STRUCTURE-FUNCTION ANALYSIS OF THE TRYPANOSOMATID SPLICED LEADER RNA

In trypanosomes, all mRNAs possess a spliced leader (SL) at their 5' e nd. SL is added to pre-mRNA via trans-splicing from a small RNA, the S L RNA. To examine structure-function aspects of the trypanosomatid SL RNA, an in vivo system was developed in the monogenetic trypanosomatid Leptomonas collosoma to analyze the function of chimeric and site-dir ected SL RNA mutants in trans-splicing. Stable cell lines expressing c himeric and mutated SL RNA from the authentic SL RNA regulatory unit w ere obtained. The chimeric RNA was expressed and assembled into an SL RNP particle, but could not serve as a substrate in splicing. Mutation s in loop II and III of L.collosoma SL RNA formed the Y structure inte rmediate. In addition, a double SL RNA mutant in loop II, and position s 7 and 8 of the intron, also formed the Y structure intermediate, sug gesting that these intron positions, although proposed to participate in the interaction of SL RNA with U5, may not be crucial for the first step of the transsplicing reaction. A mutation in the exon located in loop I was not utilized in splicing, suggesting the importance of exo n sequences for trans-splicing in trypanosomes. However, a double SL R NA mutant in loop II and exon position 31 was utilized in both steps o f splicing; the mutant thus provides a model molecule for further anal ysis of positions essential for the function of the SL RNA.