An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken β-tropomyosin pre-mRNA

摘要

Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5′ splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5′ part of a β-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently crosslinked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5′ part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.