U1 SMALL NUCLEAR RNAS WITH ALTERED SPECIFICITY CAN BE STABLY EXPRESSED IN MAMMALIAN-CELLS AND PROMOTE PERMANENT CHANGES IN PREMESSENGER RNASPLICING

Pre-mRNA 5' splice site activity depends, at least in part, on base co mplementarity to U1 small nuclear RNA. In transient coexpression assay s, 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 explo red 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 resistanc e gene. Complementation was observed in proportion to the degree of co mplementarity between transfected mutant U1 genes and different defect ive splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activa tion of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the ex pression of targeted genes in mammalian cells.