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. 2020 Sep 17;79(6):950-962.e6.
doi: 10.1016/j.molcel.2020.07.007. Epub 2020 Jul 28.

GIGYF2 and 4EHP Inhibit Translation Initiation of Defective Messenger RNAs to Assist Ribosome-Associated Quality Control

Kelsey L Hickey  1 , Kimberley Dickson  2 , J Zachery Cogan  1 , Joseph M Replogle  1 , Michael Schoof  3 , Karole N D'Orazio  4 , Niladri K Sinha  4 , Jeffrey A Hussmann  5 , Marco Jost  5 , Adam Frost  6 , Rachel Green  7 , Jonathan S Weissman  8 , Kamena K Kostova  9
Affiliations

GIGYF2 and 4EHP Inhibit Translation Initiation of Defective Messenger RNAs to Assist Ribosome-Associated Quality Control

Kelsey L Hickey et al. Mol Cell. .

Abstract

Ribosome-associated quality control (RQC) pathways protect cells from toxicity caused by incomplete protein products resulting from translation of damaged or problematic mRNAs. Extensive work in yeast has identified highly conserved mechanisms that lead to degradation of faulty mRNA and partially synthesized polypeptides. Here we used CRISPR-Cas9-based screening to search for additional RQC strategies in mammals. We found that failed translation leads to specific inhibition of translation initiation on that message. This negative feedback loop is mediated by two translation inhibitors, GIGYF2 and 4EHP. Model substrates and growth-based assays established that inhibition of additional rounds of translation acts in concert with known RQC pathways to prevent buildup of toxic proteins. Inability to block translation of faulty mRNAs and subsequent accumulation of partially synthesized polypeptides could explain the neurodevelopmental and neuropsychiatric disorders observed in mice and humans with compromised GIGYF2 function.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Fig. 1.
Fig. 1.. Genome-wide CRISPRi screen for mammalian RQC components.
(A) Diagrams of non-stop stalling reporter (GFPNon-stop) and control reporters (GFPStop and GFPpolyA) (B) Median GFP:BFP ratio of 293T cells transiently transfected with reporter constructs containing the indicated 3’ mRNA sequence (median ± SD, N=3). (C) GFP fluorescence level in cell lines stably expressing the GFPNon-stop reporter and control sgRNA or sgRNA against NEMF. (D) Workflow of FACS-based CRISPRi screen. Reporter cell line constitutively expressing GFPNon-stop is infected with the whole genome CRISPRi sgRNA library. Knockdown of genes involved in coping with ribosome stalling leads to GFP accumulation. GFP-positive and GFP-negative cells are sorted out and the sgRNAs expressed in those cells are identified via deep sequencing. (E) Volcano plot of GFP stabilization phenotype (log2(GFP high/GFP low) for 3 strongest sgRNAs) and Mann-Whitney P-values from genome-scale CRISPRi FACS screen. Negative controls are shown in lavender, targeting guides in grey, and previously characterized RQC factors are labeled with unique colors. (F) Model of the mammalian RQC pathway with screen hits highlighted.
Fig. 2.
Fig. 2.. CRISPRi genetic interaction screen for factors that affect the growth of NEMF knockdown cells.
(A) Competition assay between cells expressing a control or NEMF targeting sgRNA. (B) Workflow of growth-based CRISPRi screen. Control knockdown or NEMF knockdown cell lines were infected with the genome-scale sgRNA library and the change in the sgRNA abundance between the two cell lines following 10 cell doublings was determined by deep sequencing. (C) Expected growth phenotypes and predicted biological pathways resulting from knockdown of a hypothetical factor (X) alone or in combination with NEMF. (D) Results from genetic interaction (GI) screen for factors affecting the growth of control or NEMF knockdown cells. GI scores were derived by fitting the data to a quadratic curve (red dashed line) and calculating the distance of each point from the best-fit line. Genes exhibiting synergistic interactions with NEMF knockdown vs. control knockdown (negative GI score) are marked in blue, and genes with positive GI score (buffering interactions) are in yellow. (E) Comparison of FACS and GI CRISPRi screens. Hits from FACS screen (log2 enrichment > 1) that stabilized GFPnon-stop reporter upon individual re-testing are labeled and colored by GI score.
Fig. 3.
Fig. 3.. GIGFY2 and 4EHP are components of a pathway parallel to the RQC.
