Silencing of Sm Proteins in Trypanosoma brucei by RNA Interference Captured a Novel Cytoplasmic Intermediate in Spliced Leader RNA Biogenesis

Michal Mandelboim,Sarit Barth,Moshe Biton,Xue-Hai Liang,Shulamit Michaeli

doi:10.1074/jbc.m308997200

Abstract

In Trypanosoma brucei the small nuclear (sn) RNAs U1, U2, U4, and U5, as well as the spliced leader (SL) RNA, bind the seven Sm canonical proteins carrying the consensus Sm motif. To determine the function of these proteins in snRNA and SL RNA biogenesis, two of the Sm core proteins, SmE and SmD1, were silenced by RNAi. Surprisingly, whereas the level of all snRNAs, including U1, U2, U4, and U5 was reduced during silencing, the level of SL RNA was dramatically elevated, but the levels of U6 and spliced leader-associated RNA (SLA1) remained unchanged. The SL RNA that had accumulated in silenced cells lacked modification at the cap4 nucleotide but harbored modifications at the cap1 and cap2 nucleotides and carried the characteristic psi. This SL RNA possessed a longer tail and had accumulated in the cytoplasm in 10 and 50 S particles that were found by in situ hybridization to be present in "speckles." We propose a model for SL RNA biogenesis involving a cytoplasmic phase and suggest that the trypanosome-specific "cap4" nucleotides function as a signal for export and import of SL RNA out and into the nucleus. The SL RNA biogenesis pathway differs from that of U sn ribonucleoproteins (RNPs) in that it is the only RNA that binds Sm proteins that were stabilized under Sm depletion in a novel RNP, which we termed SL RNP-C.

Highlights

Pre-mRNA splicing in trypanosomes is an essential step in gene expression, because all mRNAs undergo trans-splicing [1]. trans-Splicing evolved to separate long polycistronically transcribed mRNAs that are dissected by the concerted action of the trans-splicing and polyadenylation [1]
Treatment with leptomycin B resulted in the accumulation of spliced leader (SL) RNA with a 3Ј tail, which lacked the modification on the cap4 position, suggesting that a cytoplasmic stage is necessary for SL RNA biogenesis [20]
An RT-PCR assay was used to monitor the level of the mature poly(A) polymerase gene (PAP)-spliced product by using oligonucleotides that are situated in the two exons; the results indicate a reduction in the level of mature PAPs (Fig. 2C, b)

Summary

Introduction

Pre-mRNA splicing in trypanosomes is an essential step in gene expression, because all mRNAs undergo trans-splicing [1]. trans-Splicing evolved to separate long polycistronically transcribed mRNAs that are dissected by the concerted action of the trans-splicing and polyadenylation [1]. Studies in L. tarentolae and Trypanosoma brucei provided evidence that the SL RNA is present in the cytoplasm and does not reside only in the nucleus [20]. Treatment of these parasites with the karyopherin-specific inhibitor leptomycin B eliminated the cytoplasmic SL RNA, and SL RNA export was shown to be mediated by the nuclear exporter, exportin 1 (XPO1). Treatment with leptomycin B resulted in the accumulation of SL RNA with a 3Ј tail, which lacked the modification on the cap position, suggesting that a cytoplasmic stage is necessary for SL RNA biogenesis [20]

Methods

Results

Conclusion