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.
DEAD-box RNA helicases are members of the SF2 superfamily of helicases, which is the largest helicase family containing 37 members in humans, 26 in yeast, and 9 in bacteria (http://www.rnahelicase.org/eukdead.htm). DEAD-box helicases use ATP to remodel RNA-RNA, RNA-DNA or RNA-protein complexes and are known to participate in all facets of the RNA metabolism1. Among these proteins, the DDX3X helicase has recently been the focus of intensive study as several recent genomic studies have identified recurrent mutations or chromosome translocations within the DDX3X gene in numerous human tumor types, including T-cell acute lymphoblastic leukemia2,3, chronic lymphoblastic leukemia4,5,6, natural killer/T-cell lymphomas7, head and neck squamous cell carcinomas8, lung carcinomas9, carcinomas of the breast corpus and uterine endometrium10, and medulloblastomas (MB)11,12,13. In MB, DDX3X is mutated in 50% and 11% of pediatric WNT- and SHH-subtypes, respectively14; and in 54% of adult SHH-subtype15 (matching the frequency of PTCH1, the most commonly mutated gene in adult MB). The roles of RNA helicases in the etiology of cancer are largely unknown, and contradictory roles have been ascribed to DDX3X as both an oncogene and tumor suppressor16,17,18. However, various recent studies have reported a positive correlation between high DDX3X levels and poor prognosis in human tumors19,20, suggesting that overexpression of DDX3X may have oncogenic implications in the etiology of this disease.
According to previous studies, DDX3X and its yeast and Drosophila orthologs (Ded1p and Belle, respectively) play multifunctional roles in cells, being implicated in transcription18,21, splicing22,23, nuclear export24,25, and mRNA translation26,27,28,29,30,31. Both DDX3X and its orthologs in other species are prominent components of cytosolic RNA granules, including stress granules (SGs)25,32,33,34,35. SGs are membrane-less cytosolic bodies composed of mRNAs and proteins that assemble when translation initiation is limiting, and are thought to represent a pool of mRNPs stalled in the process of translation initiation36,37. Importantly, DDX3X is not only present in SGs but contributes to SG assembly and regulates molecular dynamics within SGs38. A growing number of recent studies have revealed that low complexity sequence domains (LCDs) in RNA-binding proteins, including DEAD-box helicases, mediate RNA-protein assemblies such as SGs through the biophysical process of liquid-liquid phase separation35,39,40,41,42. The central helicase core of DDX3X is flanked by large N-terminal and smaller C-terminal LCDs (Fig. 1a,b). It was recently shown that the N-terminal LCD of LAF-1, the C. elegans ortholog of DDX3X, mediates the assembly of germ granules, a structure similar to mammalian SGs35, which provides insight into how DDX3X may contribute to SG assembly in mammalian cells.
All reported DDX3X mutations in MB are nonsynonymous single nucleotide variants that lie in its central helicase core, suggesting that these mutations may have deleterious consequences to the normal function of the enzyme (Fig. 1a). In agreement with this line of thought, it was recently reported that two MB-associated DDX3X mutations showed a decreased RNA unwinding activity in vitro43. In a second study, analysis of two additional MB-associated DDX3X mutants, G302V and G325E, revealed that these mutants are defective in RNA-stimulated ATP hydrolysis, and that introducing these mutations in yeasts result in defective mRNA translation of one previously known mRNA target44. However, the breadth of this putative translation defect, and its relevance to human cells expressing MB-associated mutant DDX3X are unknown.
Here, we have used complementary approaches to elucidate how MB-associated mutations interfere with the normal function of DDX3X. We demonstrate that these mutations drive the hyper-assembly of SGs in human cells, leading to impairment in mRNA translation. Additionally, using CLIP-seq, we developed a comprehensive profile of the RNA targets of endogenous DDX3X in human cells. We discovered that DDX3X is enriched in 5′-UTRs proximal to the AUG start site of a specific pool of target mRNAs, and supports their translation. MB-associated mutations do not significantly impact the ability of DDX3X to bind bona fide targets, but translation of these targets is impaired. Surprisingly, based on ribosome profiling, mutant DDX3X expression causes a broad inhibition of translation that impacts DDX3X targeted and non-targeted mRNAs alike. Importantly, this defect can be partially reversed by eliminating the N-terminal low complexity domain of DDX3X or by limiting SG assembly with a genetic approach, suggesting that SG formation mediated by MB-associated mutations contributes to the global translation defect. Thus, our study reveals novel insights into the normal function of DDX3X, uncovers the consequences of MB-associated mutations, and points to a potential strategy for therapeutic intervention for the DDX3X-mediated MB.
