MUTATIONS IN THE CONSERVED LOOP OF HUMAN U5 SNRNA GENERATE USE OF NOVEL CRYPTIC 5' SPLICE SITES IN-VIVO

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 bas es (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 con taining one of two mutations (G1-->A or T2-->A) in the essential GT di nucleotide at the 5' end of the second intron. Certain U5 loop mutants activated novel 5' splice sites only in mutant rabbit beta-globin tra nscripts. One novel site surprisingly resides in the first exon; its u se is invariably coupled to utilization of a particular cryptic 5' spl ice site in the second exon. All of the newly activated cryptic 5; spl ice sites exhibit complementarity with the mutant U5 loop in the exon 1-5 nt upstream of the cryptic site, extending previous results in yea st. However, the register of the potential pairing is not identical at the various novel cryptic 5' splice sites, indicating that the intera ction between the U5 loop and the 5' exon may be more flexible than pr eviously believed.