Expression of the RNA recognition motif protein RBP10 promotes a bloodstream‐form transcript pattern in Trypanosoma brucei

Abstract

SummaryWhen Trypanosoma brucei differentiates from the bloodstream form to the procyclic form, there are decreases in the levels of many mRNAs encoding proteins required for the glycolytic pathway, and the mRNA encoding the RNA recognition motif protein RBP10 decreases in parallel. We show that RBP10 is a cytoplasmic protein that is specific to bloodstreamform trypanosomes, where it is essential. Depletion of RBP10 caused decreases in many bloodstream-formspecific mRNAs, with increases in mRNAs associated with the early stages of differentiation. The changes were similar to, but more extensive than, those caused by glucose deprivation. Conversely, forced RBP10 expression in procyclics induced a switch towards bloodstream-form mRNA expression patterns, with concomitant growth inhibition. Forced expression of RBP10 prevented differentiation of bloodstream forms in response to cis-aconitate, but did not prevent expression of key differentiation markers in response to glucose deprivation. RBP10 was not associated with heavy polysomes, showed no detectable in vivo binding to RNA, and was not stably associated with other proteins. Tethering of RBP10 to a reporter mRNA inhibited translation, and halved the abundance of the bound mRNA. We suggest that RBP10 may prevent the expression of regulatory proteins that are specific to the procyclic form.

A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei

“…These included RBP10, which was previously shown to inhibits translation if tethered to a reporter [18] and DRBD12 (Tb927.7.5380), which was shown to destabilize ARE-containing targets [41] (Supplementary Figure S5). In total, we found 16 RBPs conferring a clear negative effect on reporter expression, from which 7 were zinc finger proteins, 8 had a RRM motif and one, PUF3, a pumilio domain (Table 2).…”

“…In trypanosomatids, although indirect results suggest that some proteins cause degradation of target transcripts this has never been shown directly (examples are UBP1 [15], PUF6 [16] and PUF5 [17].) There is evidence that one protein, RBP10, can suppress translation when attached to an mRNA [18]. With the single exception of ZC3H11 [19], the mechanisms by which trypanosome proteins determine mRNA fate are not known.…”

Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

“…There was no correlation with 3=-UTR length (R 2 value always under 0.12). ZC3H11 is required mainly for stabilization of chaperone transcripts in procyclic forms, but RNAi causes rapid death of bloodstream forms (54), while RBP10 levels correlate with expression of a subset of bloodstream-form-specific mRNAs (52). The effects of depleting ZC3H11 showed only a minor length influence (Fig.…”

“…PUF2 cross-linking to RNA followed by immunoprecipitation (CLIP) was performed. The method used was a combination of the CLIP (49) and the PAR-CLIP (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation) (50, 51) methods with minor changes and without in vivo labeling with 4-thio-uracil (52). Approximately 2 ϫ 10 9 bloodstreamform trypanosomes expressing V5-PUF2 were used for the pulldown assay.…”

The Pumilio-domain protein PUF6 contributes to SIDER2 retroposon-mediated mRNA decay in Leishmania

“…Development of an improved MS2 coat protein tethering system for identifying trans-acting factors regulating SIDER2 retroposon-mediated mRNA decay in Leishmania So far, the λN peptide has been used successfully to tether RBPs to a particular RNA of interest in the related Trypanosoma species (Delhi et al 2011;Wurst et al 2012;Droll et al 2013;Jha et al 2014;Singh et al 2014). Here, we have developed an improved MS2 coat protein tethering system adapted for use in Leishmania to attach RNA-binding proteins (RBPs) specifically to a reporter RNA.…”