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. 2017 Jul 7;45(12):7326-7338.
doi: 10.1093/nar/gkx479.

eIF5A facilitates translation termination globally and promotes the elongation of many non polyproline-specific tripeptide sequences

Vicent Pelechano  1 , Paula Alepuz  2   3
Affiliations

eIF5A facilitates translation termination globally and promotes the elongation of many non polyproline-specific tripeptide sequences

Vicent Pelechano et al. Nucleic Acids Res. .

Abstract

eIF5A is an essential protein involved in protein synthesis, cell proliferation and animal development. High eIF5A expression is observed in many tumor types and has been linked to cancer metastasis. Recent studies have shown that eIF5A facilitates the translation elongation of stretches of consecutive prolines. Activated eIF5A binds to the empty E-site of stalled ribosomes, where it is thought to interact with the peptidyl-tRNA situated at the P-site. Here, we report a genome-wide analysis of ribosome stalling in Saccharomyces cerevisiae eIF5A depleted cells using 5Pseq. We confirm that, in the absence of eIF5A, ribosomes stall at proline stretches, and extend previous studies by identifying eIF5A-dependent ribosome pauses at termination and at >200 tripeptide motifs. We show that presence of proline, glycine and charged amino acids at the peptidyl transferase center and at the beginning of the peptide exit tunnel arrest ribosomes in eIF5A-depleted cells. Lack of eIF5A also renders ribosome accumulation at the stop codons. Our data indicate specific protein functional groups under the control of eIF5A, including ER-coupled translation and GTPases in yeast and cytoskeleton organization, collagen metabolism and cell differentiation in humans. Our results support a broad mRNA-specific role of eIF5A in translation and identify the conserved motifs that affect translation elongation from yeast to humans.

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Figures

Figure 1.
Figure 1.
eIF5A depletion yields a marked pause at the stop codon. (A) Growth of wild-type BY4741 and eIF5A temperature-sensitive mutant cells tif51A-1 and tif51A-3 in YPD media at permissive (25°C) and restrictive (37°C) temperatures. (B) Western blot analysis of eIF5A depletion. Cells were grown in YPD until the exponential phase and were then transferred to the indicated temperatures. Proteins were extracted and analysed by Western blot. The eIF5A protein was visualized with a specific anti-eIF5A antibody. (C) Outline of the 5PSeq method. (D) The metagene analysis displaying the abundance 5΄P intermediates in reads per million (rpm) in relation to the ORF start (left panel) and stop (right panel) codons.
Figure 2.
Figure 2.
eIF5A depletion reveals increased pauses at proline, glycine and charged amino acids. (A) The correspondence between the ribosomal sites occupied by codons and sequencing reads, as measured by 5PSeq. (B) The differential ribosome pausing for all amino acids at –11, –14 and –17 nt from the selected codon. Significantly regulated differences (adjusted P-value < 0.01) in red. (C) The metagene that represents the 5PSeq intermediates for the indicated amino acids and positions. To facilitate the identification of codon-specific regulation and sample comparison, the total number of reads for each metagene (–80 to +20 window) was normalized.
Figure 3.
Figure 3.
Multiple tripeptide motifs influence eIF5A-dependent translation. (A) Differential ribosome pausing for all tripeptide motifs. Axes represent the raw number of reads for the metagene of each motif. Significantly regulated differences (adjusted P-value < 0.0001) in red, computed using shrunken log2 fold changes (see the Methods). (B) Hierarchical cluster depicting tripeptide motifs associated to eIF5A-dependent stall at –11nt. (C–H) The metagene that represents the 5PSeq intermediates for the selected tripeptide motifs. The total number of reads for each metagene (–80 to +20 window) was normalized.
Figure 4.
Figure 4.
5PSeq signals identify eIF5A-dependent ribosome stalling at particular genes. Coverage of the 5PSeq reads (collapsed to the single 5΄nucleotide) for the BNI1 gene in wild type (black), tif51A-1 (blue) and tif51A-3 (red). Proline codons are depicted in green.
Figure 5.
Figure 5.
Extended context of peptide motifs influence eIF5A-dependent pausing. (A–D) The metagene that represents the 5PSeq intermediates for the selected pentapeptide motifs, and a scheme of their location in the ribosome exit tunnel. The total number of reads for each metagene (–80 to +20 window) was normalized.
Figure 6.
Figure 6.
Conservation of the identified motifs in the human proteome. (A) Selected gene ontology terms significantly enriched in the human proteins with a high content (>25) of eIF5A-dependent motifs, including those tripeptides that contained PP or not. (B) Down-regulated proteins after eIF5A depletion (37) are enriched for the motifs that causer an eIF5A-dependent pause. Randomly expected proteins (green), identified (pink) and significance (according to the hypergeometric distribution, blue). Significance level for Bonferroni-adjusted multiple testing is depicted in red and indicated by *. Only the motifs present in at least 40 proteins identified by Fujimura et al. (37) were considered for the analysis. (C) Amino acid abundance in relation to the ribosome structure for those motifs associated with eIF5A-dependent pausing in S. cerevisiae.
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