Local slowdown of translation by nonoptimal codons promotes nascent-chain recognition by SRP in vivo

Abstract

The genetic code allows most amino acids a choice of optimal and nonoptimal codons. We report that synonymous codon choice is tuned to promote interaction of nascent polypeptides with the signal recognition particle (SRP), which assists in protein translocation across membranes. Cotranslational recognition by the SRP in vivo is enhanced when mRNAs contain nonoptimal codon clusters 35-40 codons downstream of the SRP-binding site, the distance that spans the ribosomal polypeptide exit tunnel. A local translation slowdown upon ribosomal exit of SRP-binding elements in mRNAs containing these nonoptimal codon clusters is supported experimentally by ribosome profiling analyses in yeast. Modulation of local elongation rates through codon choice appears to kinetically enhance recognition by ribosome-associated factors. We propose that cotranslational regulation of nascent-chain fate may be a general constraint shaping codon usage in the genome.

Figure 1

The SRP binds nascent chains in vivo with a broad distribution of specificities. (a) Schematics of selective, cotranslational SRP interaction with proteins bearing signal sequences (SS) or transmembrane (TM) segments. The SRP also binds few noncognate (NC) cytosolic and nuclear off-target proteins that do not get translocated. (b) Enrichment scores of cotranslational SRP binding for SS proteins (i.e., proteins with an N-terminal SS) and for TM proteins (i.e., proteins with TM segments as SRP-binding sites). Inset, significantly higher enrichment of SRP binding in TM proteins (n = 190) compared to SS proteins (n = 107) (P = 0.0007 by Wilcoxon rank-sum test). (c) Classification of SS proteins into noncognate (SRP-NC; n = 109), enriched (SRP-E; n = 78) and strongly enriched (SRP-SE; n = 29) substrates, on the basis of increasing SRP enrichment scores. Box plots show the data distribution through median (center line), first and third quartiles (filled box), 1.5× interquartile range (dashed line) and extreme values (circles). AU, arbitrary units. *P < 0.05.

Figure 2

An evolutionarily conserved local dip in translational efficiency characterizes the SRP-dependent translocation of the essential N-oligosaccharyl transferase (OST). (a) Four of eight OST subunits, namely OST1, OST3, SWP1 and WBP1, have a distinct topology of an SS followed by a long lumenal domain. (b) Median translational efficiency (TE) profile of the OST subunits OST1, OST3, SWP1 and WBP1, showing a distinct region of low TE (gray bar) ~40 codons downstream of the start of the hydrophobic region of the SS (yellow bar). (c) Evolutionary conservation of the region of low TE (gray bars) ~40 codons downstream of the start of the hydrophobic region of the SS (yellow bars) in OST subunits across closely related yeast species. TE profiles show predicted relative translation speeds and are in arbitrary units (AU).

Figure 3

Slowdown of translation by nonoptimal codons promotes SRP recognition. (a) Top, schematic. Bottom, median TE profiles for each class of SRP substrates, showing a dip in TE in the downstream region (gray bar) in the mRNA sequences of all SRP-SE (n = 29) substrates, but not those of SRP-E (n = 78) and SPR-NC (n = 109), just after emergence of the hydrophobic SRP-binding site from the ribosome exit tunnel. Aa, amino acids. (b) Statistical analysis showing significantly lower average TE for individual SRP-SE substrates in the translational-slowdown element encoded in the mRNA sequences downstream of the SS, compared to SRP-E substrates (P = 0.02 by Wilcoxon rank-sum test; n values as in a). (c) Enrichment in nonoptimal codons. The translational-slowdown element in the mRNA sequences of SRP-SE substrates is enriched in nonoptimal codons (P = 0.002 by Fisher’s exact test on counts of optimal and nonoptimal codons; two sided, with n values as in a). Box plots show the data distribution through median (center line), first and third quartiles (filled box), 1.5× interquartile range (dashed line) and extreme values (circles). *P < 0.05; **P < 0.01.

Figure 4

Ribosome profiling confirms a translational slowdown that associates with enhanced SRP binding. (a) Schematic for ribosome profiling. Ribosome profiling experimentally measures local translation kinetics through selective sequencing of ribosome-protected footprints. Higher footprint densities indicate slower local translation kinetics. (b) Median ribosome footprint density profiles (arbitrary units, AU) for SRP-SE (n = 16), SRP-E (n = 33) and SRP-NC (n = 20) substrates. Only SRP-SE substrates exhibit higher footprint densities in the translational-slowdown element (gray bar) downstream of the SS (yellow bar). Aa, amino acids. (c) Distributions of average relative footprint densities in the downstream translational-slowdown element for individual SRP-SE, SRP-E and SRP-NC proteins. SRP-SE substrates are locally translated more slowly compared to SRP-E and SRP-NC substrates just when the hydrophobic SS is exposed outside the ribosome (P = 0.042 by Wilcoxon rank-sum test; n values as in b). Box plots show the data distribution through median (center line), first and third quartiles (filled box), 1.5× interquartile range (dashed line) and extreme values (circles). *P < 0.05.

Figure 5

A local slowdown of translation promotes TM-helix recognition by the SRP. (a,b) Median TE (a) and median footprint density (n = 177) (b) profiles for TM proteins (n = 37), showing slower translation of TM segments (red bars) and the region ~40 codons downstream (gray bars). N-ter, N terminus. (c) Distribution of the distance d_TM1–TM2 between the start of the first and second TM segments in proteins with multiple TM helices. The most frequent distance is ~34 codons. (d) Median TE profiles for strongly enriched (TM-SE; n = 46) and not strongly enriched (TM-nSE; n = 165) TM proteins with d_TM1–TM2 > 60 codons, showing that nonoptimal codons promote SRP recognition independent of the presence of a second TM successive segment. (e) Median ribosome footprint density profiles confirming locally slower translation kinetics for TM-SE (n = 14) substrates compared to TM-nSE (n = 27) substrates. (f) Enrichment in nonoptimal codons at the translational-slowdown element in TM-nSE substrates (P = 0.030 by Fisher’s exact test on counts of optimal and nonoptimal codons; two sided). *P < 0.05.

Figure 6

REST coordinates translation elongation and SRP recognition. Translational coordination of local translation rates with in vivo SRP–nascent chain recognition. Strategically positioned nonoptimal codons form an mRNA-encoded slowdown of translation (REST) element downstream of SS or TM segments. The increased time window of the SS or TM segment at the ribosome exit promotes recognition and binding by the SRP.