Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling

Abstract

Localized protein synthesis is a fundamental mechanism for creating distinct subcellular environments. Here we developed a generalizable proximity-specific ribosome profiling strategy that enables global analysis of translation in defined subcellular locations. We applied this approach to the endoplasmic reticulum (ER) in yeast and mammals. We observed the large majority of secretory proteins to be cotranslationally translocated, including substrates capable of post-translational insertion in vitro. Distinct translocon complexes engaged nascent chains at different points during synthesis. Whereas most proteins engaged the ER immediately after or even before signal sequence (SS) emergence, a class of Sec66-dependent proteins entered with a looped SS conformation. Finally, we observed rapid ribosome exchange into the cytosol after translation termination. These data provide insights into how distinct translocation mechanisms act in concert to promote efficient cotranslational recruitment.

Autonomous translational pausing is required for XBP1u mRNA recruitment to the ER via the SRP pathway

“…Similarly, Frydman and coworkers (35) recently reported that SRP preferentially recognizes a set of proteins harboring a signal sequence with a downstream cluster of suboptimal codons, which slows translational elongation. Further, Weissman and coworkers (36) showed that artificial translational arrest by cycloheximide treatment enables the ER localization of a subset of cytosolic mRNAs encoding the proteins harboring a first transmembrane segment or a signal sequence in the C-terminal region (from 50 to 150 codons before the termination codons). Taken together, these results suggest that translational pausing or the slowdown of elongation extends the time window of the competent state in which SRP is able to recognize the RNC with an exposed signal sequence.…”

Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast

“…Experiments without CHX, on the other hand, report positive correlations of varying magnitude (Fig 1D, 0x Gerashchenko NAR points and purple labels). Experiments by Pop [36], Lareau [26], Nedialkova [29], Guydosh [35] and Gardin [33] produce weak to moderate correlations, but experiments by Gerashchenko [34], Jan [41], Williams [42], Weinberg [38], and Young [37] produce fairly strong and highly statistically significant correlations.…”

“…If downstream peaks are transient waves moving downstream after a change in the relative amounts of time ribosomes spend positioned over each codon, the total CHXinduced excess or deficit in enrichment downstream of each codon identity should exactly offset the total CHX-induced change in enrichments at the tRNA binding sites. To test whether experimental data agreed with this prediction, we analyzed several matched pair of experiments performed with and without CHX by Jan [41] (Fig 5), Williams [42], and Gerashchenko [34] (S10 Fig). For each codon identity, we compared the sum of the differences in enrichment between the experiments at the A-, P-, and E-sites (green area in insets) to the sum of the difference in enrichment across the range of downstream offsets occupied by the putative waves (red area in insets). In all matched pairs of experiments, the area of each codon's downstream peak is accurately predicted by its tRNA binding site changes (r 2 = 0.85 to 0.93, slope of best fit line β = −1.00 to −1.20).…”

“…The area of each codon identity's downstream peak is strongly predicted by changes in enrichment at the tRNA binding sites, consistent with the hypothesis that downstream peaks are transient waves caused by continued elongation with disrupted dynamics in the presence of CHX. this prediction, we examined mean relative enrichments around CGA in the full set of experiments from Jan et al [41], which involved a variety of different combinations of CHX pretreatment duration and temperature. Strikingly, when pretreatment was performed at 30°C, downstream peaks after 9 minutes of pretreatment are centered slightly more than twice as far downstream as those after 4 minutes of pretreatment (Fig 6), further supporting the hypothesis that elongation continues to occur at a slow but steady rate over the course of CHX pretreatment.…”

“…To test [41], the sum of each codon identity's changes in mean relative enrichment at the A-, P-, and E-sites between the two experiments (green area in insets) is plotted against the total excess or deficit of enrichment in the CHX experiment from 6 to 65 codons downstream (red area in insets). The area of each codon identity's downstream peak is strongly predicted by changes in enrichment at the tRNA binding sites, consistent with the hypothesis that downstream peaks are transient waves caused by continued elongation with disrupted dynamics in the presence of CHX.…”