Fig 1.
Transcriptome-wide polysome profiling upon heat shock of procyclic forms.
(A) Representative polysome profiling of procyclic cells grown at 27°C (normal culture conditions) or heat-shocked for 1h at 39°C. Fractions were pooled as described and used for the analysis (F: free, S: ribosomal subunits, L: monosomes and light polysomes, H: heavy polysomes). (B) For normalization, RNA prepared from four pools was analysed by Northern blotting. The total signal from the spliced leader RNA (present at the 5’-end of each trypanosomal mRNA) was used to calculate the distribution of total mRNA in four pools (mean, n = 2). The strong spot below 200nt is the spliced leader precursor RNA (SLRNA) and the smear above is trans spliced mRNA. (C) Quantitation of Northerns as in B, showing the percentage of total signal in each sucrose gradient fraction (2 biological replicates used for sequencing).
Fig 2.
Analysis of mRNAs in polysome gradients.
(A) For each gene in the "unique gene" set, the percentage of the mRNA in polysomes (heavy and light combined) at 27°C (cyan) and after 1h at 39°C (pink) was calculated. The results for all of these mRNAs ("All") are displayed on the left as box plots. We then examined separately mRNAs that were reproducibly at least 3x enriched in a ZC3H11 pull-down (ZC3H11 bind 3x); and mRNAs encoding ribosomal proteins (Ribprot). Boxes indicate the 25th to 75 percentiles with central median; notches represent the 95% confidence intervals for the medians; dotted lines show 1.5x the inter-quartile range and circles are outliers. (B) Effect of a 1h 39°C treatment on polysome loading and abundance for mRNAs from each gene in the list of unique genes. The x-axis shows, for each gene, the amount of mRNA after 1h at 39°C divided by the amount of mRNA at 27°C, on a log2 scale. The y-axis shows the percentage of that mRNA in the polysomes after 1h at 39°C divided by the amount of mRNA at 27°C, also on a log2 scale. The line is a regression line with the correlation coefficient (R) also displayed. Ribosomal protein mRNAs are in orange (Ribprot) and mRNAs that bind to ZC3H11 are in cyan. (C) Functional classes of proteins encoded by mRNAs that shifted away from the polysomal fraction and into the free and monosomal fractions after heat shock. For these mRNAs the proportion of the mRNA in polysomes (heavy and light combined) decreased by 1.25x or more after heat shock. The results of a Fisher test for enrichment of functional classes are shown; if no result is shown there was no significant enrichment. (D) Functional classes of proteins encoded by mRNAs that shifted towards the polysomal fraction after heat shock. "Increase" means that the proportion of the corresponding mRNA in polysomes increased by 2x or more. The results of a Fisher test for significant enrichment of functional classes are shown.
Fig 3.
Heat shock induces some developmentally regulated mRNAs.
The Venn diagrams are constructed such that all areas are proportional to the numbers of genes included. Ratios for salivary gland transcriptomes were calculated using the raw data from [28] and [29]. Categories are: (i) 246 mRNAs that increased in abundance after heat shock (Padj <0.01 and 39/27 >2, pale cyan ellipse); (ii) 1775 mRNAs that are >2x more abundant in salivary gland (SG) trypanosomes than in procyclic form (PC) trypanosomes (SG>2XPC, yellow); (iii) 2509 mRNAs that are >2x more abundant in salivary gland (SG) trypanosomes than in bloodstream form (BS) trypanosomes (SG>2XBS, orange); (iv) 985 mRNAs that are >2x more abundant in bloodstream form (BS) trypanosomes than in procyclic form (PC) trypanosomes [29] (BS>2xPC, pink); (v) mRNAs that decreased in abundance after heat shock (Padj <0.01 and 39/27 <0.5, labelled as 27>39, violet); (vi) 582 mRNAs that are >2x less abundant in salivary gland (SG) trypanosomes than in procyclic form (PC) trypanosomes (PC>2xSG, dark green), and (vii) 872 mRNAs that are >2x less abundant in bloodstream form (BS) trypanosomes than in procyclic form (PC) trypanosomes [29] (PC>2xBS, pale green). (A) The mRNAs that increased in abundance after heat shock (cyan) were compared with mRNAs that are more abundant in salivary gland trypanosomes than in procyclic form trypanosomes (yellow), and mRNAs that are more abundant in salivary gland trypanosomes than in bloodstream form trypanosomes (orange). The overlap between the last two categories indicates the 906 mRNAs that show the highest abundance in salivary gland trypanosomes. Of these, 81 were also increased by heat shock. The probability that the overlaps would arise by chance (Fisher test) are indicated using blue arrows. The probability of the overlap between 39>27 and SG>2xBS was 6 x 10−27; the probability of the overlap between 39>27 and SG>2xBS was 6 x 10−5; and the probability of the overlap between all three was 9 x 10−16. (B) The mRNAs that increased in abundance after heat shock (cyan) were compared with mRNAs that are more abundant in salivary gland trypanosomes than in procyclic form trypanosomes (yellow), and with mRNAs that are abundant in bloodstream form trypanosomes than in procyclic form trypanosomes (pink). Fischer tests are for the overlaps between 39>27 and either BS>2xPC or SG>2XPC. (C) The mRNAs that decreased in abundance after heat shock (violet) were compared with mRNAs that are less abundant in salivary gland trypanosomes than in procyclic form trypanosomes (dark green), and with mRNAs that are less abundant in bloodstream form (BS) trypanosomes than in procyclic form trypanosomes (pale green).
