Post-Transcriptional Regulation of the Trypanosome Heat Shock Response by a Zinc Finger Protein

Abstract

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3′-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures.

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

“…One PUF2 RNAi batch was sequenced on the GAII and the second with HiSeq. Data analysis, including motif searches and enrichment calculations, were performed as described previously (22,54). All data were log transformed before the correlation coefficient calculations, in order to reduce the influence of outliers and correct for a nonnormal distribution of the data.…”

The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation

“…To classify trypanosome genes, we supplemented the current annotations from GeneDB and TritrypDB with KEGG designations and a limited number of publications describing pathways and organellar or complex proteomes (Supplemental Table S2; Bringaud et al 2006;Colasante et al 2006;Michels et al 2006;Panigrahi et al 2008;Vertommena et al 2008;Acestor et al 2009;Droll et al 2013). We had already seen that, in wild-type cells, mRNAs encoding ribosomal proteins and enzymes of glucose metabolism are significantly more stable than the average (Manful et al 2011).…”

“…Supplemental Table S2 (half-lives) contains all annotated genes (coding and noncoding) with the exception of rRNAs. In order to analyze the read densities across mRNAs, we assigned predominant splice and poly(A) sites using all our available data (Manful et al 2011;Droll et al 2013). Custom PERL and R scripts were written as required.…”

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.…”

“…In T. brucei, PUF9 binds to a consensus sequence in 3 ′ UTR of transcripts whose function is important in temporal coordination of the kinetoplast and nuclear replication during late S-phase (Archer et al 2009). The T. brucei ZC3H11 binds to and stabilizes mRNAs encoding chaperones required for protein refolding following heat shock (Droll et al 2013). DRBD3, a protein containing two RRM domains, plays a role both in splicing and mRNA stability in T. brucei (Estévez 2008;Das et al 2012).…”