Conservation of stall sites across divergent eukaryotes. (A) Schematic representation of stall site analysis. (B) Number of conserved stall sites in homologous genes in yeast, fruit fly, zebrafish, mouse and human. Some stall sites are common in lower and higher vertebrates, indicating their importance in translation regulation. (C) Ribosome profile on the Xbp1 mRNA (here values for H3 library). The schematic of two isoforms is shown, the unspliced, shorter Xbp1u (solid purple) and spliced, with 3’ extension, Xbp1s (striped purple). The stall site at the 3’end of Xbp1u is indicated. (D) Alignment of C-terminal peptide sequences of Xbp1u from fruit fly, zebrafish, mouse and human. The amino acids in the P-site position where the stalling occurs are indicated in bold. Conserved amino acids are highlighted in yellow, while the ones which are most likely critical for stalling are indicated with triangles. A pink triangle indicates a position where S to A mutation has been demonstrated to increase the translational pausing (50).

Mechanisms of conserved stalling. (A) Overrepresentation of amino acids around CSSs, sorted from most to least frequent, top to bottom. (B) Fraction of positively charged amino acids in the 30 amino acids upstream of the CSSs at P-site (0), spanning the exit tunnel versus background (shaded regions show background distribution, from 5th to 95th percentile). Similarly, (C) fraction of negatively and (D) special (proline and glycine) amino acids.

Functional characterization of CSS-containing genes. (A and B) Gene ontology enrichment analysis showing molecular functions and biological processes of conserved genes that are affected by stalling. (C) Position of CSSs on exons, with exon lengths normalized to 1. (D) Coil prediction score around CSSs, compared to control. Red dashed line marks the threshold typically used for coil prediction.