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. 2016 May 16;6:25996.
doi: 10.1038/srep25996.

Cancer-associated DDX3X Mutations Drive Stress Granule Assembly and Impair Global Translation

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Free PMC article

Cancer-associated DDX3X Mutations Drive Stress Granule Assembly and Impair Global Translation

Yasmine A Valentin-Vega et al. Sci Rep. .
Free PMC article

Abstract

DDX3X is a DEAD-box RNA helicase that has been implicated in multiple aspects of RNA metabolism including translation initiation and the assembly of stress granules (SGs). Recent genomic studies have reported recurrent DDX3X mutations in numerous tumors including medulloblastoma (MB), but the physiological impact of these mutations is poorly understood. Here we show that a consistent feature of MB-associated mutations is SG hyper-assembly and concomitant translation impairment. We used CLIP-seq to obtain a comprehensive assessment of DDX3X binding targets and ribosome profiling for high-resolution assessment of global translation. Surprisingly, mutant DDX3X expression caused broad inhibition of translation that impacted DDX3X targeted and non-targeted mRNAs alike. Assessment of translation efficiency with single-cell resolution revealed that SG hyper-assembly correlated precisely with impaired global translation. SG hyper-assembly and translation impairment driven by mutant DDX3X were rescued by a genetic approach that limited SG assembly and by deletion of the N-terminal low complexity domain within DDX3X. Thus, in addition to a primary defect at the level of translation initiation caused by DDX3X mutation, SG assembly itself contributes to global translation inhibition. This work provides mechanistic insights into the consequences of cancer-related DDX3X mutations, suggesting that globally reduced translation may provide a context-dependent survival advantage that must be considered as a possible contributor to tumorigenesis.

