Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5' splice sites in vivo.
AUTOR(ES)
Cortes, J J
RESUMO
We have analyzed base pairing interactions between the U5 snRNA and 5' exon sequences during pre-mRNA splicing in a mammalian in vivo system. We constructed synthetic U5 genes with mutations that alter four bases (C3, U4, U5 and U6) within the invariant 9 nt U5 sequence GCCUUUUAC; transient transfection of HeLa cells with these U5 sequences cloned into a U1 expression vector yielded high levels of the mutant snRNAs. To test their function, we cotransfected a rabbit beta-globin gene containing one of two mutations (G1-->A or T2-->A) in the essential GT dinucleotide at the 5' end of the second intron. Certain U5 loop mutants activated novel 5' splice sites only in mutant rabbit beta-globin transcripts. One novel site surprisingly resides in the first exon; its use is invariably coupled to utilization of a particular cryptic 5' splice site in the second exon. All of the newly activated cryptic 5' splice sites exhibit complementarity with the mutant U5 loop in the exon 1-5 nt upstream of the cryptic site, extending previous results in yeast. However, the register of the potential pairing is not identical at the various novel cryptic 5' splice sites, indicating that the interaction between the U5 loop and the 5' exon may be more flexible than previously believed.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=413781Documentos Relacionados
- Mutations in U5 snRNA loop 1 influence the splicing of different genes in vivo
- Antisense oligonucleotide binding to U5 snRNP induces a conformational change that exposes the conserved loop of U5 snRNA.
- Functional characterization of X. laevis U5 snRNA genes.
- Extensive interactions of PRP8 protein with the 5' and 3' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.
- Structure and expression of the U5 snRNA gene of Arabidopsis thaliana. Conserved upstream sequence elements in plant U-RNA genes.