IN-VIVO STRUCTURAL-ANALYSIS OF SPLICED LEADER RNAS IN TRYPANOSOMA-BRUCEI AND LEPTOMONAS-COLLOSOMA - A FLEXIBLE STRUCTURE THAT IS INDEPENDENT OF CAP4 METHYLATIONS

The formation of the mRNA 5' end in trypanosomatid protozoa is carried out by trans-splicing, which transfers a spliced leader (SL) sequence and its hypermethylated cap (cap4) from the SL RNA to the pre-mRNA. P revious in vitro studies with synthetic uncapped RNAs have shown that the SL sequence of Leptomonas collosoma can assume two alternate confo rmations, Form 1 and Form 2, with Form 1 being the dominant one. To ga in information about the structure of the SL RNA in vivo, in its prote in-rich enviroment, we have used permeable Trypanosoma brucei and L. c ollosoma cells for chemical modification experiments. We introduce the use in vivo of the water-soluble reagents CMCT and kethoxal. In contr ast to the in vitro results, the Form 2 secondary structure predominat es. However, there are chemically accessible regions that suggest conf ormational flexibility in SL RNPs and a chemically inaccessible region suggestive of protection by protein or involvement in tertiary intera ctions. Using complementary 2'-O-methyl RNA oligonucleotides, we show that T. brucei SL RNA can be induced to switch conformation in vivo. S L RNA stripped of proteins and probed in vitro does not display the sa me Form 2 bias, indicating that SL RNA structure is determined, at lea st in part, by its RNP context. Finally, the methyl groups of the cap4 do not seem to affect the secondary structure of T. brucei SL RNA, as shown by chemical modification of undermethylated SL RNA probed in vi vo.