2'-O-methyl RNA oligonucleotides identify two functional elements in the trypanosome spliced leader ribonucleoprotein particle

E Ullu,C Tschudi

doi:10.1016/s0021-9258(19)38620-x

Abstract

Using permeable trypanosomes as an in vivo model system for trans-splicing, we have searched for functional elements in the Trypanosoma brucei 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-directed ribonuclease (RNase) H cleavage revealed three accessible regions: the 5' end, sequences downstream of the 5' splice site, and a putative single-stranded sequence between stem-loops II and III, which is thought to be analogous to the mammalian Sm-binding site of U small nuclear RNAs. Using antisense 2'-O-methyl RNA oligomers, two functional elements 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 addition, nucleotides +1 to +4 relative to the 5' splice site, which include the invariant GU dinucleotide were accessible to oligomer binding in the SL ribonucleoprotein particle, and their blockade resulted in complete inhibition of trans-splicing. In contrast, RNA oligomer binding to the single-stranded region between stem-loop II and III of the SL RNA had no detectable effect on trans-splicing activity of the SL RNA.

Highlights

Using permeable trypanosomes as an in uiuo model system for trans-splicing, we have searched for functional elements in the Trypanosoma brucei spliced leader (SL)RNA by masking various regions of the molecule with short antisense 2”O-methyl RNA oligomers
Nucleotides +1 to +4 relative to the 5’ splice site, which include the invariant GU dinucleotide were accessible to oligomer binding in the SL ribonucleoprotein particle, and their blockaderesulted in complete inhibition of trans-splicpairing between the SL and the 5’ splice site is not essential for trans-splicing in vitro [11].More recently, an alternate base pairing scheme for the 5’ domain of a synthetic trypanosomatid SL RNA has been proposed on the basis of a ing
We have previously shown that the SL RNP is accessible to digestion with RNA by deoxyoligonucleotide-directedribonuclease (RNase) H in the presence of complementary DNA oligonucleotides [18].The most effective digestion was obtained with Y-21, an oligomer complementary to nt 40-60of the SL RNA, which induced almost complete cleavage of the SLRNA and abolished trans-splicing completely.As an extension of these initialstudies, we wished to probe the function of the SL RNP by blocking different regions of the SL RNA with antisense 2”O-methyl RNA oligonucleotides.These RNA oligonucleotidesare resistantto RNase cleavage and upon binding to thecomplementary RNA do not induce cleavage of the target RNA by endogenous RNase H activity asit is observed with antisense DNA oligonucleotides [19]

Summary

Introduction

Using permeable trypanosomes as an in uiuo model system for trans-splicing, we have searched for functional elements in the Trypanosoma brucei spliced leader (SL)RNA by masking various regions of the molecule with short antisense 2”O-methyl RNA oligomers. DNA Oligonucleotide-directed Cleavage of Newly Synthesized SL RNA-Upon incubation of permeable T. brucei cells in a transcription mixture, the SL RNA is efficiently transcribed and utilized for trans-splicing [16].

Results

Conclusion