Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA‐binding proteome

Abstract

Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei. We describe the production of a small collection of open-reading frames encoding proteins potentially involved in mRNA metabolism. With this ORFeome collection, we used tethering to screen for proteins that play a role in post-transcriptional control. A yeast two-hybrid screen showed that several of the discovered repressors interact with components of the CAF1/NOT1 deadenylation complex. To identify the RNA-binding proteins, we obtained the mRNA-bound proteome. We identified 155 high-confidence candidates, including many not previously annotated as RNA-binding proteins. Twenty seven of these proteins affected reporter expression in the tethering screen. Our study provides novel insights into the potential trypanosome mRNPs composition, architecture and function.

Genome-wide RNAi selection identifies a regulator of transmission stage-enriched gene families and cell-type differentiation in Trypanosoma brucei

“…For example, the artificial tethering of a REG9 . 1 -lambda-N peptide fusion to a reporter gene 3’UTR bearing a BoxB lambda-N recognition sequence resulted in reporter gene repression [27, 36] consistent with the repressive effect we observed on ESAG9 transcripts. Intriguingly, however, we found that the overall abundance of REG9 .…”

The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex

“…PRO , but not TbPRMT1 ENZ , is reportedly phosphorylated (8,45), bound to mRNA (46), and trafficked to stress granules in starved trypanosomes (47). This suggests that TbPRMT1 PRO could be, in certain situations, operating independently of TbPRMT1 ENZ , possibly as a homodimer (Fig.…”

The role of the zinc finger protein ZC3H32 in bloodstream-form Trypanosoma brucei

“…Correspondingly, results from a high-throughput RNAi screen indicated that ZC3H32 is required for proliferation of bloodstream forms, but not procyclic forms [13]. CCCH zinc finger domains usually bind RNA, and indeed, ZC3H32 was detected in a mass spectrometry analysis of proteins that cross-link to polyadenylated RNA in bloodstream forms in vivo [14]. …”

“…We speculated that recruitment of the MKT1 complex by ZC3H32 should result in stablization of any mRNAs that are associated with ZC3H32. However, when ZC3H32 was artificially attached to a reporter in a "tethering" screen, results suggested instead that expression was suppressed [14]. …”