Cloning and mutational analysis of the Leptomonas seymouri U5 snRNA gene: function of the Sm site in core RNP formation and nuclear localization

We have cloned the single-copy gene for the trans-spliceosomal U5 snRNA fro m the trypanosomatid species Leptomonas seymouri, using U5 RNA affinity sel ection and cDNA cloning, Sequence comparison revealed that the trans-splice osomal U5 RNAs from trypanosomatid species share certain characteristic fea tures. Interestingly, the affinity selection procedure yielded-in addition to the bona fide U5 RNA-a closely related small RNA, which can be folded in to the same secondary structure, but carries three changes in the loop sequ ence, This raises the possibility that there may be a larger family of U5-l ike RNAs in trypanosomes. To study the U5 snRNP assembly and function in tr ypanosomes we have established a stable expression system in L.seymouri. Tw o cell lines have been generated that express U5 RNAs with mutations in the Sm site, resulting in a defect of core snRNP formation. In addition, the U 5 Sm-mutant RNAs behaved differently in cell fractionation, implying a defe ct in nuclear localization. In sum, this demonstrates for the first time th at the Sm site of trypanosome snRNAs contributes an essential element for s table core RNP assembly and may be important for nuclear localization, in a nalogy to the Sm site function of cis-spliceosomal snRNAs in higher eucaryo tes.