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. 2019 Jul 1;33(13-14):871-885.
doi: 10.1101/gad.324715.119. Epub 2019 Jun 6.

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

Michael G Kearse  1 , Daniel H Goldman  2 , Jiou Choi  1 , Chike Nwaezeapu  1 , Dongming Liang  1 , Katelyn M Green  3 , Aaron C Goldstrohm  4 , Peter K Todd  3   5 , Rachel Green  2 , Jeremy E Wilusz  1
Affiliations

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

Michael G Kearse et al. Genes Dev. .

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.

Keywords: RAN translation; cycloheximide; near-cognate; start codon; translation initiation; translational control.

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Figures

Figure 1.
Figure 1.
Development of reporters that are specific for non-AUG translation. (A) HeLa cells were transfected with nLuc reporters harboring the indicated start codons and a C-terminal 3XFlag tag. Twenty-four hours after transfection, the nLuc luminescence was quantified and normalized to a cotransfected firefly luciferase (FFLuc; pGL4.13) reporter. Signal from the AUG-nLuc reporter (black) was then used to determine the relative expression levels of the other nLuc reporters (gray). The red dashed line indicates expression above the negative control reporters. Data are shown as mean ± SD. n = 3. Signals from the non-AUG-nLuc reporters were compared with the GGG-nLuc negative control using a two-tailed unpaired t-test with Welch's correction. (*) P < 0.05. (B) Western blotting (WB) using an anti-Flag antibody was used to examine expression of the nLuc-3XFlag reporters 24 h after transfection. Vinculin was used as a loading control. AUG* denotes that 1/20th of the AUG-nLuc-3XFlag plasmid was transfected to avoid overexposure during film development. (C) Northern blotting was used to examine expression of the nLuc reporter mRNAs 24 h after transfection. 28S ribosomal RNA was used as a loading control. (D) RT-qPCR was used to quantify nLuc reporter mRNA levels 24 h after transfection. mRNA levels were first normalized to the cotransfected FFLuc (pGL4.13) reporter, and AUG-nLuc (black) was then used to determine the relative expression levels of the other nLuc reporters (gray). Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used. (n.s.) Not significant. (E) HeLa cells were transfected with FFLuc-3XFlag reporters harboring the indicated start codons. Total protein was isolated 24 h after transfection and analyzed using Western blotting. Vinculin was used as a loading control. AUG* denotes that 1/20th of the AUG-FFLuc-3XFlag plasmid was transfected to avoid overexposure during film development. (F) NSC119893 inhibits formation of the canonical TC. (G) HeLa cells were transfected with the destabilized nLuc-3XFlag-CL1/PEST reporters for 24 h and then either collected (control) or treated for 3 h with 200 µM NSC119893. Luminescence signals for each reporter were quantified and set relative to the associated control samples. The CrPV IRES does not require the TC for initiation. Data are shown as mean ± SD. n = 3.
Figure 2.
Figure 2.
Non-AUG translation reporters are resistant to inhibition by CHX. (A) HeLa cells were transfected with the nLuc-3XFlag reporters followed by treatment with 100 µg/mL CHX and quantification of luminescence signals. (B) After 1 or 24 h of treatment with 100 µg/mL CHX, HeLa cells were subjected to puromycin (PURO) labeling and Western blotting (WB) to confirm global translation inhibition. Tubulin was used as a loading control. (C) Luminescence from the nLuc-3XFlag reporters was measured at the indicated time points after 100 µg/mL CHX treatment. Data for each reporter were set relative to the 0-h time point and are shown as mean ± SD. n = 3. (D) Western blotting using an anti-Flag antibody was used to examine expression of the nLuc-3XFlag reporters before and after 24 h of 100 µg/mL CHX treatment. Vinculin was used as a loading control. AUG* denotes