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The function of U2 snRNA in splicing is mediated by the proteins of the U2 small nuclear ribonucleoprotein. To identify proteins that influence the function of U2 snRNA we carried out a screen for mutations in $accharomyces cerevisiae that suppress the cold-sensitive growth defect of a mutation in U2 stem loop IIa, a structure important for the stable association of the U2 snRNP with pre-mRNA. The screen identified three dominant suppressor genes, one of which, CUS1-54, encodes an essential splicing protein required for U2 snRNP addition to the spliceosome. The suppressor protein rescues the spliceosome assembly defect of the mutant U2 in vitro, indicating that suppression is direct. Allele specificity tests show that the suppressor does not simply bypass the requirement for U2 stem loop IIa. Extra copies of wild-type CUS1, but not CUS1-54, suppress the temperature-sensitive prpll and prp5 mutations, linking CUS1 protein to a subset of other factors required at the same step of spliceosome assembly. CUS1 is homologous to SAP 145, a component of the mammalian U2 snRNP that interacts with pre-mRNA. The yeast genome also encodes a homolog of human SAP 49, a protein that interacts strongly with both SAP 145 and pre-mRNA, underscoring the conservation of U2 snRNP proteins that function in spliceosome assembly.[Key Words: U2 snRNA; Saccharomyces cerevisiae; spliceosome assembly; pre-mRNA] Received August 25, 1995; revised version accepted October 24, 1995.The spliceosome is the large ribonucleoprotein particle responsible for removal of introns from nuclear premRNA transcripts. Spliceosome assembly and function are complex and dynamic, involving multiple transient protein-protein, protein-RNA and RNA-RNA interactions. The spliceosome is built on an intron-containing transcript by the sequential recognition of conserved sequence elements near the reactive sites. A transcript first becomes committed to the splicing pathway by stable association with the U1 small nuclear ribonucleoprotein (snRNP) to form the commitment complex, a step that does not require ATP. Stable binding of U2 snRNP to the commitment complex near the pre-mRNA branchpoint requires ATP and forms the prespliceosome. With the binding of US/U4.U6 tri-snRNP the spliceosome is assembled but must be activated before the cleavage and ligation reactions begin (for review, see Moore et al. 1993;Newman 1994;Nilsen 1994; Ares and Weiser 1995}.The recognition of an intron and choice of splice sites are likely accomplished during the early steps of spliceosome assembly and may hold the key to the regulation of splicing. As the first ATP-requiring step, the stable addition of the U2 snRNP at the branchpoint is an attractive potential site of regulation. The RNA moiety of the U2 snRNP plays two roles in this step. A region called the branchpoint interaction sequence forms base pairs with the pre-mRNA at the site of lariat formation {Parker et Wu and Manley 1989; Zhuang and Weiner 19891. A structure downstream of the branchpoint interaction sequence called stem-loop...
The function of U2 snRNA in splicing is mediated by the proteins of the U2 small nuclear ribonucleoprotein. To identify proteins that influence the function of U2 snRNA we carried out a screen for mutations in $accharomyces cerevisiae that suppress the cold-sensitive growth defect of a mutation in U2 stem loop IIa, a structure important for the stable association of the U2 snRNP with pre-mRNA. The screen identified three dominant suppressor genes, one of which, CUS1-54, encodes an essential splicing protein required for U2 snRNP addition to the spliceosome. The suppressor protein rescues the spliceosome assembly defect of the mutant U2 in vitro, indicating that suppression is direct. Allele specificity tests show that the suppressor does not simply bypass the requirement for U2 stem loop IIa. Extra copies of wild-type CUS1, but not CUS1-54, suppress the temperature-sensitive prpll and prp5 mutations, linking CUS1 protein to a subset of other factors required at the same step of spliceosome assembly. CUS1 is homologous to SAP 145, a component of the mammalian U2 snRNP that interacts with pre-mRNA. The yeast genome also encodes a homolog of human SAP 49, a protein that interacts strongly with both SAP 145 and pre-mRNA, underscoring the conservation of U2 snRNP proteins that function in spliceosome assembly.[Key Words: U2 snRNA; Saccharomyces cerevisiae; spliceosome assembly; pre-mRNA] Received August 25, 1995; revised version accepted October 24, 1995.The spliceosome is the large ribonucleoprotein particle responsible for removal of introns from nuclear premRNA transcripts. Spliceosome assembly and function are complex and dynamic, involving multiple transient protein-protein, protein-RNA and RNA-RNA interactions. The spliceosome is built on an intron-containing transcript by the sequential recognition of conserved sequence elements near the reactive sites. A transcript first becomes committed to the splicing pathway by stable association with the U1 small nuclear ribonucleoprotein (snRNP) to form the commitment complex, a step that does not require ATP. Stable binding of U2 snRNP to the commitment complex near the pre-mRNA branchpoint requires ATP and forms the prespliceosome. With the binding of US/U4.U6 tri-snRNP the spliceosome is assembled but must be activated before the cleavage and ligation reactions begin (for review, see Moore et al. 1993;Newman 1994;Nilsen 1994; Ares and Weiser 1995}.The recognition of an intron and choice of splice sites are likely accomplished during the early steps of spliceosome assembly and may hold the key to the regulation of splicing. As the first ATP-requiring step, the stable addition of the U2 snRNP at the branchpoint is an attractive potential site of regulation. The RNA moiety of the U2 snRNP plays two roles in this step. A region called the branchpoint interaction sequence forms base pairs with the pre-mRNA at the site of lariat formation {Parker et Wu and Manley 1989; Zhuang and Weiner 19891. A structure downstream of the branchpoint interaction sequence called stem-loop...
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