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

Abstract

We have cloned the single-copy gene for the trans -spliceosomal U5 snRNA from the trypanosomatid species Leptomonas seymouri, using U5 RNA affinity selection and cDNA cloning. Sequence comparison revealed that the trans -spliceosomal U5 RNAs from trypanosomatid species share certain characteristic features. Interestingly, the affinity selection procedure yielded-in addition to the bona fide U5 RNA-a closely related small RNA, which can be folded into the same secondary structure, but carries three changes in the loop sequence. This raises the possibility that there may be a larger family of U5-like RNAs in trypanosomes. To study the U5 snRNP assembly and function in trypanosomes we have established a stable expression system in L.seymouri. Two 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 U5 Sm-mutant RNAs behaved differently in cell fractionation, implying a defect in nuclear localization. In sum, this demonstrates for the first time that the Sm site of trypanosome snRNAs contributes an essential element for stable core RNP assembly and may be important for nuclear localization, in analogy to the Sm site function of cis -spliceosomal snRNAs in higher eucaryotes.