that 1/20th of the AUG-nLuc-3XFlag plasmid was transfected to avoid overexposure during film development. (E) Luminescence from the destabilized nLuc-3XFlag-PEST reporters was measured at the indicated time points after CHX treatment. Data for each reporter were set relative to the 0-h time point and are shown as mean ± SD. n = 3. (F) HeLa cells were pretreated (15 min) with vehicle (Veh.; 0.1% DMSO) or 100 µg/mL CHX followed by transfection of the nLuc-3XFlag reporters. Luminescence was measured after 24 h. (G) Raw luciferase values of nLuc-3XFlag reporters 24 h after transfection. Data are shown as mean ± SD. n = 3. (H) Luminescence signals for each reporter were quantified and set relative to the associated vehicle-treated samples. Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used. (I) Western blotting was used to examine expression of the FFLuc-3XFlag reporters before and after 24 h of 100 µg/mL CHX treatment. Vinculin was used as a loading control. AUG* denotes that 1/20th of the AUG-FFLuc-3XFlag plasmid was transfected to avoid overexposure during film development. (J) HeLa cells were transfected with expanded (CGG)100 repeat RAN translation reporters for 24 h and then collected (control) or treated for 24 h with 100 µg/mL CHX. “+1” and “+2” refer to the reading frame of the (CGG)100 repeat in relation to the nLuc-coding sequence. Luminescence signals for each reporter were quantified and set relative to the associated control samples. Data are shown as mean ± SD. n = 3.
Figure 3.
Figure 3.
Translation of endogenous DAP5 (eIF4G2/p97) is resistant to CHX. (A) HeLa cells were transfected with the GUG-nLuc-3XFlag reporter. After 24 h, cells were treated with vehicle (Veh., 0.1% DMSO) or 100 µg/mL CHX and then incubated for 15 min or 24 h before being subjected to ribosome profiling and RNA-seq. (B) A volcano plot showing changes in TE of endogenous mRNAs (24 h of CHX vs. 15 min of CHX) compared with the associated false discovery rates (FDRs). FDRs for each mRNA are listed in Supplemental Table S1. (C,D) Ribosome footprints (C) and RNA-seq fragments (D) that mapped to the GUG-encoded DAP5 locus in cells that had been treated with CHX for 15 min (blue) or 24 h (red). Raw counts are depicted, and the ribosome footprint and RNA-seq RPKMs (which account for library size and ORF length) of the DAP5-coding sequence are also given for each replicate and time point. (E) Western blotting was used to assess endogenous DAP5 protein levels in HeLa cells before (control) and after 24 h of treatment with 1, 10, or 100 µg/mL CHX. Tubulin was used as a loading control. (F) RT-qPCR was used to assess endogenous DAP5 mRNA levels before (control) and after 24 h of 1 µg/mL CHX treatment. FH and NPTX1 mRNAs (whose levels did not change during CHX treatment as determined by RNA-seq) were used as dual internal references. Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used. (G) After 1 or 24 h of treatment with 100 or 1 µg/mL CHX, HeLa cells were subjected to PURO labeling and Western blotting to confirm global translation inhibition. Tubulin was used as a loading control. (H) Western blotting was used to examine expression of the GUG- and AUG-encoded DAP5-3XFlag reporters before and after 24 h of 100 µg/mL CHX treatment. Tubulin was used as a loading control. AUG* denotes that one-fourth of the AUG-DAP5-3XFlag plasmid was transfected to avoid overexposure during film development.
Figure 4.
Figure 4.
Endogenous DAP5 mRNA cosediments with larger polysomes upon CHX treatment. (A) Polysome profiles of HeLa cells that had been treated with 100 µg/mL CHX for 15 min (blue) or 24 h (red). (B–E) RT-qPCR was used to determine the distribution of DAP5 (B), RPL28 (C), RPL30 (D), and ATF4 (E) mRNAs across 10%–50% sucrose gradients after 15 min (blue) or 24 h (red) of 100 µg/mL CHX treatment. Two biological replicates for each treatment are shown.
Figure 5.
Figure 5.
