18 research outputs found
The role of TbZC3H11 in the trypanosomal heat shock response
Zusammenfassung Der parasitĂ€re Protist Trypanosoma brucei ĂŒbt praktisch keine Kontrolle ĂŒber die Transkription aus. Dennoch muss dieser Organismus seine Genexpression an verschiedene Umgebungen anpassen, zum Beispiel den Blutstrom in SĂ€ugetieren oder den Mitteldarm des Insektenvektors (prozyklische Form). Die Regulation der Genexpression ist daher auf posttranskriptionale Mechanismen angewiesen. RNA-Bindeproteine sind wichtige posttranskriptionale Regulatoren, da sie die StabilitĂ€t, Lokalisierung und Translation ihrer Ziel-mRNAs beeinflussen können. CCCH Zinkfingerproteine, insbesondere die Tis11 Familie, sind an der Destabilisierung und Regulation von mRNAs mit AU-reichen Elementen in der 3âUTR (3â untranslatierte Region) beteiligt. ZC3H11 ist ein trypanosomales Protein mit einer Tis11-Ă€hnlichen ZinkfingerdomĂ€ne. Die Expression des ZC3H11 Proteins ist nach Hitzeschock oder Inhibition des Proteasoms erhöht und ZC3H11 ist phosphoryliert. ZC3H11-Reduktion mittels RNA-Interferenz (RNAi) ist in der Blutstromform, nicht aber in der prozyklischen Form, lethal. Die Reduktion in prozyklischen Parasiten schwĂ€cht jedoch die Fitness bei einem Hitzeschock. Mehrere mRNAs, die fĂŒr Hitzeschockproteine und Chaperone kodieren, koprĂ€zipitieren mit ZC3H11 und das Protein stabilisiert kĂŒnstlich gebundene mRNAs. AuĂerdem verringert ZC3H11-RNAi die HSP70-mRNA Menge und StabilitĂ€t. Dieser Effekt wird von der 3âUTR vermittelt, genauer gesagt von einer Region mit AUU-Wiederholungen. Dieses AUU-Motif war auch in den 3â UTRs der gebundenen Transkripte ĂŒberreprĂ€sentiert. Mittels KoimmunoprĂ€zipitation wurde ein MKT1-Homolog als Proteinbindepartner identifiziert. Da Mkt1p in Hefe mit dem PABP Bindeprotein, PBP1, interagiert, könnte die Stabilisation der ZC3H11- gebundenen Transkripte ĂŒber diese Interaktion vermittelt werden. Meine Ergebnisse legen nahe, dass ZC3H11 ein wichtiger posttranskriptionaler Regulator der trypanosomalen Hitzeschockantwort ist
Integrative analysis of the Trypanosoma brucei gene expression cascade predicts differential regulation of mRNA processing and unusual control of ribosomal protein expression
Background: Trypanosoma brucei is a unicellular parasite which multiplies in mammals (bloodstream form) and Tsetse flies (procyclic form). Trypanosome RNA polymerase II transcription is polycistronic, individual mRNAs being excised by trans splicing and polyadenylation. We previously made detailed measurements of mRNA half-lives in bloodstream and procyclic forms, and developed a mathematical model of gene expression for bloodstream forms. At the whole transcriptome level, many bloodstream-form mRNAs were less abundant than was predicted by the model. Results: We refined the published mathematical model and extended it to the procyclic form. We used the model, together with known mRNA half-lives, to predict the abundances of individual mRNAs, assuming rapid, unregulated mRNA processing; then we compared the results with measured mRNA abundances. Remarkably, the abundances of most mRNAs in procyclic forms are predicted quite well by the model, being largely explained by variations in mRNA decay rates and length. In bloodstream forms substantially more mRNAs are less abundant than predicted. We list mRNAs that are likely to show particularly slow or inefficient processing, either in both forms or with developmental regulation. We also measured ribosome occupancies of all mRNAs in trypanosomes grown in the same conditions as were used to measure mRNA turnover. In procyclic forms there was a weak positive correlation between ribosome density and mRNA half-life, suggesting cross-talk between translation and mRNA decay; ribosome density was related to the proportion of the mRNA on polysomes, indicating control of translation initiation. Ribosomal protein mRNAs in procyclics appeared to be exceptionally rapidly processed but poorly translated. Conclusions: Levels of mRNAs in procyclic form trypanosomes are determined mainly by length and mRNA decay, with some control of precursor processing. In bloodstream forms variations in nuclear events play a larger role in transcriptome regulation, suggesting aquisition of new control mechanisms during adaptation to mammalian parasitism
SUMOylation in Trypanosoma brucei
Small ubiquitin like modifier (SUMO) proteins are involved in many processes in eukaryotes. We here show that Trypanosoma brucei SUMO (Tb927.5.3210) modifies many proteins. The levels of SUMOylation were unaffected by temperature changes but were increased by severe oxidative stress. We obtained evidence that trypanosome homologues of the SUMO conjugating enzyme Ubc9 (Tb927.2.2460) and the SUMO-specific protease SENP (Tb927.9.2220) are involved in SUMOylation and SUMO removal, respectively
The suppressive cap-binding complex factor 4EIP is required for normal differentiation
Trypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low messenger RNA (mRNA) content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA, but while tethered EIF4E1 suppresses only when 4EIP is present, suppression by tethered 4EIP does not require the interaction with EIF4E1. In growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Bloodstream-or procyclic-form trypanosomes lacking 4EIP have only a marginal growth disadvantage. Bloodstream forms without 4EIP are, however, defective in translation suppression during stumpy-form differentiation and cannot subsequently convert to growing procyclic forms. Intriguingly, the differentiation defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. In contrast, bloodstream forms lacking EIF4E1 have a growth defect, stumpy formation seems normal, but they appear unable to grow as procyclic forms. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP contributes to translation suppression during differentiation to the stumpy form
Disruption of the RNA exosome reveals the hidden face of the malaria parasite transcriptome
International audienceAntisense transcription emerges as a key regulator of important biological processes in the human malaria parasite Plasmodium falciparum. RNA-processing factors, however, remain poorly characterized in this pathogen. Here, we purified the multiprotein RNA exosome complex of malaria parasites by affinity chromatography, using HA-tagged PfRrp4 and PfDis3 as the ligands. Seven distinct core exosome subunits (PfRrp41, PfMtr3, PfRrp42, PfRrp45, PfRrp4, PfRrp40, PfCsl4) and two exoribonuclease proteins PfRrp6 and PfDis3 are identified by mass spectrometry. Western blot analysis detects Dis3 and Rrp4 predominantly in the cytoplasmic fraction during asexual blood stage development. An inducible gene knock out of the PfDis3 subunit reveals the upregulation of structural and coding RNA, but the vast majority belongs to antisense RNA. Furthermore, we detect numerous types of cryptic unstable transcripts (CUTs) linked to virulence gene families including antisense RNA in the rif gene family. Our work highlights the limitations of steady-state RNA analysis to predict transcriptional activity and link the RNA surveillance machinery directly with post-transcriptional control and gene expression in malaria parasites
Disruption of the RNA exosome reveals the hidden face of the malaria parasite transcriptome
Post-Transcriptional Regulation of the Trypanosome Heat Shock Response by a Zinc Finger Protein
<div><p>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, <i>Trypanosoma brucei</i> heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of <i>HSP70</i> mRNA turnover. We here show that the <i>T. brucei</i> 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 <i>HSP70</i> mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3âČ-untranslated region. ZC3H11 bound preferentially to AUU repeats <i>in vitro</i>, and ZC3H11 regulation of <i>HSP70</i> 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.</p> </div
Expression of ZC3H11 is induced by stress.
<p><b>A.</b> Alignment of ZC3H11 with members of the Tis11 family of several species. The conserved amino acid signature (marked in red) precedes the zinc finger domain (cysteines and histidine shaded in blue). Conserved residues are shaded in yellow and chemically similar ones in orange. Amino acids at the positions marked on the top with # and * are involved in RNA binding by hydrogen bonds or base stacking respectively <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003286#ppat.1003286-Hudson1" target="_blank">[31]</a>. <b>B.</b> Expression of <i>in situ</i> tagged V5-ZC3H11 in procyclic cells under different conditions. The incubation temperature is shown above along with the duration of the incubation. Treatment with puromycin was for one hour (lanes 11â13). 10<sup>7</sup>cells were loaded per lane; detection was with anti-V5 antibody and with anti-aldolase as loading control. <b>C.</b> Expression of <i>in situ</i> tagged V5-ZC3H11 in bloodstream-form trypanosomes under different conditions. Details are as for (B) <b>D.</b> Effect of proteasome inhibition by treatment with MG132 (10 ”g/ml, 1 h) on V5-ZC3H11 expression. <b>E.</b> ZC3H11-myc is phosphorylated. Extracts from 5Ă10<sup>6</sup> cells were incubated with lambda phosphatase, in the presence or absence of inhibitors <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003286#ppat.1003286-Benz1" target="_blank">[64]</a>. Both the full-length protein (upper panel) and the N-terminal fragment of ZC3H11 (first 128 amino acids; lower panel) are phosphorylated. <b>F.</b> ZC3H11-TAP is localized in the cytoplasm. TAP-tagged protein was detected by immunofluorescence. DAPI - DNA stain, detecting nucleus and kinetoplast (mitochondrial DNA); DIC - differential interference contrast.</p
ZC3H11 depletion kills bloodstream forms but not procyclics.
