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Ribonucleoproteins (RNPs) mediate key cellular functions such as gene expression and its regulation. Whereas most RNP enzymes are stable in composition and harbor preformed active sites, the spliceosome, which removes noncoding introns from precursor messenger RNAs (pre-mRNAs), follows fundamentally different strategies. In order to provide both accuracy to(More)
Exactly how specific splice sites are recognized during the processing of complex precursor messenger RNAs is not clear. Small nuclear ribonucleoprotein particles (snRNPs) are involved, but are not sufficient by themselves to define splice sites. Now a human protein essential for splicing in vitro, called alternative splicing factor/splicing factor 2, is(More)
Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of(More)
Splicing of mammalian precursor transfer RNA (tRNA) molecules involves two enzymatic steps. First, intron removal by the tRNA splicing endonuclease generates separate 5' and 3' exons. In animals, the second step predominantly entails direct exon ligation by an elusive RNA ligase. Using activity-guided purification of tRNA ligase from HeLa cell extracts, we(More)
In metazoans, two distinct spliceosomes catalyzing pre-messenger RNA splicing have been identified. Here, the human U11/U12 small nuclear ribonucleoprotein (snRNP), a subunit of the minor (U12-dependent) spliceosome, was isolated. Twenty U11/U12 proteins were identified, including subsets unique to the minor spliceosome or common to both spliceosomes.(More)
The first step in splicing of pre-mRNAs with long introns is exon definition, where U1 and U2 snRNPs bind at opposite ends of an exon. After exon definition, these snRNPs must form a complex across the upstream intron to allow splicing catalysis. Exon definition and conversion of cross-exon to cross-intron spliceosomal complexes are poorly understood. Here(More)
In the U12-dependent spliceosome, the U4atac/U6atac snRNP represents the functional analogue of the major U4/U6 snRNP. Little information is available presently regarding the protein composition of the former snRNP and its association with other snRNPs. In this report we show that human U4atac/U6atac di-snRNPs associate with U5 snRNPs to form a 25S(More)
The conserved Prp19 (pre-RNA processing 19) complex is required for pre-mRNA splicing in eukaryotic nuclei. Recent RNAi screens indicated that knockdown of Prp19 complex subunits strongly delays cell proliferation. Here we show that knockdown of the smallest subunit, BCAS2/Spf27, destabilizes the entire complex and leads to specific mitotic defects in human(More)
The splicing factor SF3b is a multiprotein complex essential for the accurate excision of introns from pre-messenger RNA. As an integral component of the U2 small nuclear ribonucleoprotein (snRNP) and the U11/U12 di-snRNP, SF3b is involved in the recognition of the pre-messenger RNA's branch site within the major and minor spliceosomes. We have determined(More)
Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S(More)