Table 1.
Selected mRNAs that show increased translation or abundance at 39°C.
For increased abundance the threshold was a 2-fold relative increase, with adjusted P value of less than 0.01 in DESeq. The ratios have not been corrected for the mRNA content of heat-shocked cells (57% relative to 27°C). Additional mRNAs showed a minimum (min) increase of 2-fold in the percentage of that RNA that was in polysomes. RNA abundance ratios for Droll et al. [16] are indicated by "DD". Salivary gland transcriptomes (SG) and ratios were calculated from the raw data from [28] and ratios for bloodstream form (BS) versus procyclic form (PC) are from [29]. Ratios are shown to 2 significant figures. HS gran: percentage in heat shock granules at 41°C. na: no data available. "PPCTI" = peptidyl-prolyl cis-trans isomerase; "Protein phos" = protein with protein phosphatase domain. A complete list of ORFs that were regulated at the level of total RNA in at least 2 experiments is in S2 Table, sheet 11.
Fig 4.
Confirmation of results for individual mRNAs.
(A) Extracts from cells grown only at 27°C, or subjected to a 39°C heat shock, were separated on sucrose gradients: the absorbance profiles of two typical gradients are shown. (B) Northern blots of chosen mRNAs (gene IDs on the right) that moved from the non-polysomal to the polysomal fraction after heat shock. A typical rRNA profile is shown as a control. In vitro transcribed human β-globin RNA was added to each fraction before RNA preparation, and is shown as a control of equal RNA isolation efficiency. (C) ZC3H11, PIP39 and RBP6 protein levels before and after chronic mild heat shock (16h at 37°C) analyzed by Western blotting. An unspecific band (*) recognized by the anti-ZC3H11 antibody is shown as loading control.
Fig 5.
Granule formation at 27°C and 41°C.
(A) The percentage of an mRNA in the small granule fraction increases with mRNA length. Both axes are on log2 scales, but the percentage labels on the y axis are not log-transformed in order to make them easier to understand. The correlation coefficient (R) was calculated using log-transformed values. Ribosomal protein mRNAs (ribprot) are in orange and the formula for the regression line is shown. (B) The percentage of each mRNA in polysomes was plotted against the percentage in both granule fractions. The ribosomal protein mRNAs are in orange, ZC3H11 mRNA is in green and the mRNAs bound by ZC3H11 (at least 3x enriched in a ZC3H11 pull-down) are in cyan. (C) Distribution of unique gene list mRNAs in different granule fractions at 41°C. The mRNAs were divided into five categories according to their percentage in large or small granules. The percentage of mRNAs in each of those categories is shown. For example, nearly 70% of genes and mRNAs for which less than 20% was in small granules (blue); for about 65% of genes, between 20% and 40% of the mRNA was in large granules (violet). Adding the small and large granule fractions together, just over 40% of genes encoded mRNas for which 40–60% of the mRNA was in small or large granules. (D) Relationship between mRNA abundance and % in granules at 41°C. The x-axis shows the reads per million after sequencing, on a log2 scale. The y axis is again on a log scale, but the labels are not log-transformed. Colour coding is as in (B). Selected mRNAs are indicated. (E) Relationship between the percentage of each mRNA in polysomes at 39°C (y-axis) and the percentage in both granule fractions at 41°C (x-axis). Colour coding as in (B).
Fig 6.
Binding to ZC3H11 related to polysome association and granule incorporation.
The ability of mRNAs to associate with ZC3H11 was previously assessed by co-immunoprecipitation [16]. The extent of binding was expressed as the read count per million (RPM) in the immunoprecipitated preparation, divided by the RPM in the input. Here, the mRNAs encoding ribosomal proteins were first extracted (Rib), then the remainder of the mRNAs were sorted according to the bound: input ratio (indicated in the line "ZC3H11 binding"). The number of open reading frames (ORFs) in each group is shown below this. The bottom line shows the results of a Student T-test comparing results for each category with those for mRNAs with bound:input ratios of less than 0.5. (A) Box plot showing the percentages of the mRNAs in granules (large and small combined) at 41°C. (B) Box plot showing the proportions of the mRNAs in the polysomal fractions at 39°C.
Fig 7.
Binding to ZC3H11 protects against granule association after heat shock.
(A) Cells without induction of RNAi were separated into sedimentable granules (large granules, LG); mRNA trapped inside cytoskeletons but not sedimented at 20000g, 10min (small granules, SG); and soluble supernatants (SN1-3). RNA was prepared from these as well as from unfractionated total cell lysate (T). All preparations are from 5×108 control or heat-shocked (1 hour at 41°C) cells. RNA was analysed by Northern blotting, probing for Tb927.10.16100 (FKBP) and the major cytosolic HSP70 mRNA (Tb927.11.11330). The variations in mobility are not reproducible and might be due to different amounts of RNA and salt. (B) As (A) but RNAi was induced for 48h. (C) Western blot showing the amount of ZC3H11, measured in cytoskeleton-depleted extracts [13]. At 27°C ZC3H11 is not detectable but a band is seen from tubulin, which cross-reacts with the antibody [13]. The loading control is another cross-reacting band.