2'-O-METHYL RNA OLIGONUCLEOTIDES IDENTIFY 2 FUNCTIONAL ELEMENTS IN THE TRYPANOSOME SPLICED LEADER RIBONUCLEOPROTEIN PARTICLE

Using permeable trypanosomes as an in vivo model system for trans-spli cing, we have searched for functional elements in the Trypanosoma bruc ei spliced leader (SL) RNA by masking various regions of the molecule with short antisense 2'-O-methyl RNA oligomers. Initial probing of the structure of newly synthesized SL RNA by deoxyoligonucleotide-directe d ribonuclease (RNase) H cleavage revealed three accessible regions: t he 5' end, sequences downstream of the 5' splice site, and a putative single-stranded sequence between stem-loops II and III, which is thoug ht to be analogous to the mammalian Sm-binding site of U small nuclear RNAs. Using antisense 2'-O-methyl RNA oligomers, two functional eleme nts of the SL RNA became apparent. Masking of positions 1-18 inhibited modification of the cap 4 structure of newly synthesized SL RNA and, thereby, blocked utilization of the SL RNA in trans-splicing. In addit ion, nucleotides +1 to +4 relative to the 5' splice site, which includ e the invariant GU dinucleotide were accessible to oligomer binding in the SL ribonucleoprotein particle, and their blockade resulted in com plete inhibition of trans-splicing. In contrast, RNA oligomer binding to the single-stranded region between stem-loop II and III of the SL R NA had no detectable effect on trans-splicing activity of the SL RNA.