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. 2015 Jun 9;16(1):443.
doi: 10.1186/s12864-015-1563-8.

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi

Pablo Smircich  1   2 , Guillermo Eastman  3 , Saloe Bispo  4 , María Ana Duhagon  5   6 , Eloise P Guerra-Slompo  7 , Beatriz Garat  8 , Samuel Goldenberg  9 , David J Munroe  10 , Bruno Dallagiovanna  11 , Fabiola Holetz  12 , Jose R Sotelo-Silveira  13   14
Affiliations

Ribosome profiling reveals translation control as a key mechanism generating differential gene expression in Trypanosoma cruzi

Pablo Smircich et al. BMC Genomics. .

Abstract

Background: Due to the absence of transcription initiation regulation of protein coding genes transcribed by RNA polymerase II, posttranscriptional regulation is responsible for the majority of gene expression changes in trypanosomatids. Therefore, cataloging the abundance of mRNAs (transcriptome) and the level of their translation (translatome) is a key step to understand control of gene expression in these organisms.

Results: Here we assess the extent of regulation of the transcriptome and the translatome in the Chagas disease causing agent, Trypanosoma cruzi, in both the non-infective (epimastigote) and infective (metacyclic trypomastigote) insect's life stages using RNA-seq and ribosome profiling. The observed steady state transcript levels support constitutive transcription and maturation implying the existence of distinctive posttranscriptional regulatory mechanisms controlling gene expression levels at those parasite stages. Meanwhile, the downregulation of a large proportion of the translatome indicates a key role of translation control in differentiation into the infective form. The previously described proteomic data correlate better with the translatomes than with the transcriptomes and translational efficiency analysis shows a wide dynamic range, reinforcing the importance of translatability as a regulatory step. Translation efficiencies for protein families like ribosomal components are diminished while translation of the transialidase virulence factors is upregulated in the quiescent infective metacyclic trypomastigote stage.

Conclusions: A large subset of genes is modulated at the translation level in two different stages of Trypanosoma cruzi life cycle. Translation upregulation of virulence factors and downregulation of ribosomal proteins indicates different degrees of control operating to prepare the parasite for an infective life form. Taking together our results show that translational regulation, in addition to regulation of steady state level of mRNA, is a major factor playing a role during the parasite differentiation.

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Figures

Figure 1
Figure 1
mRNA levels are regulated both in epimastigote (E) and metacyclic trypomastiogote (MT) life cycle stages. ( A ) Venn diagram indicating, at each stage, the number of detected genes (see Methods for detection criteria). The intersection is colored. ( B ) Scatter plot of the estimated expression levels as nRPK for both stages. Differentially expressed genes are shown in red (FC > 2, FDR < 0.05). ( C ) Heat map showing the variation of expression for the genes showing differences at the mRNA level at each stage.
Figure 2
Figure 2
Translation is regulated in epimastigotes (E) and metacyclic trypomastigotes (MT). ( A ) Venn diagram indicating, at each stage, the number of detected genes (see Methods for detection criteria). The intersection is colored. ( B ) Scatter plot of the estimated translated levels as nRPK for both stages. Differentially expressed genes are shown in red (FC > 2, FDR < 0.05). ( C ) Heat map showing the variation of expression for the genes showing differences at the translation level at each stage.
Figure 3
Figure 3
Regulation is higher at the translatome level than at the transcriptome level. Box plots of the fold change (MT/E expression estimates) distribution for regulated genes at the mRNA steady state level (light grey) or the translatome level (dark grey).
Figure 4
Figure 4
Translatome is better correlated with the proteome than the transcriptome. Inter sample correlations. ( A ) Pearson correlation coefficients for the different samples are shown. Upper values correspond to the ones observed in the E stage experiments. Lower values are as before for the MT stage. ( B ) Log-log scatter plots of the expression estimates in the E and MT stages. Upper panel: Left: Correlation of the proteome to the transcriptome in the E stage. Right: Correlation of the proteome to the translatome in the E stage. AU: Arbitrary units. Lower panel: Same as above for the MT stage.
Figure 5
Figure 5
Translation efficiency (TE) varies between the epimastigote (E) and metacyclic trypomastigote (MT) T. cruzi stages. Upper panel: ( A ) Scatter plot of the fold change (MT/E expression estimates) in the translatome vs the transcriptome. ( B ) Scatter plot of the TE (RFP/Total RNA expression estimates) in the MT vs the E stage for genes detected in all samples. Genes exhibiting non proportional changes (FC > 2, FDR < 0.05) are colored. Lower panel: ( C ) TE histograms for epimastigotes (light grey) and metacyclic trypomastigotes (dark grey). Median efficiency values are 0.51 and 0.69 respectively.
Figure 6
Figure 6
Trans-sialidase (TS) family genes increase their TE upon differentiation. Expression profiles for the TS genes in T. cruzi epimastigotes (E) and metacyclic trypomastigotes (MT) are shown. ( A ) Box plots of the TS family translational efficiency in E and MT. Statistically significant differences among populations are indicated by asterisks (Wilcoxon p < 0.05). ( B ) Scatter plot of the fold change (MT/E expression estimates) in the translatome vs the transcriptome. TS genes are shown in red.
Figure 7
Figure 7
Ribosomal proteins (RP) genes decrease their TE upon differentiation. Expression profiles for the RP genes are shown in T. cruzi epimastigotes (E) and metacyclic trypomastigotes (MT). Upper panel: (A) Bar plot of the E stage transcriptome and translatome levels for the RP and for all T. cruzi genes. Each bar marks the population median while the whiskers represent the interquartile range. (B) Bar plot of the translation efficiency (TE) for the RP and for all T. cruzi genes. Statistically significant differences among populations are indicated by asterisks (Wilcoxon p < 0.05). Lower panel: (C) and (D) are the same type of bar plots as A and B respectively, but for the MT stage. Note the decrease in translation efficiency of RP (D) as a result of a major decrease in RP translation (C). Y-axis scales in figures A and C are different.
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