ACCURATE MODIFICATION OF THE TRYPANOSOME SPLICED LEADER CAP STRUCTUREIN A HOMOLOGOUS CELL-FREE SYSTEM

During RNA maturation in trypanosomatid protozoa, trans-splicing trans fers the spliced leader (SL) sequence and its cap from the SL RNA to t he 5' end of all mRNAs. In Trypanosoma brucei and Crithidia fasciculat a the SL RNA has an unusual cap structure with four methylated nucleot ides following the 7-methylguanosine residue (cap 4). Since modificati on of the 5' end of the SL RNA is a pre-requisite for trans-splicing a ctivity in T. brucei, we have begun to characterize the enzyme(s) invo lved in this process. Here we report the development of a T. brucei ce ll-free system for modification of the cap of the SL RNA. Analysis of the nucleotide composition of the in vitro generated cap structure by two-dimensional thin layer chromatography established that the in vitr o reaction is accurate. Cap 4 formation requires the SL RNA to be in a ribonucleoprotein particle and can be inhibited by annealing a comple mentary 2'-O-methyl RNA oligonucleotide to nucleotides 7-18 of the SL RNA. Methylation of the 5' end of the SL RNA is also required for tran s-splicing in T. cruzi and Leishmania amazonensis and cell-free extrac ts from C. fasciculata and L. amazonensis are capable of modifying the cap structure on the T. brucei SL ribonucleoprotein particle.