<p><b>A.</b> Effect of <i>ZC3H11</i> RNAi on growth of bloodstream forms. Tetracycline was added at day 0, and cells were diluted as required. Cumulative growth curves are shown. Results are for four independent cell lines, immediately after isolation. Results from two experiments are included. In experiment one, three lines were tested over three days of tetracycline treatment. In experiment 2, the same lines plus another were examined for two days with tetracycline. The control is from experiment 1, and shows pooled results for 11 trypanosome lines with RNAi targeting a non-essential gene, growth without tetracycline for three days. Results are shown as arithmetic mean ± standard deviation. <b>B.</b> Effect of <i>ZC3H11</i> RNAi on growth of procyclic forms, cumulative growth curve. Results are shown from triplicate measurements from a line with stem-loop RNAi, as arithmetic mean ± standard deviation. The error bars are not visible because they are smaller than the symbols.</p
ZC3H11 is required for recovery of procyclic trypanosomes from heat shock.
<p><b>A.</b> Effects of incubating procyclic cells at 37°C, with or without <i>ZC3H11</i> RNAi. Cells were diluted to 1Ă10<sup>6</sup>/ml as required to avoid densities above 5Ă10<sup>6</sup>/ml. Results are for three experiments, expressed as arithmetic mean ± standard deviation. If no error bars are visible, they were smaller than the symbols. <b>B.</b> Recovery of procyclic cells with or without <i>ZC3H11</i> RNAi from a one-hour heat shock at 41°C. On day 0, cells were incubated at 41°C for one hour then returned to 27°C. They were diluted on subsequent days as required. Cells with no heat shock served as controls. Results are for three experiments, expressed as arithmetic mean ± standard deviation. <b>C.</b> Analysis of DNA content of procyclic trypanosomes recovering from heat shock, with and without <i>ZC3H11</i> RNAi by FACS. The growth curve for these particular cells was included in those used to make the graph B. <b>D.</b> Effect of <i>ZC3H11</i> RNAi on heat-shock protein synthesis in procyclic forms. Cells were shocked at 41°C for one hour, then pulsed with [<sup>35</sup>S]-methionine. Labelled proteins were separated by SDS-PAGE and detected by autoradiography. <b>E.</b> Effect of heat shock on total mRNA abundance. RNA was prepared and analysed by Northern blotting after a one-hour heat shock at 41°C. The total mRNA was detected by hybridisation with the spliced leader (present at the 5âČ-end of each mRNA). The numbers below show the total signal from spliced mRNA (without the <i>SLRNA</i> itself), normalized to ribosomal RNA (measured by methylene blue staining of the blot). <b>F. </b><i>ZC3H11</i> RNAi abolishes the specific stabilization of <i>HSP70</i> mRNA upon heat shock. Northern blots are shown, as in (E) but also including a one-hour incubation at 37°C. The quantitation, normalised to the <i>7SL</i> RNA signal, is the average of 2 independent experiments. <b>G.</b> The UAU-rich region of the <i>HSP70</i> 3âČ-UTR is able to stablise a reporter mRNA after heat shock. Procyclic trypanosomes expressing <i>CAT</i> reporters with segments of the <i>HSP70</i> 3âČ-UTR were incubated at 41°C for one hour, then RNA was prepared and analysed by Northern blotting. Constructs contained either full-length <i>HSP70</i> 3âČ-UTR or the fragments âdelAUâ or âonlyAUâ (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003286#ppat-1003286-g004" target="_blank">Figure 4</a>) and the reporter with full-length actin 3âČ-UTR served as control. For each reporter, <i>CAT</i> RNA levels were quantified relative to the non-heat shock level and normalized using the <i>7SL</i> signal.</p