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The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei - PubMed

The post-transcriptional trans-acting regulator, TbZFP3, co-ordinates transmission-stage enriched mRNAs in Trypanosoma brucei

Pegine B Walrad et al. Nucleic Acids Res. 2012 Apr.

Abstract

Post-transcriptional gene regulation is essential to eukaryotic development. This is particularly emphasized in trypanosome parasites where genes are co-transcribed in polycistronic arrays but not necessarily co-regulated. The small CCCH protein, TbZFP3, has been identified as a trans-acting post-transcriptional regulator of Procyclin surface antigen expression in Trypanosoma brucei. To investigate the wider role of TbZFP3 in parasite transmission, a global analysis of associating transcripts was carried out. Examination of a subset of the selected transcripts revealed their increased abundance through mRNA stabilization upon TbZFP3 ectopic overexpression, dependent upon the integrity of the CCCH zinc finger domain. Reporter assays demonstrated that this regulation was mediated through 3'-UTR sequences for two target transcripts. Global developmental expression profiling of the cohort of TbZFP3-selected transcripts revealed their significant enrichment in transmissible stumpy forms of the parasite. This analysis of the specific mRNAs selected by the TbZFP3mRNP provides evidence for a developmental regulon with the potential to co-ordinate genes important in parasite transmission.

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Figures

Figure 1.
Figure 1.

TbZFP3 selects a specific subset of mRNAs. (A) Schematic representation of the selection of TbZFP3mRNP associating transcripts. Cell extracts were immunoprecipitated (IP) using TbZFP3-specific anti-peptide antibody, specificity being confirmed using a parallel IP in the presence of blocking peptide. Four separate α-TbZFP3 IPs were initially validated for their specificity of selection, and the RNA isolated from the pooled material. PolyA+ mRNA was then used to generate cDNA and the population subjected to Illumina Digital Tag sequencing, with the resulting reads compared with unselected procyclic mRNA to identify enriched transcripts in the TbZFP3-selected pool. (B) RT-PCR analysis of TbZFP3-associated mRNAs isolated in the presence or absence of a TbZFP3 antibody peptide block. The relative enrichment of the individual transcripts is shown when the combined ORF and UTR data sets were considered. A negative control transcript (‘nmd3’) was not enriched in the TbZFP3 selected mRNA pool, despite its detection in total RNA.

Figure 2.
Figure 2.

TbZFP3-selected transcripts are specifically upregulated by ectopic TbZFP3, dependent upon the predicted RNA binding domain. (A) Expression of ectopic TbZFP3-TY and TbZFP3ccAh-TY in cells induced, or not, with tetracycline. The expressed proteins were detected using BB2 antibody (‘anti-TY’), specific for the incorporated Ty1 epitope tag in each protein. A schematic representation of the ectopically expressed proteins is shown to the right. (B) Detection of the transcript abundance for three target mRNAs selected by TbZFP3. The abundance of each transcript is shown when the following cells lines were induced with tetracycline: lanes 1 and 2, parental procyclic forms; lanes 3 and 4, ectopic TbZFP3-TY; lanes 5 and 6, ectopic TbZFP3 CCAH-TY. (C) As in B, for three negative control transcripts that are not selected by TbZFP3. Note that a small increase in actin transcript was observed upon tetracycline induction in this experiment, but that this was not clearly reproducible between independent experiments.

Figure 3.
Figure 3.

Cell lines created to investigate the regulation by TbZFP3 of target mRNA 3′-UTRs. (A) Chloramphenicol acetyltransferase (CAT) Reporter strategy. Intergenic sequences downstream of Rbp23, SmB and GrpE coding regions were inserted individually into a reporter construct adjacent to the CAT coding region. In each case, the inserted sequence length is indicated in the Table in Panel A, as is the predicted endogenous 3′-UTR length for each transcript. Each reporter was transfected into parental ‘wild-type’ procyclic form cells and those capable of the inducible ectopic expression of untagged TbZFP3 (ZFP3-No Tag), TbZFP3-TY or ZFP3 CCAH-TY. The respective cells lines generated in each case are summarized in the chart. ND = not done. (B) Western blots of inducible ectopic TbZFP3 expression in lines transfected with the respective CAT reporter constructs. Note that for the CAT-SmB/ZFP3-No tag cell line, the level of ectopic TbZFP3 expression was equivalent in uninduced and induced cells. Hence, for this cell line expression comparisons of the CAT reporter were made with the parental CAT-SmB transfected line. Relative loading is indicated by the Coomassie stained gel images in each case. ND =Not done.

Figure 4.
Figure 4.

