TbCPSF30 Depletion by RNA Interference Disrupts Polycistronic RNA Processing in Trypanosoma brucei

Abstract

Gene expression in eukaryotes requires the post-transcriptional cleavage of mRNA precursors into mature mRNAs. In Trypanosoma brucei, mRNA processing is of particular importance, since most transcripts are derived from polycistronic transcription units. This organization dictates that regulated gene expression is promoter-independent and governed at the posttranscriptional level. We have identified tbCPSF30, a protein containing five CCCH zinc finger motifs, which is a homologue of the cleavage and polyadenylation specificity factor (CPSF) 30-kDa subunit, a component of the machinery required for 3'-end formation in yeast and mammals. Using gene silencing of tbCPSF30 by RNA interference, we demonstrate that this gene is essential in bloodstream and procyclic forms of T. brucei. Interestingly, tbCPSF30-specific RNA interference results in the accumulation of an aberrant tbCPSF30 mRNA species concomitant with depletion of tbCPSF30 protein. tbCPSF30 protein depletion is accompanied by the accumulation of unprocessed tubulin RNAs, implicating tbCPSF30 in polycistronic RNA processing. By genome data base mining, we also identify several other putative components of the T. brucei cleavage and polyadenylation machinery, indicating their conservation throughout eukaryotic evolution. This study is the first to identify and characterize a core component of the T. brucei CPSF and show its involvement in polycistronic RNA processing.