Figures

Figure 1
Figure 1. MB-associated mutations in DDX3X drive the assembly of stress granules.
(a) Schematic representation of DDX3X missense mutation occurring in pediatric MB. Shown is the structural organization of DDX3X protein (ATPase/helicase and RNA binding functional motifs are denoted in orange and dark green boxes; N- and C-terminal low complexity domains are denoted in light gray). MB-associated mutation positions (yellow and red boxes) are based on three genomic studies. Cancer mutations analyzed in this study are denoted in the diagram (red boxes). (b) Low complexity domains (LCD1 and LCD2) of DDX3X are predicted to be disordered. Scores above 0.5 indicate predicted disorder by the meta-predictor PONDR-FIT. (c) Photomicrographs of DDX3X immunofluorescence in three pediatric MBs carrying mutations in DDX3X and normal brain (cerebellum and cortex). Green and blue colors represent DDX3X and DAPI staining, respectively. Boxes denote magnified area shown at the bottom of the indicated sample. (d) Quantification of protein levels in MB tumors and normal brain. Green squares represent tumors carrying mutations in DDX3X (panel c), while red represent tumors carrying the wild-type form. Mean ± SEM is shown. **P = 0.0068, Student’s t-test comparison between normal brain tissues and MB tumors. (e) Co-localization of the indicated EGFP-tagged DDX3X with the SG marker eIF4G in HeLa cells under normal culture conditions. EGFP-tagged DDX3X plasmids were transfected in HeLa cells for 24 h followed by immunofluorescence against the SG marker eIF4G (red). (f) Quantification of SGs in HeLa cells transfected with wild-type and MB-associated mutant DDX3X forms for 24 h under normal conditions. Mean ± SEM values are based on a minimum of three replicated experiments. Student’s t-test comparison between wild-type DDX3X and each MB-associated mutants is shown.
Figure 2
Figure 2. MB-associated DDX3X mutations impair mRNA translation.
(a) 35S-Met/Cys labeling of nascent polypeptides in HEK293T cells transfected for 36 h with the indicated FLAG-tagged DDX3X variants or empty vector (control). Values are the mean counts per minute (CPM) of a minimum of three replicates per sample, normalized to total protein level as a function of labeling time (min) with 35S-Met/Cys. Error bars represent SEM at each time point. Two-way ANOVA: *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Western blot (right) shows DDX3X levels in pooled replicates at each time point; β-actin served as the loading control. (b) Puromycin incorporation analysis in HEK293T cells expressing EGFP-tagged wild-type or cancer-related mutant DDX3X (G302V, G325E, M370R). Puromycin incorporation was conducted as indicated in Material and Methods. Translation is monitored by staining against puromycin (red), while SGs are detected by staining against eIF4G (magenta). Green color represents EGFP-tagged DDX3X, nuclei are stained with DAPI. Dashed lines denote cells with impaired translation as monitored by lack of puromycin; yellow boxes show magnified area at the right.
Figure 3
Figure 3. CLIP-seq of endogenous DDX3X identified RNA targets.
(a) Western blot analyses of immunoprecipitated endogenous DDX3X used in the CLIP-seq experiment. A higher-mobility protein band (indicated by *) of 150 kDa represents an oligomeric form of DDX3X identified by mass spectrometry (data not shown). (b) Radiography of γ32P-labeled DDX3X/RNA complexes migrating near the size of DDX3X. RNA material extracted for CLIP-seq is denoted by red boxes. (c) Peak density, represented by number of peaks per Kb to annotated genomic regions (TTS, transcription termination sites; Pseudo, pseudogenes; ncRNA, non-coding RNA; 5′-UTR, 5′ untranslated regions; 3′-UTR, 3′ untranslated regions; CDS, coding sequences). (d) Metagene analysis of DDX3X binding density across coding and non-coding regions of mRNA targets. To normalize differences in gene size, 5′-UTR, CDS and 3′-UTR (marked at the top) for each gene is divided into 50 bins, with 0 and 50 corresponding to the start and end of each region, respectively. The number of peaks in each bin was summarized across all genes and the density marks the number of peaks per 1Kb in each bin. (e) Examples of DDX3X CLIP targets. Coding sequences for GNB2L1 and EEFA1 mRNAs are shown in gray (intronless). Blue color denotes 5′- and 3′-UTRs. Red color above the mRNA structure denotes DDX3X binding to mRNAs. (f) Gene ontology analyses of DDX3X CLIP-seq mRNA targets obtained by DAVID Bioinformatics Resource 6.7. False discovery rate (FDR) is shown for each category.
Figure 4
Figure 4. DDX3X regulates its mRNA targets by modulating their translation, not their levels.
(a) MA (log ratio vs. abundance) plot comparing expression profiles of HEK293T cells treated with control or DDX3X siRNAs. FPKM values from RNAseq data was used in this analysis. DDX3X targets are represented as red dots, while non-target RNAs are represented as gray dots. Efficient knockdown is confirmed by detection of low mRNA levels of DDX3X (denoted by black arrow). (b) Reverse transcription-qPCR analysis of 33 DDX3X CLIP-seq targets plus two non-targets (DDX10 and TPR) in HEK293T cells treated with control or DDX3X siRNAs. GAPDH was used to normalize the samples. Mean ± SEM values are based on a minimum of two replicated experiments. Only two putative targets showed significantly altered mRNA levels (student’s t-test comparison: *P = 0.01; **P = 0.008). (c) Western blot analysis of proteins encoded by DDX3X-targeted mRNAs (SET, HELLS, GNB2L1, hnRNPA2B1, PNN) identified by CLIP-seq in HEK293T cells treated with control or DDX3X siRNAs. β-actin was used as the loading control. Reduced protein expression is observed for DDX3X and its targets. (d) Quantification of relative protein levels in HEK293T cells treated with control or DDX3X siRNAs. Values are the means of two biological replicates. Error bars represent SEM. Student t-test comparisons: **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.
Figure 5
Figure 5. MB-associated mutations in DDX3X impact the translation of mRNA targets.
