Abstract
Primary gene transcripts of eukaryotes contain introns, which are removed during processing by splicing machinery. Biochemical studies In vitro have identified a specific pathway in which introns are recognised and spliced out. This occurs by progressive formation of spliceosomal complexes designated as E, A, B, and C. The composition and structure of these spliceosomal conformations have been characterised in many detail. In contrast, transitions between the complexes and the intermediates of these reactions are currently less clear. We have previously isolated a novel 35S U5 snRNP from HeLa nuclear extracts. The protein composition of this particle differed from the canonical 20S U5 snRNPs but was remarkably similar to the activated B* spliceosomes. Based on this observation we have proposed a hypothesis that 35S U5 snRNPs represent a dissociation product of the spliceosome after both transesterification reactions are completed. Here we provide experimental evidence that 35S U5 snRNPs are generated from the activated B* spliceosomes during In vitro splicing.
Highlights
The majority of protein coding genes in eukaryotes are interrupted by introns, which are removed from mRNA precursors via a process of pre-mRNA splicing to produce mature mRNA for protein translation
We have shown that the activated spliceosomes eluted from beads were catalytically active and spliced pre-mRNA in the presence of micrococcal nuclease (MN)-treated extract [7]
The 20S U5 snRNP associates with the 10S U4/U6 snRNP to form stable 25S U4/U6.U5 trisnRNP complex [15], which is integrated into the spliceosome during transition from the A to B complex [16]
Summary
The majority of protein coding genes in eukaryotes are interrupted by introns, which are removed from mRNA precursors (pre-mRNA) via a process of pre-mRNA splicing to produce mature mRNA for protein translation. Intron removal and joining of exons is carried out by the spliceosome which catalyses two sequential trans-esterification reactions with single nucleotide precision to generate a correct message. The spliceosome is a very complex and dynamic cellular machine comprising five small nuclear (sn) RNAs (U1, U2, U4, U5, U6) and many, but still an uncertain number of proteins, around 200, which interact with premRNA in a temporally ordered manner (reviewed in [1]). The spliceosome assembly is initiated by the recognition of the 5’ splice site (ss) by the U1 snRNP and the 3’ss by the protein factors SF1/mBBP and U2AF to form the Early (E) complex. The fully assembled spliceosome contains the U4/U6.U5 tri-snRNP
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