Dynamic regulation of translation quality control associated with ribosome stalling

Abstract

Translation of problematic mRNA sequences induces ribosome stalling. Collided ribosomes at the stall site are recognized by cellular quality control machinery, resulting in dissociation of the ribosome from the mRNA and subsequent degradation of the nascent polypeptide and in some organisms, decay of the mRNA. However, the timing and regulation of these processes are unclear. We developed a SunTag-based reporter to monitor translation in real time on single mRNAs harboring difficult-to-translate poly(A) stretches. This reporter recapitulates previous findings in human cells that an internal poly(A) stretch reduces protein output ~10-fold, while mRNA levels are relatively unaffected. Long-term imaging of translation indicates that poly(A)-containing mRNAs are robustly translated in the absence of detectable mRNA cleavage. However, quantification of ribosome density reveals a ~3-fold increase in the number of ribosomes on poly(A)-containing mRNAs compared to a control mRNA, consistent with queues of many stalled ribosomes. Using single-molecule harringtonine runoff experiments, we observe the resolution of these queues in real-time by the cellular quality control machinery, and find that rescue is very slow compared to both elongation and termination. We propose that the very slow clearance of stalled ribosomes provides the basis for the cell to distinguish between

Puromycin reactivity does not accurately localize translation at the subcellular level

“…smFISH Probe Labeling smFISH probes targeting the SunTag region of the mRNA reporter transcript were synthesized as described (39,40). 20-mer olgionucleotides were ordered from IDT in an arrayed format, pooled, and labeled on the 3'-end with amino-11-12 ddUTP (Lumiprobe A5040) using deoxynucleotidyl transferase (TdT, Thermo Fisher EP0162).…”

“…smFISH-IF was performed similarly as described (39,41). smFISH-IF was performed on cell stably expressing the SunTag mRNA reporter and scFV-sfGFP.…”

“…Fixed cell image analysis was performed as described (39) with existing or custom MATLAB software. Spot detection of the mRNA and proteins channels were performed independently in FISH-Quant (42).…”

Rqc1 and other yeast proteins containing highly positively charged sequences are not targets of the RQC complex

“…However, recent studies on the global analysis of disome formation suggested that collisions are relatively common, and most of these events are resolved without translation termination [29]. What determines whether a collision event will become a target of RQC or not is still an open question, but the disome structure, the time required to resume translation, and/or exposure of an empty A site, seem to play an important role in this decision [30].…”

Puromycin reactivity does not accurately localize translation at the subcellular level

“…smFISH Probe Labeling smFISH probes targeting the SunTag region of the mRNA reporter transcript were synthesized as described(41,42). 20-mer oligonucleotides (Supplementary table 1) were ordered from IDT in an arrayed format, pooled, and labeled on the 3'-end with amino-11-12 ddUTP (Lumiprobe A5040) using deoxynucleotidyl transferase (TdT, Thermo Fisher EP0162).…”