Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5′ splice sites in vivo.

Abstract

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 (C 3 , U 4 , U 5 and U 6 ) within the invariant 9 nt U5 sequence GCCUUUUAC; transient transfection of HeLa cells with these US sequences cloned into a U1 expression vector yielded high levels of the mutant snRNAs. To test their function, we cotransfected a rabbit β-globin gene containing one of two mutations (G 1 → A or T 2 → A) in the essential GT dinucleotide at the 5′ end of the second intron. Certain US loop mutants activated novel 5′ splice sites only in mutant rabbit β-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 US 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. 5′ exon recognition/pre-mRNA splicing/rabbit β-globin pre-mRNA/U5 small nuclear RNA.