Evidence from complementation assays in vitro that U5 snRNP is required for both steps of mRNA splicing.

摘要: We have established an in vitro complementation system that has allowed us to investigate the role of individual purified snRNPs splicing pre-mRNA molecules. For preparation snRNP-depleted nuclear extracts we first removed majority endogenous from by one passage over anti-m3G column and then degraded remaining with micrococcal nuclease. The mixture U1, U2, U4/U6 U5, obtained immuno-affinity chromatography, was functionally active able restore extracts. Mono-Q chromatography used for further fractionation U1-U6. This produced three fractions were highly enriched U1 U5 respectively. Conditions found where addition [U1, U2] snRNP gave rise formation splice intermediates absence any 3' cleavage/exon 1-exon 2 product formation. Only when 20S added did both steps reaction occur efficiently. Our data suggest is absolutely required second step needed efficient initiation reaction. requirement corroborated glycerol gradient sedimentation analysis respective reconstituted pre-mRNP complexes. Stable 50-60S spliceosomes observed only presence all snRNPs.