(A) Immunoprecipitation (IP) and immunoblot (IB) for endogenously tagged GIGYF2, and its binding partners, ZNF598 and 4EHP. (B) Outline of growth competition experiments. K562 cells were infected with RFP labeled construct carrying two sgRNAs (targeting + control or two targeting guides). The abundance of RFP positive cells is measured over time via flow cytometry. (C) Competition assay among cells expressing sgRNA targeting GIGYF2 and NEMF alone or in combination (mean ± SD, N=2). Dotted grey line highlights the expected growth phenotype and the black line represents the observed growth defect of the double knockdown cells. The deviation from the expected phenotype is indicative of synergistic growth interaction. (D) Competition assay of double knockdown cell lines. The bar graph represents the difference in the measured (actual) growth defect and expected phenotype (additive value of single growth defects) on day 16 of the competition assay.
Fig. 4.
Fig. 4.. GIGYF2 and 4EHP selectively inhibit translation of faulty messages harboring stalled ribosomes.
(A) BFP and GFP fold change upon knockdown of various RQC factors measured by flow cytometry (median ± SD, N=3). (B) Relative GFPNon-stop reporter mRNA levels measured by qPCR upon RQC factors knockdown (mean ± SD, N=3). (C) Translation efficiency (TE) of GFP non-stop reporter in knockdown cell (bar plot represents the mean, with data points shown, N=2). (D) Histogram of TE change in GIGYF2 knockdown compared to control. GFPNon-stop reporter is highlighted with a red line. (E) Bidirectional promoters were used to express GFP with stop codon and polyA tail, and RFP with or without stop codon (diagramed above). Cumulative distribution plot of RFP/GFP protein ratios measured by flow cytometry in RQC knockdown cells lines. Cells expressing RFPNon-stop reporters are shown as solid lines, whereas cells harboring RFPstop are shown as dashed lines. (F) Cumulative distribution plot of GFP protein levels measured by flow cytometry in RQC knockdown cells lines. Solid and dashed lines represent GFP signal from reporter also containing RFPNon-stop or RFPstop respectively. (G) BFP and GFP protein fold change of K20 stalling reporter upon RQC factors knockdown (median ± SD, N=2). (H) Cells expressing GFPNon-stop reporter were treated with the proteasome inhibitor Bortezomib for 3h and the BFP and GFP protein stabilization was measured by flow cytometry (median ± SD, N=2).
Fig. 5.
Fig. 5.. The yeast homologs of GIGYF2, Smy2p and Syh1p, are RQC factors.
(A) Schematic of control (ctr) reporter and no-go decay (NGD) reporter containing a stretch of 12 non-optimal CGA codons in the middle of the HIS3 open reading frame (top). Box plot of GFP/RFP ratios for NGD or control reporter levels in knockout yeast strains measured by flow cytometry (bottom). (B) Relative mRNA levels for ctr or NGD reporter in wild type or knockout strains measured by qPCR (mean ± SD, N=3). (C) Alignment of regions of GIGYF2, Syh1p, and Smy2p highlighting the 4EHP-binding and GYF domains.
Fig. 6.
Fig. 6.. Translational inhibition by ZNF598 is mediated through GIGYF2 and 4EHP in a ubiquitination-independent manner.
(A) Schematic of MS2-mediated recruitment of putative silencing factors to a fluorescent reporter. (B) GFP fluorescence of GFPMS2-stop reporter transiently expressed in HEK293T cells alone or with MS2BP-fusion proteins. (C) GFP fluorescence of K562 cells stably expressing GFPMS2-stop reporter alone or in combination with MS2BP-fusion proteins. (D, G) MS2-fusion protein is recruited to the GFPMS2-stop reporter in control cells or knockdown cells for ZNF598, GIGYF2 or 4EHP. The effect of the knockdown on the ability of the fusion protein to silence translation from the reporter is measured via the change in GFP fluorescence. (E) Model for GIGYF2 and 4EHP recruitment to mRNA by ZNF598. (F) GFP fluorescence from GFPMS2-stop reporter upon MS2-mediated recruitment of wild type or ubiquitination incompetent ZNF598 (ZNF598C29A or ZNF598ΔRING).
Fig. 7.
Fig. 7.. ZNF598-dependent and independent pathways for GIGYF2–4EHP recruitment and Ribosome-associated Quality Control.
(A) BFP and GFP fold change upon single or double RQC factors knockdown measured by flow cytometry (median ± SD, N=2). (B) Immunoblot (IB) of ZNF598 in wildtype, ZNF598 knockdown, and ZNF598 knockout cell lines. (C) BFP and GFP fold change upon RQC factors knockdown in a ZNF598 knockout cell line measured by flow cytometry (median ± SD, N=2). (D) Ribosome collision is detected by the collision sensor ZNF598. Its binding triggers a cascade of events that ultimately leads to the release of the stalled ribosome, and the degradation of the faulty mRNA and stalled nascent peptide. In addition, ZNF598 recruits the translation inhibitors GIGYF2 and 4EHP to the defective message, which blocks further ribosome initiation. Recruitment of GIGYF2 and 4EHP to defective messages could be mediated by factors other than ZNF598.
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