Trypanosoma brucei RRM1 Is a Nuclear RNA-Binding Protein and Modulator of Chromatin Structure

Abstract

TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure.

The proteome and transcriptome of the infectious metacyclic form of Trypanosoma brucei define quiescent cells primed for mammalian invasion

“…Five RBPs (Tb927.2.4710, Tb927.6.4440, Tb927.8.6650, Tb927.8.710, Tb927.11.350), which includes RBP42 previously shown to target mRNAs involved in energy metabolism (Das et al ., ), and two putative Zn‐finger proteins (Tb927.7.2670, Tb927.7.2680) were down‐regulated at the transcript level. Tb927.2.4710 may have an influence on transcription as it is a chromatin modulator (Naguleswaran et al ., ). However, at the protein level, and perhaps consistent with the contracted total RNA and mRNA content in metacyclics, fifteen RBPs and twelve Zn‐finger proteins were down‐regulated (Fig.…”

The Trypanosoma brucei RNA‐Binding Protein TbRRM1 is Involved in the Transcription of a Subset of RNA Pol II‐Dependent Genes

“…D and (Naguleswaran et al. )). These results suggest that TbRRM1 might be involved in transcription repression of the SL‐RNA genes, as depletion of TbRRM1 increased their transcription.…”

“…Although it has been previously suggested that TbRRM1 does not play a role in global trans ‐splicing (Manger and Boothroyd ; Naguleswaran et al. ), it could be involved in the splicing processing of a small set of transcripts. We found that trans ‐splicing of both transcripts was unaffected (Fig.…”

“…It has previously shown that depletion of TbRRM1 upregulates the levels of approximately 300 transcripts (Naguleswaran et al. ), among them, a polyadenylated version of the SL‐RNA , while the levels of regular SL‐RNA remained unchanged. Here, we also observed a slightly, but significant increase in the SL‐RNA levels (Fig.…”

“…It has previously been shown by an RNA‐seq analysis that TbRRM1 depletion decreases the abundance of about one‐third of the T. brucei transcriptome (Naguleswaran et al. ). Among them, we found several genes involved in transcription, such as the transcription elongation factor TFIIS1 (Uzureau et al.…”