Interactions of the Trypanosoma brucei brucei zinc-finger-domain protein ZC3H28

Fig. 1.. ZC3H28 is essential for the normal growth of trypanosome bloodstream forms, and it increases the abundance of an attached mRNA. (A) Structure of ZC3H28, drawn to scale. The positions of the zinc finger domain, poly(His) and poly(Gln) sequences are indicated. (B) Effects of tethering lambdaN-tagged ZC3H28 on a boxB-bearing CAT reporter mRNA. This panel shows the expression of the CAT mRNA (2 different exposures), with a beta-tubulin control, in the upper four panels; the lower two panels show the expression of the lambdaN-tagged protein, which has a myc tag at the C-terminus. For the Northern blots, the first two lanes after the markers are cells with no CAT gene (WT) and cells with the reporter but no inducible lambdaN-tagged protein. These lanes are not present in the lowest two (Western blot) panels. (C) Effects of tethering on CAT activity and mRNA. For each cell line in (B), the amount of CAT mRNA or CAT activity in the presence of tetracycline was divided by the mRNA level or CAT activity in the absence of tetracycline. The symbols correspond to those in (B). (D). Effect of ZC3H28 depletion on cell numbers over 5 days. Expression of a dsRNA corresponding to part of the…

Fig. 2.. Protein interactions of ZC3H28. (A) Interactions of ZC3H28 in the yeast two-hybrid assay. Saccharomyces cerevisiae AH109 was transformed with combinations of plasmids used as bait (pBD, DNA binding domain) and prey (pAD, transcription activating domain). Growth on media lacking adenine, histidine, leucine and tryptophan (SD-WLAH) indicates protein interactions while growth in media lacking leucine and tryptophan (SD-WL) controls for expression of the bait and prey. The interaction between SV40 large T-antigen (T) and p53 (first row, last column) serves as positive control and the combination of LaminC and SV40 large T-antigen (first row, third column) serves as negative control. PBP1 and XAC fused with the DNA-binding domain both activate when expressed alone (Nascimento et al., 2020), therefore they are used here only as activation domain fusions, which give appropriate negative control results (Nascimento et al., 2020). (B) Volcano plot of proteins interacting with TAP-ZC3H28, compared with GFP-TAP which served as the negative control. The figure was generated using Perseus software (Tyanova et al., 2016). The y-axis is the negative log10 of the P value for significant…

Fig. 3.. Interactions of ZC3H28 with mRNA. (A) Characteristics of mRNAs that were at least 3-fold enriched with TAP-ZC3H28 relative to the unbound fraction. Results for the whole transcriptome are shown for comparison. All results in this figure are for the set of unique genes, to avoid over-counting repeated genes. In these blots, the boxes extend from the 25th to 75th percentiles, and the whiskers to 1.5x the inter-quartile length; dots are outliers. The notches are an indicator of variability, ±1.57x the inter-quartile range divided by the square root of the number of data points. Median and mean values are shown underneath the labels. (B) Plot for the whole dataset: enrichment with ZC3H28 (y-axis) relative to mRNA length (x-axis). (C) The set of unique genes was placed in functional categories (Supplementary Table S4) and binding to ZC3H28 was plotted for each category. The black dotted line indicates equal distribution in both fractions. The magenta dotted line is the median and the green background box indicates the range from the 25th to the 75th percentiles. (D) Motifs enriched in the 3′-UTRs of ZC3H28-bound mRNAs relative to size-matched controls showing less than 1-fold average…

Fig. 4.. Effects of ZC3H28 depletion on the transcriptome. (A) Amounts of ZC3H28 mRNA with and without tetracycline treatment. The amounts of RNA (reads per million reads from the coding region) were all compared with the arithmetic mean for untreated cells (time = 0). Results for the two control sets are colour-coded as in Fig. 4B. (B) Principal component analysis. Samples without induction are in black and grey; others are colour-coded according to the time with tetracycline. All results in this figure are for the set of unique genes, to avoid over-counting repeated genes. (C) The set of unique genes was placed in functional categories (Supplementary Table S4) and the effects of ZC3H28 depletion (14 h RNAi) were plotted for each category. The green background shows the range between 25th and 75th percentiles for the total dataset and the magenta dotted line is the median. The category with a median above the 75th percentile is coloured in pink while those with medians below the 25th percentile are in blue. (D) The effect of RNAi (y-axis) was plotted against the annotated mRNA length (x-axis). Only mRNAs with two annotated untranslated regions were included. Correlation coefficients were…

Fig. 5.. ZC3H28 and translation. In A–C, each dot represents a single mRNA coding region. (A) Binding of the mRNA to ZC3H28 (data from Supplementary Table S2) is on the x-axis. The y-axis shows the number of ribosomes per kilobase of coding region, taken from Antwi et al. (2016). Ribosomal protein mRNAs are highlighted in orange. (B) The log2 of annotated 3′-UTR length is on the x-axis and the ribosome density is on the y-axis. mRNAs that were at least 3-fold enriched with ZC3H28 in all 3 replicate pull-downs are highlighted in green. (C) Binding of mRNAs to ZC3H28 is on the x-axis (Supplementary Table S2). (Note that these results, like all others in this paper, are from bloodstream forms.) The y-axis shows the proportion of the mRNA that is found in granules after starvation of procyclic forms (Fritz et al., 2015). (D) Box plots showing properties of the mRNAs that were enriched with ZC3H28: ribosome density (as in A); half-lives (Fadda et al., 2014); and the codon adaptation index (de Freitas Nascimento et al., 2018). Student t-test results for the log-transformed data are shown on each plot.