Analysis of small nuclear ribonucleoproteins (RNPs) in Trypanosoma brucei: structural organization and protein components of the spliced leader RNP.

M Cross,Z Palfi,A Bindereif,A Günzl

doi:10.1128/mcb.11.11.5516

Abstract

trans splicing in Trypanosoma brucei involves the ligation of the 40-nucleotide spliced leader (SL) to each of the exons of large, polycistronic pre-mRNAs and requires the function of small nuclear ribonucleoproteins (snRNPs). We have identified and characterized snRNP complexes of SL, U2, U4, and U6 RNAs in T. brucei extracts by a combination of glycerol gradient sedimentation, CsCl density centrifugation, and anti-m3G immunoprecipitation. Both the SL RNP and the U4/U6 snRNP contain salt-stable cores; the U2 snRNP, in contrast to other eucaryotic snRNPs, is not stable under stringent ionic conditions. Two distinct complexes of U6 RNA were found, a U6 snRNP and a U4/U6 snRNP. The structure of the SL RNP was analyzed in detail by oligonucleotide-directed RNase H protection and by in vitro reconstitution. Our results indicate that the 3' half of SL RNA constitutes the core protein-binding domain and that protein components of the SL RNP also bind to the U2 and U4 RNAs. Using antisense RNA affinity chromatography, we identified a set of low-molecular-mass proteins (14.8, 14, 12.5, and 10 kDa) as components of the core SL RNP.

Highlights

In trypanosomes, all nuclear mRNAs analyzed to date are generated through a trans-splicing reaction whereby a noncoding 40-nucleotide spliced leader (SL) is joined to each protein-coding exon
Both of these RNAs are independently transcribed as precursors, an SL RNA of approximately 140 nucleotides with the SL portion at the 5' end and premRNAs derived from long, polycistronic transcription units. trans splicing proceeds through a two-step mechanism of cleavage-ligation reactions formally analogous to conventional cis splicing of other eucaryotic systems: the first step results in a branched RNA intermediate corresponding to the lariat intermediate RNA of the cis-splicing reaction; in the second step, the SL exon is ligated to the protein-coding exon and the intron portion is released in the form of a Y-shaped RNA molecule (38, 47; for reviews, see references 1 and 7)
We have obtained further evidence for a special core structure of the trypanosomal U2 small nuclear ribonucleoproteins (snRNPs): only the U2 snRNP was unstable at high ionic strength, whereas the SL RNP and U4/U6 snRNP were in the form of core particles, as detected by native gel electrophoresis and Northern hybridization analysis [17]

Summary

Introduction

All nuclear mRNAs analyzed to date are generated through a trans-splicing reaction whereby a noncoding 40-nucleotide spliced leader (SL) is joined to each protein-coding exon. No Ul and U5 RNA homologs are known in trypanosomes; the SL RNA, has been proposed to function both as a Ul-like snRNP and as a donor of its own 5'-terminal 40 nucleotides [11, 54] This has recently been directly demonstrated in a mammalian in vitro system, in which the 5'-terminal domain of Leptomonas collosoma SL RNA could substitute for the 5' end of Ul snRNA in pre-mRNA cis splicing [10]. In support of such a functional role, the SL sequence as well as the overall secondary structure of the SL RNA are strongly conserved between different trypanosome and nematode species (11, 22, 32, 40; for the T. brucei SL RNA structure, see Fig. 3A). It is the underlying rationale of our study that structural deviations of trypanosomal snRNPs from the otherwise highly conserved eucaryotic snRNPs reflect important functional differences between the cis- and trans-splicing mechanisms

Methods

Results

Conclusion