High levels and punctated distribution of DDX3X in pediatric medulloblastomas and other tumors
To investigate the expression level of DDX3X in pediatric MB, we performed immunofluorescence against DDX3X in a panel of these tumors with or without DDX3X mutations. Whereas DDX3X expression was relatively low in normal cerebellar or cortical tissue, DDX3X expression showed frequent up-regulation regardless of its mutational status in MB tumors (Fig. 1c,d, and Supplementary Figure S1a), consistent with prior reports in other tumor types19,20. All 3 MB samples carrying DDX3X mutations showed a significant increase in DDX3X expression relative to normal brain. Interestingly, we observed a cytoplasmic punctated distribution of DDX3X in these tumors (Fig. 1c and Supplementary Figure S1a). We also used a publically available database (http://www.proteinatlas.org/)45 to investigate the expression levels and cellular distribution of DDX3X in other tumor types and observed a similar high levels and punctate distribution in a wide range of human cancers including glioma, melanoma and breast carcinoma tumors compared to normal tissues (Supplementary Figure S1b). Thus, these data suggest that high DDX3X expression in a punctate distribution is frequent in human tumors.
MB-associated DDX3X mutations drive the hyper-assembly of stress granules
The punctated distribution of DDX3X in human tumors prompted us to investigate the subcellular localization of MB-associated DDX3X mutants in cell lines. Epitope-tagged wild-type DDX3X was expressed diffusely in the cytoplasm with rare accumulation in cytoplasmic puncta in HeLa cells (Fig. 1e). This subcellular expression pattern was consistent with the distribution of endogenous DDX3X in HeLa cells as well as in lower rhombic lip progenitor (LRLP) cells, which are the cells of origin of WNT-subtype MB (Supplementary Figure S2a). In contrast, despite comparable or lower levels of expression, MB-associated DDX3X mutants were found to assemble in discrete cytoplasmic puncta with significantly greater frequency than wild-type DDX3X (Supplementary Figure S2b, Fig. 1e). Immunofluorescence staining for the SG marker eIF4G confirmed that DDX3X mutants accumulated in SGs (Fig. 1e). Quantitative analyses showed that MB-associated mutations in DDX3X drove SG assembly to a significantly greater extent than wild-type in the absence of stress (Fig. 1f). To eliminate the potential contribution of transient transfection, which may result in DDX3X expression to a greater extent than endogenous levels, we generated Tet-On-inducible HEK293T cells expressing wild-type or the MB-associated mutant DDX3X G325E (Supplementary Figure S2c). When exogenous DDX3X expression was induced comparable to endogenous levels, wild-type DDX3X remained diffuse in the cytoplasm, whereas mutant DDX3X accumulated in SGs (Supplementary Figures 2d,e) under normal culture conditions, confirming significant mutation-dependent augmentation of SG assembly. Both, wild-type or the MB-associated mutant DDX3X were almost completely relocalized to SGs under stress conditions (Supplementary Figure 2d,e). Finally, to investigate the impact of mutations on DDX3X stability, we examined protein half-life using our Tet-on-inducible HEK293T cells. We observed that MB-associated mutant DDX3X G325E shows comparable or modestly reduced stability relative to wild-type DDX3X, consistent with comparable or modestly reduced steady state levels of mutant DDX3X (Supplementary Figure 2f). Thus, SG assembly mediated by MB-associated mutations in DDX3X is not a consequence of a greater stability of the protein.
MB-associated DDX3X mutations bind RNA and impair mRNA translation
The presence of MB-associated mutant DDX3X in SG implies that these mutants retain the ability to bind RNA. However, it has been proposed that these mutations would interfere with normal nucleic acid binding capabilities based on structural modeling predictions12. Thus, we next sought to examine the impact of MB-associated DDX3X mutations on RNA binding in intact cells. We performed immunoprecipitation (IP) of wild-type or mutant DDX3X that had been ultraviolet (UV) cross-linked to its native RNA targets. We included two additional mutants in our analysis: a point mutation within the DEAD motif of DDX3X (DDX3XDQAD), which is expected to impair the helicase/ATPase activities of the protein and to prevent release of ATP hydrolysis products46,47, and a deletion mutant within motif IVa (DDX3X∆IVa), which is expected to impair nucleic acid binding capabilities48. As expected, DDX3XDQAD showed enhanced RNA-binding capacity relative to wild-type DDX3X, whereas DDX3X∆IVa displayed impaired ability to bind RNA (Supplementary Figure S3a–c). Consistent with a role in driving SG hyper-assembly, which implies retained RNA-binding ability, we found that MB-associated DDX3X mutants effectively precipitated target RNAs in amounts similar or only modestly reduced relative to wild-type DDX3X (Supplementary Figure S3a–c).
