicing and must also interact specifically with the conserved 3' splice site sequence. Using our 3' splice site cross-linking assay and a genetic screen for mutants that affect 3' splice site recognition, we are also identifying individual nucleotides in the U2 and U6 snRNAs that specifically bind to the conserved AG dinucleotide of the 3' splice site.
The precise recognition of intron boundaries in nuclear pre-mRNAs by the spliceosome is absolutely essential to avoid exon skipping and the introduction of frameshift errors. The high fidelity of nuclear pre-mRNA splicing appears to be achieved through sequential recognition steps involving multiple, weak specific RNA-protein and RNA-RNA interactions. Whereas many of the factors involved in the sequence-specific recognition of the 5' splice site and branch site sequences of nuclear pre-mRNAs are now fairly well understood, the mechanisms underlying the sequence-specific recognition of the 3' splice site remain enigmatic.
Selected Publications
Yang, C., McPheeters, D.S., and Yu, Y-T. 2005. Psuedouridine 35 in the branch site recognition region of U2 snRNA is important for pre-mRNA splicing in Saccharomyces cerevisiae, Journal of Biological Chemistry 280, 6655-6662. [PubMed]
McPheeters, D.S. and Muhlenkamp, P. 2003. Spatial organisation of protein-RNA interactions in the branch site-3’ splice site region during pre-mRNA splicing in yeast. Molecular and Cellular Biology 23, 4174-4186. [PubMed]
Chang, J.-S. and McPheeters, D.S. 2000. Identification of a U2/U6 Helix Ia mutant that influences 3' splice site selection during nuclear pre-mRNA splicing. RNA 6(8):1120-30. [PubMed]
Pauling, M.H., McPheeters, D.S. and Ares, M. 2000. Association of U2 RNA, Hsh155p, and Hsh49p with functional Cus1p reveals conserved features of Splicing Factor 3b action. Molecular and Cellular Biology 20, 2176-2185. [PubMed]
McPheeters, D.S., Schwer, B. and Muhlenkamp, P. 2000 Cross-linking of the yeast DExH-box RNA Helicase, Prp22, to the 3' splice site during pre-mRNA splicing, Nucleic Acids Research 28, 1313-1321. [PubMed]
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