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. Author manuscript; available in PMC: 2019 Dec 17.
Published in final edited form as: Cell Rep. 2019 Dec 3;29(10):3134–3146.e6. doi: 10.1016/j.celrep.2019.10.129

Figure 2. eIF5A Relieves Ribosomal Pausing at PP and PG Dipeptides during Translation Elongation in Human Cells.

Figure 2.

(A and B) Cumulative distribution function (CDF) plots of coding sequence (CDS) ribosome occupancy on transcripts that have either ≤1 or >1 PP dipeptide in their ORF in EIF5A−/− (A) or DOHH−/−

(B) cells relative to cells expressing non-target (NT) sgRNA. CDS occupancy is defined as ribosome occupancy in CDS (RPKM) normalized to transcript expression level derived from RNA-seq (FPKM). Number of genes in the analysis (that fulfilled minimum coverage criteria; see STAR Methods): in (A), 2,467 (≤1 PP) and 2,309 (> 1 PP); in (B), 2,597 (≤1PP) and 2,482 (>1 PP). p values for this and all subsequent CDF plots were calculated using one-sided Kolmogorov-Smirnov tests.

(C) Meta-codon plot of ribosome occupancy in a 100 nt window surrounding all PP dipeptides (28,571 total) in EIF5A−/−, DOHH−/−, and control sgNT cells. Mean ribosome occupancy of two biological replicates is shown.

(D and E) Motif analysis of amino acid enrichment within the E, P, and A sites of paused ribosomes in EIF5A−/− (D) and DOHH−/− (E) cells, compared with sgNT control cells.

(F) Top tripeptide pause motifs identified in EIF5A−/− cells (having pause scores > 3 and ranked by ratio of pause score in EIF5A−/− versus sgNT). PP/PG-containing tripeptides are indicated with red text. Ribosome pause sites identified in both EIF5A−/− and DOHH−/− cells are shaded gray.

(G) Meta-codon plot of ribosome occupancy in a 100 nt window surrounding the top 20 tripeptide pause motifs in EIF5A−/− cells compared with sgNT (32,856 total). Mean ribosome occupancy of two biological replicates is shown.

See also Figure S3.