Since SG assembly typically correlates with translational impairment49, we next sought to determine whether the SG induction by MB-associated DDX3X mutations was associated with altered translation. First, we assessed the impact of DDX3X on translation by 35S-Met/Cys metabolic labeling. Over-expression of wild-type DDX3X resulted in modest impairment of translation consistent, and this observation was concordant with the low rate of SG assembly initiated by over-expression of wild-type DDX3X (Figs 1e,f and 2a). By contrast, over-expression of DDX3X mutants, G325E and M370R, at equivalent levels resulted in significantly greater translation impairment (Fig. 2a), correlating with the high frequency of SG assembly driven by these proteins (Fig. 1e,f). Second, we monitored mRNA translation in cells expressing wild-type or mutant DDX3X by pulse-labeling with puromycin for 30 minutes. Puromycin immunostaining permits visualization of newly synthesized proteins50 and the relationship to SG assembly in individual cells. This analysis showed a striking correlation between assembly of SGs and profound impairment of translation, which was significantly more frequent when DDX3X mutants where expressed (Fig. 2b). Thus, we consistently observe impairment in mRNA translation associated with MB-associated mutations in DDX3X by two independent assays.
DDX3X binds predominantly to mature mRNAs
The translation defect mediated by MB-associated mutations in DDX3X was anticipated based on the finding that (i) mutant DDX3X binds RNA and induces SG hyper-assembly (Fig. 1 and Supplementary Figure S3), and (ii) these mutants are defective in RNA-stimulated ATPase-activity44. What was surprising, however, was the apparently broad inhibition of translation as revealed by two distinct metabolic labeling approaches given the presumed limited role of DDX3X in facilitating translation of a discrete subset of mRNA targets harboring long and highly structured 5′-UTRs25. This paradox motivated a comprehensive investigation of DDX3X RNA targets by CLIP-seq51.
First, we examined the specificity and efficiency of 5 different anti-DDX3X antibodies for IP followed by mass spectrometry (MS) of the recovered protein to confirm the specificity of the interaction (Supplementary Figure S4a,b). The best of these antibodies was used for IP of endogenous DDX3X that had been cross-linked to its native RNA targets by UV irradiation. Recovery of endogenous DDX3X was monitored by Western blot (Fig. 3a), whereas recovery of cross-linked target RNA was monitored by autoradiography after RNase treatment and end-labeling with γ32P (Fig. 3b). Recovered RNA was amplified and sequenced as previously described51. DDX3X CLIP-seq was performed in duplicates and a control experiment using IgG antibody that does not recognize DDX3X was included to monitor background signal. From each DDX3X CLIP experiment, we obtained 2.4–2.6 × 106 mapped reads in the DDX3X CLIP-seq data set compared to <18,000 in control, showing high signal to noise ratio in the CLIP-seq data (Supplementary Figure S4c). Peak calling was performed to identify DDX3X target RNAs (the complete list of DDX3X target RNAs is included in Supplementary Table S1). Two biological replicates showed high reproducibility (Pearson correlation coefficient 0.934, Supplementary Figure S4d), therefore these data were pooled for subsequent analyses.
Assessment of DDX3X peak density across defined genomic regions revealed that DDX3X binding was enriched in coding regions as well as the 5′- and 3′-UTRs, and exhibited very low binding to intronic and intergenic regions (Fig. 3c). The very high enrichment in exonic compared to intronic regions suggests that DDX3X primarily binds to mature mRNAs. Metagene analysis of DDX3X occupancy showed that within mRNA species, DDX3X binding spanned the entire transcript with a prominent enrichment in the 5′-UTR proximal to the AUG start codon (Fig. 3d,e, Supplementary Figure S4e). This binding pattern of DDX3X at 5′-UTR suggests a role for DDX3X in assisting the ribosome during late events of translation initiation, whereas binding to coding regions suggests that this helicase may also participate in subsequent elongation steps.
Standard motif-finding algorithms failed to identify a favored binding motif within the mRNA targets, suggesting that either DDX3X recognizes RNA structural features rather than a primary nucleotide sequences or that the association of DDX3X with protein partners may also account for the positional preferences of this helicase towards its target mRNAs. While DDX3X binds to abroad range of mRNA targets, gene ontology analyses using DAVID illustrated that DDX3X targets were significantly enriched in genes encoding proteins involved in RNA metabolism, particularly the process of translation, although other cellular processes such as viral reproduction, chromosome organization, and cellular trafficking were also represented (Fig. 3f and Supplementary Table S2).
DDX3X regulates the translation of its mRNA targets
With the identity of DDX3X target mRNAs in hand for the first time, we next sought to elucidate the role of DDX3X in the metabolism of these mRNAs. Our assessment began with RNAseq-based expression profiling in HEK293T cells with or without RNAi-mediated knockdown of endogenous DDX3X. Depletion of endogenous DDX3X had no significant impact on the expression level or splicing of DDX3X mRNA targets (red) and non-targets (grey) (Fig. 4a), a result we confirmed by quantitative PCR analysis for 33 individual RNA targets of DDX3X (Fig. 4b). In contrast, we found that the steady state levels of the proteins produced by DDX3X-targeted mRNAs was consistently and significantly reduced in DDX3X depleted cells (Fig. 4c,d). This observation strongly implies that DDX3X positively regulates expression of its target mRNAs at the levels of translation.