Ribosome queuing likely occurs upon CHX treatment. (A) A model of CHX-resistant translation via ribosome queuing. Scanning PICs inefficiently recognize non-AUG start codons, and thus most PICs continue to scan downstream in search of a start codon. When an elongating 80S ribosome is slowed by CHX (top), PICs continue to be loaded and ultimately start to queue (or stack) upstream of the slowed 80S ribosome (middle). (Bottom) This places a PIC near a non-AUG start codon for an extended period of time, allowing for increased non-AUG translation. (4A) eIF4A. (B) A stable hairpin (HP) was inserted within the 5′ leader of the AUG-nLuc-3XFlag and GUG-nLuc-3XFlag reporters. Northern blotting was used to examine expression of the nLuc reporter mRNAs in HeLa cells 24 h after transfection. 28S ribosomal RNA was used as a loading control. (C) Raw luciferase values of nLuc-3XFlag reporters with and without the hairpin (HP) 24 h after transfection. Data are shown as mean ± SD. n = 3. (D) HeLa cells were transfected with the indicated nLuc-3XFlag reporters for 24 h and then either collected (control) or treated for 24 h with 100 µg/mL CHX. Luminescence signals for each reporter were quantified and set relative to the associated control samples. Data are shown as mean ± SD. n = 3. (E) HeLa cells were transfected with the destabilized nLuc-3XFlag-CL1/PEST reporters for 24 h and then collected (control) or treated for 3 h with 100 µg/mL CHX, 200 µM NSC119893, or 100 µg/mL CHX + 200 µM NSC119893. Luminescence signals for each reporter were quantified and set relative to the associated control samples. Data are shown as mean ± SD. n = 3.
Figure 6.
Figure 6.
Inhibition of scanning PICs by RocA blocks CHX-resistant translation. (A) RocA inhibits scanning PICs by causing the eIF4A helicase to clamp onto AGAGAG-rich sequence motifs. (B) nLuc-3XFlag reporters harboring either a (CAA)26 5′ leader, which is insensitive to RocA, or a (CAA)12 + (AGAGAG)7 5′ leader, which is sensitive to RocA. (C) RT-qPCR was used to quantify nLuc reporter mRNA levels 24 h after transfection. mRNA levels were first normalized to the cotransfected FFLuc (pGL4.13) reporter. (CAA)26 5′ leader AUG-nLuc (black) was then used to determine the relative expression levels of the other nLuc reporters (gray). Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used. (D) HeLa cells were transfected with the indicated nLuc-3XFlag reporters and treated with 0.1% DMSO (control) or 0.3 µM RocA for 24 h. Luminescence signals for each reporter were quantified and set relative to the associated control samples. Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used. (E) HeLa cells were transfected with the indicated nLuc-3XFlag reporters for 24 h and then collected (control) or treated for 24 h with 100 µg/mL CHX ± 0.3 µM RocA. Luminescence signals for each reporter were quantified and set relative to the associated control samples. Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used.
Figure 7.
Figure 7.
Slowing elongation within coding sequences stimulates non-AUG translation. (A) Multiple translation inhibitors, including CHX, ANS, DIDB, and BVD, that can inhibit elongating ribosomes within the coding sequence are predicted to generate a PIC queue over the non-AUG-start codon. (B) HHT and LTM preferentially inhibit 80S ribosomes shortly after initiation and thus should generate a PIC queue upstream of the non-AUG start codon. (C) HeLa cells were transfected with the destabilized nLuc-3XFlag-CL1/PEST reporters for 24 h and then collected (control) or treated for 3 h with vehicle (0.1% DMSO) or one of the following protein synthesis inhibitors: 100 µg/mL CHX, 0.5 µg/mL ANS, 1 µM DIDB, 1 µM BVD, 1 µg/mL HHT, or 5 µM LTM. Luminescence signals for each reporter were quantified and set relative to the associated control samples. Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used to compare expression levels with the control treatment samples. (*) P < 0.01.
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