Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages - PubMed (original) (raw)

Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages

Juan-José Vasquez et al. Nucleic Acids Res. 2014 Apr.

Abstract

While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5' UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei.

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Figures

Figure 1.

Figure 1.

Ribosome footprints reveal coding sequences at sub-codon resolution. (A) Ribosome footprints are strongly enriched across CDSs. mRNA densities and ribosome densities are shown as reads per nucleotide per million reads (RPM) to normalize for differences in library size. (B) Alignment of the 5′ nucleotide from ribosome footprint reads that map close to translation start or translation termination sites. Blue boxes mark the approximate size of the ribosome footprint. (C) Percentage of position of sequence reads relative to reading frame.

Figure 2.

Figure 2.

Ribosome footprints reveal translated regions. (A) Ribosome profiles for the two intron-containing genes. Introns are represented as a dashed line. (B) Ribosome profiles of two possibly mis-annotated CDSs. Black bars mark annotated CDSs. Grey bars mark CDSs predicted based on ribosome profiles. Green boxes represent ATG codons and red boxes represent stop codons.

Figure 3.

Figure 3.

Translational efficiency is regulated. (A) Histograms of mRNA abundance and ribosome density (rate of protein synthesis) for BF (left panel) and PF parasites (right panel). (B) Histogram of translational efficiency (ratio of ribosome footprint density to mRNA abundance) for BF (left panel) and PF parasites (right panel). (C) Pair-wise comparisons of translational efficiency in PF and BF. CDSs were ranked based on translational efficiency (1 = highest translational efficiency, 7782 = lowest translational efficiency) in BF and PF. Ranks between life cycle stages show a correlation of R = 0.7428. Gene families with developmentally regulated translational efficiencies are colour coded: Pumillio genes (red), cytochromes oxidase (blue) genes required for glycolysis (63) (green) and the alternative oxidase (Tb927.10.7090, black). (D) Ribosome footprint profile of a gene with developmentally regulated translational efficiency. Green arrow indicates direction of transcription.

Figure 4.

Figure 4.

Ribosome footprints reveal translation of uORFs. (A) Alignment of the 5′ nucleotides from ribosome footprint reads that map close to translation start or translation termination sites of uORFs. (B) Percentage of position of sequence reads relative to reading frame. (C) Ribosome profiles of two genes with uORF (left panel) and without uORF (right panel). Narrow grey boxes represent 5′ UTRs, green box represents AUG-codon, red box represents termination codon.

Figure 5.

Figure 5.

Ribosome footprints reveal previously un-annotated CDSs. (A) Alignment of the 5′ nucleotides from ribosome footprint reads that map close to translation start or translation termination sites of candidate CDSs. (B) Percentage of position of sequence reads relative to reading frame. (C) Ribosome, mRNA and RIT-seq (RNAi target sequencing) profiles of two previously un-annotated putative CDSs.

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