MB-associated mutations in DDX3X impair the translation of its mRNA targets
We next assessed the ability of DDX3X mutants to bind 34 specific target RNAs using IP followed by qPCR (RIP-qPCR). Consistent with the data indicating that mutant DDX3X retains RNA-binding ability (Supplemental Figure S3), this analysis confirmed that mutant DDX3X bound well to specific target mRNAs. With rare exception, target binding by DDX3X mutants did not differ significantly from target binding by wild-type DDX3X (Fig. 5a). We also evaluated the consequence of mutant DDX3X on the stability of its target RNAs by performing RNA-seq and comparison of exon-level expression in HEK293T cells transfected with wild-type or mutant DDX3X. Similar to our observations with depletion of endogenous DDX3X, over-expression of neither wild-type nor mutant DDX3X had a significant impact on the expression levels of mRNA targets of DDX3X (black) or non-targets (grey) (Fig. 5b). This result was confirmed by quantitative PCR analysis of 34 individual RNA targets of DDX3X revealed only rare changes in expression level in association with mutant DDX3X, with modest down-regulation of one mRNA (PTMA) (Fig. 5c). Thus, mutant DDX3X retains the ability to interact with target mRNAs and this does not significantly alter the expression levels of these targets. In stark contrast, over-expression of mutant but not wild-type DDX3X resulted in consistently and significantly reduced steady state levels of proteins produced by DDX3X target mRNAs (Fig. 5d,e). These results indicate that DDX3X mutations do not significantly impair RNA binding or stability, but impair translation of its target mRNAs.
Ribosome profiling illustrates that MB-associated mutations in DDX3X impair translation initiation of both targeted- and non-targeted mRNAs
To deeply investigate the translation defect caused by DDX3X mutations we employed ribosome profiling in cells expressing wild-type or mutant DDX3X. Combined with knowledge of DDX3X targeted mRNAs as revealed by CLIPseq, ribosome profiling permits assessment of DDX3X mutation on the translation of individual targeted and non-targeted mRNAs. Ribosome profiling involves purification and quantitative assessment of RNA fragments bound and protected by ribosomes in intact cells, which yields high resolution ‘footprints’ of ribosomes on actively translating messages52. Thus, we transfected HEK293T cells with empty vector, wild-type or mutant DDX3X, followed by isolation and high-throughput sequencing of ribosome-protected mRNA fragments (Supplementary Figure S5a). As expected, ribosome binding was highly enriched in 5′-UTRs and coding regions (Fig. 6a). The obtained ribosome profiles had additional characteristics consistent with accurate capture of bone fide ribosome-protected mRNA fragment, such as elevated ribosome density at the beginning and end of coding sequences, reflecting slower ribosome processivity at initiation and termination sites, and rapid drop-off in ribosome densities immediately after termination codons53.
We performed metagene analysis to analyze ribosome occupancy on a global scale. This analysis showed minimal difference in ribosome occupancy profiles between cells expressing empty vector and those expressing wild-type DDX3X (Fig. 6b), whereas cells expressing mutant DDX3X showed a significant decrease in ribosomal density within coding regions relative to cells expressing empty vector or wild-type DDX3X, consistent with a substantial global defect in translation (Fig. 6b). Surprisingly, we noted that expression of mutant DDX3X impacted targeted and non-targeted mRNAs alike, consistent with the broad impact of mutant DDX3X on translation (Fig. 6c).
Concomitant with altered ribosome density in the coding regions in cells expressing mutant DDX3X there was also a striking increase in ribosomal density in the 5′-UTR relative to cells expressing empty vector or wild-type DDX3X (Fig. 6b and Supplementary Figure S5b). This pattern of ribosome occupancy suggests mutation-dependent impairment of a DDX3X function in late events translation initiation, consistent with the occupancy pattern of mutant DDX3X on target mRNAs (Fig. 3d). Whereas these results strongly suggest a mutation-dependent defect in translation initiation, a failure of translation elongation could also contribute to the global impairment in protein synthesis we observed. To rule out this possibility, we examined ribosome half-transit times, which reflect translation elongation efficiency. This analysis showed that expression of mutant DDX3X did not significantly affect elongation rates compared to cells expressing wild-type DDX3X or vector control, suggesting no mutation-dependent defect in translation elongation (Fig. 6d).
