Mutagenesis of the Yeast Gene PRP8 Reveals Domains Governing the Specificity and Fidelity of 3' Splice Site Selection

INVESTIGATIONS

Mutagenesis of the Yeast Gene PRP8 Reveals Domains Governing the Specificity and Fidelity of 3' Splice Site Selection

J. G. Umen and C. Guthrie
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448

PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and later steps of pre-mRNA splicing. We recently identified a novel allele, prp8-101, that specifically impairs recognition of the uridine tract that precedes most yeast 3' slice sites. We carried out extensive mutagenesis of the gene and selected for new alleles that confer a phenotype similar to that of prp8-101. The strongest alleles cause changes in one of two amino acids in the C-terminal portion of the protein. We also identified a second class of PRP8 mutant that affects the fidelity of 3' splice site utilization. These alleles suppress point mutations in the PyAG motif at the 3' splice site and do not alter uridine tract recognition. The strongest of these alleles map to a region directly upstream of the prp8-101-like mutations. These new PRP8 alleles define two separable functions of Prp8p, required for specificity of 3' splice site selection and fidelity of 3' splice site utilization, respectively. Taken together with other recent biochemical and genetic data, our results suggest that Prp8p plays a functional role at the active site of the spliceosome during the second catalytic step of splicing.

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				D. S. McPheeters and P. Muhlenkamp Spatial Organization of Protein-RNA Interactions in the Branch Site-3' Splice Site Region during pre-mRNA Splicing in Yeast Mol. Cell. Biol., June 15, 2003; 23(12): 4174 - 4186. [Abstract] [Full Text] [PDF]

				A. N. Kuhn, E. M. Reichl, and D. A. Brow Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation PNAS, July 9, 2002; 99(14): 9145 - 9149. [Abstract] [Full Text] [PDF]

				A. B. McKie, J. C. McHale, T. J. Keen, E. E. Tarttelin, R. Goliath, J. J.C. van Lith-Verhoeven, J. Greenberg, R. S. Ramesar, C. B. Hoyng, F. P.M. Cremers, et al. Mutations in the pre-mRNA splicing factor gene PRPC8 in autosomal dominant retinitis pigmentosa (RP13) Hum. Mol. Genet., July 1, 2001; 10(15): 1555 - 1562. [Abstract] [Full Text] [PDF]

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				W. H. McDonald, R. Ohi, N. Smelkova, D. Frendewey, and K. L. Gould Myb-Related Fission Yeast cdc5p Is a Component of a 40S snRNP-Containing Complex and Is Essential for Pre-mRNA Splicing Mol. Cell. Biol., August 1, 1999; 19(8): 5352 - 5362. [Abstract] [Full Text] [PDF]

				C. A. Collins and C. Guthrie Allele-specific genetic interactions between Prp8 and RNA active site residues suggest a function for Prp8 at the catalytic core of the spliceosome Genes & Dev., August 1, 1999; 13(15): 1970 - 1982. [Abstract] [Full Text]

				M. Siatecka, J. L. Reyes, and M. M. Konarska Functional interactions of Prp8 with both splice sites at the spliceosomal catalytic center Genes & Dev., August 1, 1999; 13(15): 1983 - 1993. [Abstract] [Full Text]

				V. Segault, C. L. Will, M. Polycarpou-Schwarz, I. W. Mattaj, C. Branlant, and R. Luhrmann Conserved Loop I of U5 Small Nuclear RNA Is Dispensable for Both Catalytic Steps of Pre-mRNA Splicing in HeLa Nuclear Extracts Mol. Cell. Biol., April 1, 1999; 19(4): 2782 - 2790. [Abstract] [Full Text] [PDF]

				L. A. Lindsey and M. A. Garcia-Blanco Functional Conservation of the Human Homolog of the Yeast Pre-mRNA Splicing Factor Prp17p J. Biol. Chem., December 4, 1998; 273(49): 32771 - 32775. [Abstract] [Full Text] [PDF]

				D. Xu, D. J. Field, S.-J. Tang, A. Moris, B. P. Bobechko, and J. D. Friesen Synthetic Lethality of Yeast slt Mutations with U2 Small Nuclear RNA Mutations Suggests Functional Interactions between U2 and U5 snRNPs That Are Important for Both Steps of Pre-mRNA Splicing Mol. Cell. Biol., April 1, 1998; 18(4): 2055 - 2066. [Abstract] [Full Text]

				H. Urlaub, K. Hartmuth, S. Kostka, G. Grelle, and R. Luhrmann A General Approach for Identification of RNA-Protein Cross-linking Sites within Native Human Spliceosomal Small Nuclear Ribonucleoproteins (snRNPs). ANALYSIS OF RNA-PROTEIN CONTACTS IN NATIVE U1 AND U4/U6.U5 snRNPs J. Biol. Chem., December 22, 2000; 275(52): 41458 - 41468. [Abstract] [Full Text] [PDF]