The 3′-UTR's of SmB and Rbp23 are sufficient for regulation by TbZFP3. (A) Ectopic overexpression of TbZFP3-TY (left panel) or untagged TbZFP3 (right panel) increases levels of CAT-Rbp23 and CAT-SmB, matching the effect on the endogenous mRNAs. This upregulation is dependent upon the CCCH predicted RNA-binding domain of TbZFP3. In contrast, the CAT-GrpE transcript was not significantly changed in response to ectopic TbZFP3 expression, unlike endogenous GrpE mRNA (

Supplementary Figure S3

). In each case, a northern blot is shown detecting CAT mRNA. Relative loading is indicated by EtBr stained rRNA. The relative fold increases for each reporter mRNA, based on their chemifluorescent signal, are highlighted above the lane numbers used for comparison, these being derived from an independent experiment. For the CAT-SmB reporter in the ‘TbZFP3 No Tag’ line, the leakiness of ectopic protein expression necessitated comparison with the PCF control line (lanes 3 and 4). (B) Corresponding CAT protein levels are not significantly upregulated in response to ectopic TbZFP3-TY (left-hand panel) or TbZFP3 (right-hand panel) overexpression. In each case CAT protein levels were determined by CAT-ELISA assay and normalized to the uninduced ZFP3-TY line (left-hand panel) or PCF (without tetracycline) containing the reporter constructs, but no ectopic TbZFP3.

Figure 5.
Figure 5.

TbZFP3 stabilizes associating transcripts. (A) mRNA quantitation derived from triplicate qRT-PCR assays of TbSm15K mRNA (not selected by TbZFP3) at time points after treatment with actinomycin D. Samples were derived from cells induced, or not, to ectopically express TbZFP3, with values expressed as a percentage of the starting abundance. Using a GLM with % of starting transcript as the response variable, the presence/absence of TET was not a significant factor (F1 = 0.10, P = 0.759). (B) As in A, but assayed for the abundance of the SmB (selected by TbZFP3). The steady state abundance of SmB is enhanced upon TbZFP3 ectopic expression, hence comparisons between induced and uninduced samples are expressed as a percentage of the abundance at time = 0 h in each case. In this case, the presence/absence of TET was a significant factor (F1 = 5.62, P = 0.050).

Figure 6.
Figure 6.

TbZFP3-associated transcripts are enriched in stumpy forms. (A) EdgeR representation of the ratio of transcript concentrations in stumpy (ST) versus slender (SL) stage parasites of the total transcript pool (light grey) versus the 100 most enriched transcripts in the TbZFP3-selected pool (black). The distribution of the top 100 enriched transcripts in the TbZFP3-RIP selected material with respect to their relative expression in slender forms (<0) or stumpy forms (>0) is shown. The selected transcripts are predominantly (67%) in the stumpy-enriched cohort. (B) Relative expression of the top 100, top 200, top 300, top 400 and top 500 (‘100 ZFRIP’, etc.) TbZFP3-RIP selected transcripts ranked in order of their enrichment in slender or stumpy forms with respect to unselected material. The selected transcripts are more predominant amongst those transcripts enriched in stumpy forms (‘Up in stumpy’), and less predominant in those transcripts enriched in slender forms (‘Up in slender’). The relative enrichment after TbZFP3-RIP correlates with the extent of enrichment in stumpy forms (one-way ANOVA analysis, F11 = 6.18, P = 0.023) although the correlation was less significant for transcripts >2-fold enriched in stumpy forms (F11 = 3.27, P = 0.091). This inversely correlates with the extent of enrichment in slender forms (F11 = 6.18, P = 0.023) and for transcripts >2-fold enriched in slender forms compared to stumpy (F11 = 14.93, P = 0.002). Post hoc Tukey's tests indicate that the Top100, Top200 and Total categories are the important factors in those ANOVA that are significant. Data represent the analysis of two independent pleomorphic slender and stumpy samples derived from different strains of T. brucei. Error bars for each transcript group are shown.

Figure 7.
Figure 7.

GO analysis of the TbZFP3mRNP associating transcripts. Representation of the GO terms enriched in the TbZFP3-RIP selected transcripts (A) versus the relative abundance of the same GO classes in the total (B) or stumpy enriched (C) gene set. P-values were calculated using the GOStat package (40).

Figure 8.
Figure 8.

TbZFP3 association in cytoplasmic P granules. Panels (A) and (B) show Procyclic form cells engineered to express TbSCD6-YFP were incubated in SDM-79 (A) or PBS for 2 h. (B) and then the location of TbSCD6 YFP (green) or TbZFP3 (red) determined by immunofluorescence. The third panel in each case represents the image derived from DAPI staining to visualize the parasite nucleus and kinetoplast, whereas the fourth panel shows a merge of the TbSCD6-YFP, TbZFP3 and DAPI panels. After PBS starvation the TbSCD6-YFP and TbZFP3 signals colocalize in discrete cytoplasmic granules or P Bodies (arrowed). Panels (C) and (D) show the signal generated when slender (SL) and stumpy (ST) cells are probed for the location of ep procyclin mRNA (panel C, anti-sense probe, panel D, sense probe) and TbZFP3, with DAPI staining of the cells being shown in the third panel and a merge of all staining in the fourth panel. The TbZFP3 signals do not localize into enlarged P bodies, unlike serum starved procyclic forms. However, some concentration, in particular regions is observed (arrowed).

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