Removing the N-terminal low complexity domain of DDX3X prevents cancer-associated mutant from inducing SG assembly and repressing mRNA translation
Ribosome profiling indicates that cancer-associated mutation impacted the translation of DDX3X targeted and non-targeted mRNAs alike (Fig. 6), consistent with the global impairment of translation revealed by metabolic labeling studies (Fig. 2). The perfect correlation between SG formation and global translation impairment at the level of single cell resolution (Fig. 2b) suggested that SG assembly itself could be the basis of translation impairment that extends beyond DDX3X targets, by sequestering non-target mRNAs and/or essential translation factors in SGs. To test this hypothesis we took advantage of recent insights into the contribution of low complexity domains (LCDs) to the assembly of SGs and related non-membrane bound organelles35,39,40,41,42. The central helicase core of DDX3X is flanked by large N-terminal and smaller C-terminal LCDs (Fig. 1a). Deletion of the N-terminal LCD of LAF-1, the C. elegans ortholog of DDX3X, significantly impedes the assembly of germ granules, a structure similar to mammalian SGs35. Thus, we engineered EGFP-tagged wild-type and mutant DDX3X lacking either N- or C-terminal LCDs (Fig. 7a). Immunofluorescence of HeLa cells transfected with these DDX3X constructs revealed that whereas the full-length proteins are efficiently recruited to SGs in response to oxidative stress, deletion of N-terminal LCD (ΔLCD1) significantly limited the recruitment of these proteins to SG under these conditions (Supplementary Figure S6a,b). Deletion of C-terminal LCD (ΔLCD2) however, showed limited impact on SG assembly (Supplementary Figure S6a,b), similar to observations with LAF-135. To further analyze the requirement of LCD1 in driving SG assembly of DDX3X, we performed live imaging in HeLa cells treated with sodium arsenite for a period of 25 min. This analysis confirmed that deletion of LCD1 limited the recruitment of both wild-type and G325E-DDX3X to SGs in response to oxidative stress (Fig. 7b). Quantitative analyses confirmed that deleting LCD1 of DDX3X significantly prevented SG hyper-assembly mediated by both wild-type DDX3X and the MB-associated mutant G325E under normal and stress conditions (Fig. 7c,d). Importantly, deletion of LCD1 also reversed the translation defect that accompanied DDX3X-depended SG formation as monitored by puromycin incorporation (Fig. 7c,e). These results suggest that SG hyper-assembly contributes to the global translation defect caused by cancer-associated DDX3X mutations.
SG hyper-assembly mediated by MB-associated DDX3X mutations are independent of eIF2α phosphorylation
A possible mechanism whereby MB-associated mutations in DDX3X could induce SG assembly and affect global translation is induction of a stress response, perhaps through accumulation of abnormal mRNA, leading to phosphorylation of eukaryotic translation initiation factor alpha (eIF2alpha) and activation of an integrated stress response that includes SG assembly and global translation impairment54. To investigate this possibility, we treated HeLa cells with the small molecule inhibitor of the integrated stress response, ISRIB, which potently inhibits stress granule assembly downstream of eIF2alpha phosphorylation55. Whereas ISRIB significantly inhibited SG assembly induced by arsenite treatment (Supplementary Figure S7a,b), it did not prevent the formation of SG initiated by MB-associated DDX3X mutants (Supplementary Figure S7a,c). Similarly, the PERK inhibitor GSK260641456 did not prevent the formation of SGs mediated by MB-associated mutations (Supplementary Figure S8). Together, these data indicate that the initiation of SG assembly by DDX3X is independent of eIF2alpha phosphorylation.
Depletion of the SG assembly factors G3BP1/G3BP2 prevents DDX3X-induced SG formation and allows normal rates of mRNA translation
To further evaluate the role of SG hyper-assembly in the translation defect caused by cancer-associated DDX3X mutations we took advantage of two RNA-binding proteins that are essential to SG assembly, G3BP1 and G3BP257. As expected, concomitant depletion of endogenous G3BP1 and G3BP2 in HeLa cells prevented arsenite-induced SG assembly as monitored by immunostaining against the SG marker eIF4G (Supplementary Figure S9). We next assessed how inhibition of SG assembly impacted the translation defect associated with mutant DDX3X. We determined that depletion of endogenous G3BP1/G3BP2 significantly diminished MB-associated mutant DDX3X-induced SG formation (Fig. 8a,b, and Supplementary Figure S9), and, remarkably, also reversed the global translation defect (Fig. 8a,c). These results confirm that SG hyper-assembly is largely responsible for the global translation defect caused by cancer-associated DDX3X mutations and points towards a strategy of therapeutic intervention for the downstream consequences of DDX3X mutations.