(a) RNA-immunoprecipitation analysis of numerous DDX3X targets using exogenous FLAG-tagged wild-type DDX3X and two cancer mutants (G302V and G325E). Two non-target RNAs were analyzed as negative control (DDX10 and TPR). Cells transfected with empty vector and immunoprecipitated with the anti-FLAG antibody M2 served as control. 87.5% of DDX3X mRNA targets were validated by the assay (horizontal black-dotted line represents cut-off based on non-target RNAs). (b) MA plot comparing RNA-seq data sets from HEK293T cells transfected with empty vector or a DDX3X-expressing vector (wild-type and two mutants: G302V and G325E). Transfections efficiencies are confirmed by the detection of high mRNA levels of DDX3X itself (denoted by the black arrows). Black dots represent DDX3X targets, gray dots represent non-targets. (c) Reverse transcription–qPCR analysis of several DDX3X CLIP-seq targets plus two non-targets (DDX10 and TPR) in HEK293T cells in cells expressing either wild-type DDX3X or two cancer-related mutant (G302V and G325E). Mean ± SEM values are based on a minimum of two replicated experiments. ~91% of DDX3X mRNA targets showed insignificant changes in their levels between cells expressing vector and those expressing DDX3X variants (Multifactorial ANOVA: *P ≤ 0.01). (d) Western blot analyses against FLAG and CLIP-seq targets HELLS, GNB2L1, and SET in HEK293T cells transfected with FLAG-tagged wild-type DDX3X or two cancer-related mutants (G325E and M370R). (e) The mean protein levels of three biological replicates of samples shown in (d) was graphed. Error bars represent mean values ± SEM (Student’s t-test between control or cells expressing DDX3X variants; *P ≤ 0.05; **P ≤ 0.01).
Figure 6
Figure 6. Ribosome profiling illustrates that expression of MB-associated DDX3X mutant G325E results in accumulation of ribosomes at 5′-UTR of mRNAs and impairs global translation.
(a) Ribosomal density per thousand nucleotides uniquely mapped to various annotated genomic regions (TTS, transcription termination sites; Pseudo, pseudogenes; ncRNA, non-coding RNAs; 5′-UTR, 5′- untranslated regions; 3′-UTR, 3′ untranslated regions; CDS, coding sequences). (b) Metagene analyses of ribosomal densities across the mRNA structure. Shown are the relative ribosomal density curves calculated for each of the 50-binned positions among three regions of the mRNA (5′-UTR, CDS, and 3′-UTR). Vertical lines separate the three regions of the mRNA. (c) MB-associated DDX3X mutant G325E impairs global translation. Metagene analyses of ribosomal densities across the mRNA structure for total mRNA (dashed-blue line), mRNAs identified as DDX3X targets (red lines), and mRNAs not identified as DDX3X targets (green lines) by CLIP-seq. The analysis was done in control cells or cells expressing wild-type DDX3X or the G325E mutant form. Shown are the relative ribosomal density curves calculated for each of the 20-binned positions among three regions of the mRNA (5′-UTR, CDS, and 3′-UTR). Vertical lines separate the three regions of the mRNA. The P-values for significant changes of ribosomal distribution between target or non-target mRNAs in the coding sequence was calculated using Kolmogorov-Smirnov equality-of-distribution test. (d) Ribosome half-transit analyses for cells expressing vector or FLAG-tagged wild-type DDX3X or MB-associated mutant DDX3X-G325E for 24 h in HEK293T cells. 35S-Met/Cys labeling incorporation into all polypeptides (postmitochondrial supernatant, PMS) and into polypeptide released from ribosomes (postribosomal supernatant, PRS) was obtained by linear regression analysis. Mean ± SEM values are based on a minimum of four replicated experiments. Ribosome half-transit time (1/2τ) were relatively equal in all samples.
Figure 7
Figure 7. Deletion of N-terminal low complexity domain prevents cancer-related DDX3X mutant from inducing SG assembly and repressing mRNA translation.
(a) Schematic representation of full-length DDX3X or DDX3X with deletion of either N-terminal low complexity domain lacking amino acids 1–114 (ΔLCD1) or C-terminal low complexity domain lacking amino acids 553–662 (ΔLCD2). The ATPase/helicase and RNA binding functional motifs are denoted in orange and dark green boxes. The location of the MB-associated mutant G325E is shown in the scheme. (b) Live imaging of HeLa cells transfected with the indicated EGFP-tagged DDX3X constructs for 24 h and treated with sodium arsenite for a period of 25 min. Images were collected every 30 seconds. Representative images of the indicated time-points are shown. (c) Puromycin incorporation assay in HeLa cells transfected with EGFP alone or the indicated EGFP-tagged DDX3X constructs for 24 h. After pulse labeling of newly synthesized proteins using puromycin for 30 min, cells were fixed and immmunostained against G3BP1 (magenta) and puromycin (PURO, red). EGFP signal was used to visualize the desired tagged proteins (denoted by white curve lines). Nuclei were stained with DAPI (blue). (d) Quantification of SGs in HeLa cells transfected with EGFP alone or the indicated EGFP-tagged DDX3X constructs for 24 h under normal conditions. Mean ± SEM values are based on a minimum of three replicated experiments. Student’s t-test comparison between full-length DDX3X and either ΔLCD1 or ΔLCD2 mutants is shown (***P ≤ 0.001). (e) Quantification of cells expressing EGFP plasmids (EGFP+) that are concomitantly devoid of PURO incorporation [PURO(−)] from the experiment shown in (c). Mean ± SEM values are based on a minimum of three biological replicates. Student’s t-test comparison between full-length DDX3X and either ΔLCD1 or ΔLCD2 mutants is shown (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; n.s., not significant).
Figure 8
Figure 8. Knocking-down the SG nucleator factors G3BP1/G3BP2 prevents DDX3X-induced SG formation and allows normal rates of mRNA translation.
(a) Puromycin incorporation assay in HeLa cells treated with control siRNAs (left panel) or G3BP1 and G3BP2 siRNAs (right panel) for 24 h followed by transfection with the indicated EGFP plasmids for another 24 h. Immunofluorescence was performed against G3BP1 (magenta) and puromycin (PURO, red). GFP signal was used to visualize the desired tagged proteins. Shown are representative images for each sample. Dashed lines denote cells displaying DDX3X granules and are devoid of PURO incorporation. Nuclei were stained with DAPI (blue). (b) Quantification of SG formation in cells expressing EGFP (control) or EGFP-DDX3X variants (wild-type, G302V, or G325E) from the experiment shown in (a). Error bars represent mean values ± SEM (N = 3, Two-way ANOVA; **P ≤ 0.01, ***P ≤ 0.001). (c) Quantification of cells expressing EGFP plasmids (GFP+) that are concomitantly devoid of PURO incorporation [PURO(−)] from the experiment shown in (a). Error bars represent mean values ± SEM (N = 3, Two-way ANOVA; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

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