Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors

Michael G. Kearse, Daniel H. Goldman, Jiou Choi, Chike Nwaezeapu, Dongming Liang, Katelyn M. Green, Aaron C. Goldstrohm, Peter K. Todd, Rachel Green, Jeremy E. Wilusz

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

Original languageEnglish (US)
Pages (from-to)871-885
Number of pages15
JournalGenes and Development
Volume33
Issue number13-14
DOIs
StatePublished - Jul 2019

Bibliographical note

Funding Information:
We thank Gideon Dreyfuss, Ben Garcia, Zissimos Mourelatos, and members of the Wilusz laboratory for valuable discussions and advice. Tin Tin Su (University of Colorado at Boulder) and Malia Potts (St. Jude Children’s Research Hospital) kindly provided BVD and DIDB, respectively. This work was supported by National Institutes of Health grants K99-GM126064 (to M.G.K.), R35-GM119735 (to J.E.W.), NCI-P30-CA016520 (to J.E.W.), T32-GM007315 (to K.M.G.), F31-NS100302 (to K.M.G.), R01-GM105707 (to A.C.G.), R01-NS099280 (to P.K.T.), R01-NS086810 (to P.K.T.), and R37-GM059425 (to R.G.). D.H.G. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2280-16). P.K.T. is supported by Veterans Affairs Biomedical Laboratory Research and Development grants 1I21BX001841 and 1I01BX003231 and the Michigan Discovery Fund. R.G. is a Howard Hughes Medical Institute Investigator. J.E.W. is a Rita Allen Foundation Scholar. Illumina sequencing was conducted at the Genetic Resources Core Facility at the Johns Hopkins Institute of Genetic Medicine.

Funding Information:
We thank Gideon Dreyfuss, Ben Garcia, Zissimos Mourelatos, and members of the Wilusz laboratory for valuable discussions and advice. Tin Tin Su (University of Colorado at Boulder) and Malia Potts (St. Jude Children?s Research Hospital) kindly provided BVD and DIDB, respectively. This work was supported by National Institutes of Health grants K99-GM126064 (to M.G.K.), R35-GM119735 (to J.E.W.), NCI-P30-CA016520 (to J.E.W.), T32-GM007315 (to K.M.G.), F31-NS100302 (to K.M.G.), R01- GM105707 (to A.C.G.), R01-NS099280 (to P.K.T.), R01-NS086810 (to P.K.T.), and R37-GM059425 (to R.G.). D.H.G. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2280-16). P.K.T. is supported by Veterans Affairs Biomedical Laboratory Research and Development grants 1I21BX001841 and 1I01BX003231 and the Michigan Discovery Fund. R.G. is a Howard Hughes Medical Institute Investigator. J.E.W. is a Rita Allen Foundation Scholar. Illumina sequencingwas conducted at the Genetic Resources Core Facility at the Johns Hopkins Institute of Genetic Medicine.

Publisher Copyright:
© 2019 Nostramo and Hopper.

Keywords

  • Cycloheximide
  • Near-cognate
  • RAN translation
  • Start codon
  • Translation initiation
  • Translational control

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