The DEAD-box RNA helicase DDX3X has recently emerged a protein commonly mutated in multiple tumor types2,3,4,5,6,7,8,9,10, and it is among the most frequently mutated genes in pediatric and adult MB11,12,13,14,15. We undertook a comprehensive, unbiased evaluation of DDX3X function and the consequences of DDX3X missense mutations in cancer by analyzing a spectrum of these mutations occurring in MB tumors using cell biological, biochemical, genomic, and proteomic approaches. We found that cancer-related DDX3X mutations were associated with hyper-assembly of SGs that was accompanied by a striking impairment in mRNA translation (Figs 1 and 2). We performed a comprehensive assessment of the RNA targets bound by endogenous DDX3X, which revealed that DDX3X bound a specific pool of mature mRNAs with a particular enrichment toward 5′-UTRs and supported their translation (Figs 3 and 4). We also determined that cancer-associated variants did not substantially impact the ability of DDX3X to bind mRNA targets, although translation of target mRNAs was significantly impaired (Fig. 5). The significant reduction in global protein synthesis associated with expression of mutant DDX3X seemed greater than could be accounted for by the specific pool of DDX3X target mRNAs (Fig. 2). Thus, we examined global translation directly by ribosome profiling, a technique which provides a snapshot of ribosome occupancy across the breadth of the translatome52. This analysis indicated that expression of a MB-associated DDX3X mutant resulted in a global decrease of ribosome occupancy at coding sequences of mRNAs, with a concomitant accumulation of ribosomes at 5′-UTRs, consistent with a defect in translation initiation (Fig. 6). Remarkably, this analysis revealed that translation impairment was not limited to DDX3X targets, but also extended to mRNAs not bound by DDX3X (Fig. 6), indicating that MB-associated mutations in DDX3X cause a global impairment in translation. This broad impairment of translation in cells expressing mutant DDX3X as shown by ribosome profiling correlates with mutation-dependent hyper-assembly of SGs (Fig. 1). Indeed, a puromycin incorporation assay that permits cell-by-cell assessment of translation showed strong correlation between mutant DDX3X-dependent SG assembly and profoundly impaired translation (Fig. 2).
We propose that cancer-associated missense mutations in DDX3X results in a global impairment of protein synthesis by (i) directly binding and blocking translation initiation of target mRNAs that leads to the nucleation of SGs and the sequestration of these targets into SGs, and (ii) indirectly promoting the recruitment of non-target mRNAs and essential translation factors to SGs. This model predicts that global translation impairment caused by expression of mutant DDX3X could be limited or potentially reversed by inhibiting SG assembly. We investigated this possibility first by deleting the N- and C-terminal LCD sequences within DDX3X that contribute to the concentration-dependent liquid-liquid phase separation that underlies SG assembly35,39,40,41,42. Second, we inhibited SG formation by genetic depletion of G3BP1/2 factors that are essential for SG assembly. Indeed, deletion of N-terminal low complexity domain of DDX3X or genetic depletion of G3BP1/2, significantly reversed DDX3X-mediated SG hyper-assembly and restored translation as assessed by monitoring puromycin incorporation in nascent proteins and assessment of global protein synthesis (Figs 7 and 8). Thus, our data suggest that the SG hyper-assembly mediated by MB-associated mutations in DDX3X contributes to global translation inhibition independent of a primary effect at the level of translation initiation. Importantly, the fact that cancer-associated mutant DDX3 forms drive hyper-assembly of SGs even under non-stress conditions strongly suggest that the mutant forms of the protein may possess gain of function alterations that somehow bestow stress granule nucleator activity. Together, these results suggest that drugs that limit SG assembly and/or promote stress granule disassembly might reverse translation defects caused by cancer-associated DDX3X mutations and potentially contribute treatment strategies for tumors that harbor DDX3X mutations.
Mechanistic insights into DDX3X role in mRNA translation
CLIP-seq showed that DDX3X preferentially binds 5′-UTR proximal to the AUG start codon (Fig. 3); and coincidentally, ribosome profiling revealed that expression of a MB-associated mutant DDX3X led to a significant accumulation of ribosomes within 5′-UTRs (Fig. 6b). Thus, we speculate that DDX3X may play an important role during late events of translation initiation, perhaps by resolving secondary structures at regions proximal to AUG start sites on mRNA targets or by positioning competent monosomes at AUG start sites and priming translation elongation. However, additional experimental data must be obtained to support these conclusions. Another interestingly finding from our CLIP-seq data was that DDX3X not only bound 5′-UTR regions on mRNAs, but unexpectedly, there was substantial binding within coding sequences and 3′-UTRs (Fig. 3c,d). These observations suggest the possibility that DDX3X participates in additional steps of the mRNA metabolism, including translation elongation or mRNA decay. Nevertheless, we did not detect significant changes in translation elongation upon expression of MB-associated mutations in DDX3X (Fig. 6). It will be interesting to elucidate the biological significance of DDX3X binding in coding regions and 3′-UTRs in future studies.
