In trypanosomatid protozoa the biogenesis of mature mRNA involves addition of the spliced leader (SL) sequence from the SL RNA to polycistronic pre-mRNA via trans-splicing. Here we present a mutational analysis of the trypanosomatid Leptomonas collosoma SL RNA to further our understanding of its functional domains important for trans-splicing utilization. Mutant SL RNAs were analyzed for defects in modification of the hypermethylated cap structure (cap 4) characteristic of trypanosomatid SL RNAs, for defects in the first step of the reaction and overall utilization in trans-splicing. Single substitution of the cap 4 nucleotides led to undermethylation of the cap 4 structure, and these mutants were all impaired in their utilization in trans-splicing. Abrogation of the sequence of the Sm-like site and sequences downstream to it also showed cap modification and trans-splicing defects, thus providing further support for a functional linkage between cap modifications and trans-splicing. Further, we report that in L. collosoma both the exon and intron of the SL RNA contribute information for efficient function of the SL RNA in trans-splicing. This study, however, did not provide support for the putative SL RNA-U6 small nuclear RNA (snRNA) interaction at the Sm site like in the nematodes, suggesting differences in the bridging role of U6 in the two trans-splicing systems.