Title: Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5′ splice sites in vivo.
Abstract: Research Article15 December 1993free access Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5′ splice sites in vivo. J.J. Cortes J.J. Cortes Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author E.J. Sontheimer E.J. Sontheimer Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author S.D. Seiwert S.D. Seiwert Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author J.A. Steitz J.A. Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author J.J. Cortes J.J. Cortes Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author E.J. Sontheimer E.J. Sontheimer Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author S.D. Seiwert S.D. Seiwert Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author J.A. Steitz J.A. Steitz Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. Search for more papers by this author Author Information J.J. Cortes1, E.J. Sontheimer1, S.D. Seiwert1 and J.A. Steitz1 1Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, CT 06536-0812. The EMBO Journal (1993)12:5181-5189https://doi.org/10.1002/j.1460-2075.1993.tb06213.x PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Previous ArticleNext Article Volume 12Issue 131 December 1993In this issue RelatedDetailsLoading ...