Contribution of cancer-associated missense mutations in DDX3X to the etiology of the disease
The high recurrence of somatic DDX3X mutations in numerous cancer types suggests that perturbation of translational regulation by DDX3X plays an important role in tumor development. While it is possible that DDX3X missense mutations contribute to tumorigenesis through inhibited expression of a specific tumor suppressor, an alternative possibility is that altered translation due to DDX3X mutations contributes to tumor development as an adaptive mechanism to metabolic stress. This notion is consistent with a growing body of evidence indicating that deregulation of translation is a common mechanism by which diverse oncogenic pathways promote cellular transformation and tumor development58. Deregulation of translation is presumed to confer growth or survival advantages to tumor cells, in many cases by augmenting protein synthesis59. However, in certain circumstances inhibition of translation can confer survival advantages to tumor cells, and is particularly important for metabolic adaptation during nutrient or oxygen deprivation59. An excellent example of translation-related metabolic adaptation is the recent report that eukaryotic elongation factor 2 kinase (eEF2K), which is activated by AMP-kinase (AMPK), confers survival of MB tumor cells under acute nutrient depletion by blocking translation elongation60. We propose that DDX3X mutations act similarly, inhibiting global translation to support metabolic adaptation of MB cells; and it achieves this by promoting hyper-assembly of SGs during metabolic adaptation. However, we cannot exclude the possibility that mutation-dependent SG formation plays a role in the regulation of specific signaling pathways, either by inhibiting specific mRNAs or sequestering key molecules in SGs, thus contributing to tumorigenesis. Either case, drugs that disassemble SGs may disarm cancer cells in MB patients. Our data is consistent with recent findings that demonstrate that SGs play critical roles during tumor progression by promoting invasion and metastasis in vivo61. It is worth mentioning that despite we observe DDX3X aggregates resembling SGs in MB and other tumor types (Fig. 1 and Supplementary Figure S1), it remains to be determined whether these structures reflect an accumulation of SGs. Elucidating the precise mechanism whereby induction of SG assembly and inhibition of translation by mutations in DDX3X contribute to cancer remains to be determined but is only growing in importance.
While in MB tumors nearly all DDX3X mutations are nonsynonymous single nucleotide variants that are capable of expressing stable DDX3X proteins harboring single amino acid substitutions (Fig. 1a), other tumor types such as various blood cancers often display other DNA changes leading to frameshifts, spliced variants, and chromosome rearrangements between the DDX3X and other loci2,3,5,6. Thus, elucidating the precise mechanism whereby these genomic variations in DDX3X interfere with the normal function of this helicase in each individual tumor type and how they contribute to tumorigenesis remains to be determined but is only of growing importance.
Material and Methods
Full-length human DDX3X cDNA clone was purchased from Thermo Scientific (Accession BC011819, Clone ID 3617040). Point mutations on DDX3X (T275M, G302V, G325E, M370R) that resembled mutations found in patients with MB were made using the QuickChange II XL Site-Directed Mutagenesis kit (Agilent Technologies). DDX3X point mutants A222P, R351W, L353F, D354V, and P568P were kindly provided by Dr. Eric Enemark (SJCRH). N-terminal FLAG- or GFP-tagged DDX3XWT and the respective mutants were PCR-generated and cloned into EcoRI and XhoI sites in the mammalian expression vector pCDNA 3.1. Plasmid pCDNA3.1 FLAG-tagged DDX3XDQAD, which directs the expression of the DDX3X mutant harboring a mutation in the DEAD motif, was constructed with an E348Q substitution by QuickChange II XL Site-Directed Mutagenesis method. The pCDNA3.1 FLAG-tagged DDX3XΔIVa construct, which directs the expression of DDX3X lacking motif IVa, was done by deleting HGDRSQRDRE472-481 residues of human DDX3X by using the overlap-extension PCR method. All plasmids were verified by restriction digestion and sequencing methods. HEK293T cells stably expressing FLAG-DDX3XWT or FLAG-DDX3XG325E were generated by lentiviral transduction of a pCLEG-Tet2-FLAG DDX3X plasmid. The pCLEG-Tet2-FLAG-DDX3X plasmid was constructed by releasing the dsRedEX2 from the pCLEG-Tet2-dsRedEx1 plasmid (Vector Development and Production, SJCRH) and inserting the FLAG-wild-type or the DDX3X mutant G325E into the EcoRI and NotI positions. HEK293T cells were transduced with the lentiviral plasmid, plated at 1 × 105 cells per well in a 6-well dish in DMEM +10% fetal calf serum. Transduced cells were selected by sorting GFP+ (which is expressed by a separate EF1a promoter within the pCLEG-Tet2-FLAG-DDX3X plasmid) cells by flow cytometry. For EGFP-DDX3XWT/G325E-ΔLCD1, PCR amplification was carried out from amino acid 115 to 661 with stop codon. For delta For EGFP-DDX3XWT/G325E-ΔLCD2, PCR amplification was carried out from amino acid 1 to 581 with stop codon. Overlap PCR extension method was followed using Pfu Ultra Hotstart enzyme to create EGFP-DDX3XWT/G325E-ΔLCD1/ΔLCD2. These fragments were cloned into pCDNA 3.1 at BamH1 and Xho1 sites.
