An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.
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 beta-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 cross-linked 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.
Sirand-Pugnet
P
P
Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique associé Université de Paris VI, Gif-sur-Yvette, France.
Durosay
P
P
Brody
E
E
Marie
J
J
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D007438:Introns; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012322:RNA Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017381:Spliceosomes / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014335:Tropomyosin / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D012322:RNA Precursors; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D014335:Tropomyosin