U4 small nuclear RNA genes of trypanosomes: cloning of the Leptomonas seymouri gene and mutational analysis of core snRNP assembly

Trans mRNA splicing in trypanosomatids requires as cofactors small nuclear RNAs (snRNAs) U2, U4, U5, and U6, in addition to the spliced leader (SL) RNA. To allow a phylogenetic comparison and functional analysis of trypanosomatid U4 snRNAs, we have cloned the single-copy gene for the Leptomonas seymouri U4 snRNA. In addition, a putative U4 snRNA gene from Leishmania tarentolae was identified by database searching. Using an episomal expression system, we introduced mutations into the conserved Sm region of the L. seymouri U4, which is the putative binding site for the common proteins that are present in each of the trans -spliceosomal snRNPs. As demonstrated by CsCl density gradient centrifugation, Sm mutant U4 snRNAs are non-functional in core RNP assembly. Furthermore, we present evidence by cell fractionation that U4 snRNAs with Sm mutations are partially defective in nuclear–cytoplasmic translocation. Taken together this indicates that the Sm site of U4 snRNA is responsible for stable core RNP assembly and nuclear localization.