Stress susceptibility in Trypanosoma brucei lacking the RNA-binding protein ZC3H30

Figures

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  Fig 1. Tethering of ZC3H30 to a reporter mRNA represses expression. (A) Protein and reporter. a) Diagram of ZC3H30 protein, to scale, with zinc fingers in dark magenta and predicted disordered regions in orange. The zinc fingers are shown with the Cys and His residues in colour. b) lambdaN-ZC3H30-myc protein; c) The CAT-boxB mRNA. (B). RNA and protein from cells expressing CAT reporters with or without BoxB and λN-ZC3H30-myc. Tetracycline (100ng/mL) was added for 24h prior to analysis. For CAT mRNA quantification the rRNA bands (stained with methylene blue) were used as the loading control. (C) Quantification of CAT activity and CAT mRNA for 2 independent experiments, each with two different clones each with or without boxB (4 data points per condition). Results are expressed relative to cells with no lambdaN-ZC3H30-myc, as arithmentic mean and standard deviation for the four measurements.

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  Fig 2. Cell lines used in this paper. Each frame represents a trypanosome nucleus with the relevant loci shown. The different panels A, B, C, D and E are labelled on the Figure and are described in the text. The diagrams are not to scale. Evidence for various lines is shown in S2 Fig and S3 Fig.

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  Fig 3. Trypanosomes lacking ZC3H30 are sensitive to stress. Each panel shows cell numbers after application of a physical or chemical stress. Cells were incubated without selective drugs and were all in exponential growth at the start of the experiments. The DKO+ cell cultures included tetracycline for three days prior to, and for all of the time during, the experiments. Stresses were applied, then the cells were centrifuged and resuspended in fresh medium. In some experiments cells were diluted as necessary. Additional stresses are shown in S4A–S4C Fig. In individual experiments, the replicates for DKO and DKO+ were for independent cultures, usually of different clones. When experiments were repeated, 2 clones were usually used. (A) Bloodstream forms, 1 h at 42°C. This is a single experiment and individual replicates are shown. For a similar experiment at 42°C see S4A Fig. (B) Bloodstream forms, 1 h with 0.1 mM DTT. This is a single experiment and individual replicates are shown. (C) Procyclic forms, 1 h at 37°C. This is a single experiment with 3x WT, 3x DKO (each in triplicate), and 3x DKO+. Standard deviations are not visible beneath the data points. (D) Procyclic forms, 1 h at…

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  Fig 4. Protein synthesis. Trypanosomes were subjected to heat shock for 30 min in medium lacking methionine, then [35S] methionine was added for 30 min. Cells were washed once then the pellets were boiled in sample buffer and subjected to SDS-PAGE followed by autoradiography. (A) shows results for procyclic forms: the positions at which HSP83 and HSP70 may migrate are indicated. (B) shows results from bloodstream forms. The identity of the prominent band (marked ?) is not known but from its size, it might be variant surface glycoprotein.

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  Fig 5. HSP70 mRNA in bloodstream forms lacking ZC3H30. RNA was isolated from bloodstream-form trypanosomes with and without a 1h 41°C heat shock. HSP70 mRNA ws detected with a radioactive probe and the signal was quantitated relative to the SRP RNA. The lower panel shows quantitation for three biological replicates, as indicated on the graph. The signal for WT cells at 27°C was set at 1.0. Additional replicates are shown in S4F and S4G Fig.

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  Fig 6. Transcriptomes of ZC3H30 DKO and DKO+ procyclic forms are similar. (A) Principal component analysis for ZC3H30 DKO and DKO+ cells. The key is on the Figure. Note that 90% of the variance is on the x-axis. The two replicates are numbered. (B) Effect of heat shock on transcriptomes. Each point represents one gene. The log2 fold change for DKO+ cells is on the x-axis and the log2 fold change for DKO cells is on the y-axis.

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  Fig 7. The association of mRNAs with ZC3H30 is weakly correlated with mRNA length. (A) The average enrichment of each mRNA (eluate RPM / input RPM) was plotted (y-axis) relative to the annotated mRNA length (x-axis). Both are on a log2 scale. Ribosomal protein mRNAs are in orange. Note that some annotated mRNA lengths are too short, usually due to underestimation of 3'-untranslated regions. (B) The lengths of mRNAs that were reproducibly enriched in the ZC3H30 pull-down (all ratios >2) and those that reproducibly were not enriched (all ratios less than 1) are shown as box plots. For gene lists see S3 Table, sheet 3. (C) Relationship between RNA binding by ZC3H30 and ZC3H32. The x-axis shows binding to ZC3H32 (average of three bound/eluate) and the y-axis shows the average of bound/DKO+ (total) for ZC3H30, both on a log2 scale. The cyan spots represent mRNAs that were reproducibly enriched in the ZC3H30 pull-down and for which the enrichment ratio was at least 2-fold higher with ZC3H30 than with ZC3H32.

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  Fig 8. ZC3H30 interacts with the protein encoded by Tb927.8.3820. Proteins were immunoprecipitated from cell lines expressing V5-ZC3H30 and/or YFP-927.8.3820, then analysed by Western blotting with the indicated antibodies. Ribosomal protein S9 served as a negative control. I = Input; Un = Unbound; B = Bound. Left panel: immunoprecipitation with anti-V5 (for V5-ZC3H30); right panel: immunoprecipitation with anti-GFP (for YFP-927.8.3820). For RNase treatment, the lysis and wash buffers contained 200 μg/mL of RNase A. Both anti-V5 and anti-GFP antibodies detected degradation products in addition to the full-length fusion proteins (S8 Fig); here, only the full-length bands are shown.

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  Fig 9. ZC3H30 is in a granule fraction after heat shock. A) In procyclic trypanosomes grown at 27°C, ZC3H30 is in the cytosolic fraction. Trypanosomes were disrupted using silicon carbide, and fractionated by centrifugation. 5 x 106 cell-equivalents were loaded on each lane of an SDS-PAGE gel and the proteins were detected by Western blotting. Tot: total lysate; C: cytosol: Sup: detergent-soluble organellar proteins; Pel: IGEPAL-insoluble pellet. TR is trypanothione reductase, a cytosolic protein, and XRND is in the nucleus. (B) ZC3H30 moves to the granule fraction after heat shock. Trypanosomes expressing V5-ZC3H30, with or without a 1h heat shock at 41°C, were fractionated into various soluble and granular fractions, using the cytoskeleton as a molecular sieve to trap stress granules. T: Unfractionated total cell lysate; SN1-3: soluble supernatants; SN4: molecules trapped inside cytoskeletons but not sedimented at 20000g (10min); G: sedimentable granules. Proteins were detected from equal proportions of each fraction. DHH1 and SCD6 are stress granule markers and trypanothione reductase (TR) is in the soluble fraction. (C) Loss of GFP-Tb927.8.3820 after 24h RNAi induction in procyclic…