DIRECT INTERACTIONS BETWEEN PRE-MESSENGER-RNA AND 6 U2 SMALL NUCLEAR RIBONUCLEOPROTEINS DURING SPLICEOSOME ASSEMBLY

被引:85
作者
STAKNIS, D [1 ]
REED, R [1 ]
机构
[1] HARVARD UNIV, SCH MED, DEPT CELL BIOL, BOSTON, MA 02115 USA
关键词
D O I
10.1128/MCB.14.5.2994
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF(65), which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF(65) and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.
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页码:2994 / 3005
页数:12
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