35S-Met/Cys metabolic labeling
HEK293T or LRLPs cells seeded on poly-L-lysine treated 60 mm plates (~50% confluent) were transfected with 10 μg FLAG-DDX3X plasmids for 48 h or 100 nmol control/DDX3X siRNA oligos for 48 h. Media was removed and cells were washed with pre-warmed 1× PBS twice. Cells were incubated for 30 min in labeling media (DMEM, Corning, cat. no. 17-204-CL) supplemented with dialyzed FBS (w/o glutamine and methionine). 100 μCi 35S-Met/Cys (EasyTag EXPRESS Protein Labeling Mix, Perkin Elmer, cat. no. NEG772) diluted in labeling media was then added to plates for the desired time points. After each time point, cells were washed with 1× cold PBS twice, and lyzed in NP-40 buffer on ice for 20 min. Lysates were spun down at 14,000 r.p.m. at 4 °C for 10 min. 35S-Met/Cys incorporation was measured using liquid scintillation counting.
Puromycin incorporation assay
For Figs 2 and 7, HEK293T cells were transfected with with 0.25 μg/well (4-well glass slide, Millipore) of control plasmid, or with the indicated EGFP-DDX3X fusion constructs for 24 h using FuGENE 6 as indicated by the company. For Fig. 8, HeLa cells were first treated with ON-TARGETplus human G3BP1 and G3BP2 siRNAs (Thermo Scientific, cat. no. L-012099-00 or LU-015329-01-0002), or ON-TARGETplus non-targeting control pool (Thermo Scientific, cat. no. D-001810-10) to a final concentration of 100 nmol using Dharmafect 1 transfection reagent for 24 h as indicated by the company. Then, cells were transfected with EGFP plasmids as indicated above. After transfection, pulse labeling was done with Puromycin (InvivoGen, cat. no. ant-pr-1) diluted in DMEM media (1 μg/mL) for 30 min at 37 °C and with a 5% CO2 concentration. Cells were then washed with 1× PBS and immunofluorescence was performed next against Puromycin and eIF4G or G3BP1/2. Images were captured using a LSM510 (Zeiss) confocal microscope with a 63X or 20X objectives and Zeiss ZEN software.
For RNAseq analyses, DDX3X was knock-down by treating HEK293T cells with control or DDX3X siRNA oligos (for information of siRNA oligos refer to supplementary information) for 48 h, whereas for ectopic expression of DDX3X cells were transfected with 5 μg GFP alone or GFP-DDX3X variants for 32 h. GFP+ cells were then collected by flow cytometry (BD Biosciences). RNA-sequencing libraries were prepared using approximately 1 μg total RNA using TruSeq RNA Sample Prep v2 kits per the manufacturer’s instructions (Illumina). Details of statistical procedures are provided in supplemental information.
See supplemental information for detailed CLIP-seq. Briefly, for CLIP-seq HEK293T cells were UV treated and then lysed with NP-40 buffer. Immunoprecipitation of endogenous DDX3X was performed using the mouse monoclonal anti-DDX3X antibody (clone 2253C5, Santa Cruz) in samples previously treated with DNAse and various concentrations of RNAse. RNA was labeled at the 5′ end with γP32 ATP by using T4 polynucleotide kinase. RNA/DDX3X complexes were resolved by SDS-PAGE and transferred to nitrocellulose membranes. RNA was then extracted, and the library was prepared. Details of statistical procedures are provided in supplemental information.
HEK293T cells plated at low confluency were transfected with either empty vector or FLAG-tagged DDX3X constructs for 24 h. To stall ribosomes to mRNA, cycloheximide (100 μg ml−1; Sigma-Aldrich) was added to cells for 5 min. Cell lysis, nuclease footprinting, ribosome recovery, and RNA-library preparation were done as previously described52. After the library construction, deep sequencing was performed as for CLIP-seq. Details of statistical procedures are provided in supplemental information.
How to cite this article: Valentin-Vega, Y. A. et al. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation. Sci. Rep. 6, 25996; doi: 10.1038/srep25996 (2016).
Gene Expression Omnibus
We thank E. Enemark for the generous gift of plasmid reagents, the Hartwell Center of St Jude Children’s Research Hospital for assistance with high throughput sequencing, and N. Badders and J. Otero for helpful discussion. This work was supported by the American-Lebanese-Syrian Associated Charities and a Development Award from the St. Jude Comprehensive Cancer Center.