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tbCPSF30 depletion by RNA interference disrupts polycistronic RNA processing in Trypanosoma brucei
Edward F Hendriks1, Ammar Abdul-Razak, Keith R Matthews
1School of Biological Sciences, Division of Biochemistry, 2.205 Stopford Building, University of Manchester, Oxford Road, Manchester M13 9PT, United Kingdom.
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.
Insights
This study identifies tbCPSF30, essential for mRNA processing in Trypanosoma brucei. Depleting this protein disrupts polycistronic RNA processing, crucial for gene expression regulation in this parasite.
Area of Science:
- Molecular Biology
- Eukaryotic Gene Expression
- Parasitology
Background:
- Eukaryotic gene expression relies on mRNA cleavage and processing.
- Trypanosoma brucei exhibits polycistronic transcription, making post-transcriptional regulation vital.
- Most T. brucei transcripts originate from polycistronic units, necessitating promoter-independent gene regulation.
Purpose of the Study:
- To identify and characterize components of the mRNA 3'-end processing machinery in Trypanosoma brucei.
- To investigate the role of tbCPSF30, a homolog of CPSF30, in T. brucei gene expression.
- To elucidate the involvement of tbCPSF30 in processing polycistronic transcripts.
Main Methods:
- Gene silencing using RNA interference (RNAi) to deplete tbCPSF30.
- Analysis of mRNA species and protein levels following tbCPSF30 depletion.
- Genome database mining to identify other cleavage and polyadenylation factors.
Main Results:
- tbCPSF30 is essential for both bloodstream and procyclic forms of T. brucei.
- RNAi-mediated depletion of tbCPSF30 led to aberrant tbCPSF30 mRNA accumulation and protein depletion.
- Depletion of tbCPSF30 resulted in unprocessed tubulin RNAs, indicating its role in polycistronic RNA processing.
Conclusions:
- tbCPSF30 is a core component of the cleavage and polyadenylation specificity factor (CPSF) machinery in T. brucei.
- This study demonstrates tbCPSF30's essential function in polycistronic RNA processing in T. brucei.
- The findings suggest conservation of the mRNA 3'-end formation machinery across eukaryotes.