Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing.
Although both U1 and U2 snRNPs have been implicated in the splicing process, their respective roles in the earliest stages of intron recognition and spliceosome assembly are uncertain. To address this issue, we developed a new strategy to prepare snRNP-depleted splicing extracts using Saccharomyces cerevisiae cells conditionally expressing U1 or U2 snRNP. Complementation analyses and chase experiments show that a stable complex, committed to the splicing pathway, forms in the absence of U2 snRNP. U1 snRNP and a substrate containing both a 5' splice site and a branchpoint sequence are required for optimal formation of this commitment complex. We developed new gel electrophoresis conditions to identify these committed complexes and to show that they contain U1 snRNA. Chase experiments demonstrated that these complexes are functional intermediates in spliceosome assembly and splicing. Our results have implications for the process of splice site selection.
Seraphin
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Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, Massachusetts 02254.
Rosbash
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D001483:Base Sequence; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008957:Models, Genetic; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000235:genetics; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D015345:Oligonucleotide Probes; D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear