Abstract
The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs). Both RBPs have reduced expression in insect procyclic forms (PCFs) compared with bloodstream forms (BSFs). Upon overexpression in PCFs, both proteins were recruited to cytoplasmic foci, co-localizing with the processing body marker SCD6. Further, both RBPs altered the transcriptome from a PCF- to a BSF-like pattern. Notably, upon expression of BirA*-RBP9 and BirA*-RBP10, BioID yielded more than 200 high confidence protein interactors (more than 10-fold enriched); 45 (RBP9) and 31 (RBP10) were directly related to mRNA metabolism. This study validates the use of BioID for investigating mRNP components but also illustrates the complexity of mRNP function.
Highlights
The fate of an mRNA is determined by its interaction with proteins and small RNAs, which control its stability, translatability and localization within the cell
We report the combination of biotin identification (BioID) and RNA-seq as an approach to defining the composition of mRNPs implicated in developmental regulation in T. brucei
The analysis showed that many mRNAs that were upregulated after transgene induction had low initial mRNA copy numbers for both fast- and slow-regulated transcripts (RBP9: fast, 1.2/cell and slow, 1.6/cell; RBP10: fast, 1.3/cell and slow, 1.1/cell), whereas downregulated mRNA had a higher copy number for both fast- and slow-regulated transcripts (RBP9: fast, 7.5/cell and slow, 6.8/cell; RBP10, fast, 5.1/cell and slow, 8/cell)
Summary
The fate of an mRNA is determined by its interaction with proteins and small RNAs, which control its stability, translatability and localization within the cell These proteins are either RNA-binding (RBPs) or part of a complex that contains RBPs. The complement of proteins bound to any mRNA is dynamic and alters on export from the nucleus to the cytoplasm and in response to changes, for example external stress [1,2,3], internal cell cycle rhythms [4] or circadian rhythms [5,6]. Trypanosoma brucei has a complex life cycle with 10 or more different developmental forms in the insect vector and the mammalian host. The T. brucei genome is organized into long polycistronic units which are constitutively transcribed [13 –15] and co-transcriptionally processed into monocistronic mRNA molecules by a trans-splicing reaction that involves the addition of a 39 nt spliced leader sequence and the
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
