title: U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.
abstract: Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.
authors.affiliation: Department of Cell Genetics, Genentech, Inc., South San Francisco, California 94080.
authors.forename: J B
authors.initials: JB
authors.lastname: Cohen
authors.forename: S D
authors.initials: SD
authors.lastname: Broz
authors.forename: A D
authors.initials: AD
authors.lastname: Levinson
mesh_terms: D017398:Alternative Splicing; D001483:Base Sequence; D002460:Cell Line; D003850:Deoxyribonuclease I / Q000235:genetics; D015870:Gene Expression; D005796:Genes; D011905:Genes, ras; D005822:Genetic Vectors; D013006:Growth Hormone / Q000235:genetics*; D006801:Humans; D007438:Introns; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010770:Phosphotransferases / Q000235:genetics*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010957:Plasmids; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D014162:Transfection
chemical_list: D012322:RNA Precursors; D012342:RNA, Small Nuclear; D012313:RNA; D013006:Growth Hormone; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D019868:Kanamycin Kinase; D003850:Deoxyribonuclease I; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)