Medulloblastoma-associated mutations in the DEAD-box RNA helicase DDX3X/DED1 cause specific defects in translation
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100296
ISSN1083-351X
AutoresNicolette P. Brown, Ashley M. Vergara, Alisha B. Whelan, Paolo Guerra, Timothy A. Bolger,
Tópico(s)RNA and protein synthesis mechanisms
ResumoMedulloblastoma is the most common pediatric brain cancer, and sequencing studies identified frequent mutations in DDX3X, a DEAD-box RNA helicase primarily implicated in translation. Forty-two different sites were identified, suggesting that the functional effects of the mutations are complex. To investigate how these mutations are affecting DDX3X cellular function, we constructed a full set of equivalent mutant alleles in DED1, the Saccharomyces cerevisiae ortholog of DDX3X, and characterized their effects in vivo and in vitro. Most of the medulloblastoma-associated mutants in DDX3X/DED1 (ded1-mam) showed substantial growth defects, indicating that functional effects are conserved in yeast. Further, while translation was affected in some mutants, translation defects affecting bulk mRNA were neither consistent nor correlated with the growth phenotypes. Likewise, increased formation of stress granules in ded1-mam mutants was common but did not correspond to the severity of the mutants’ growth defects. In contrast, defects in translating mRNAs containing secondary structure in their 5’ untranslated regions (UTRs) were found in almost all ded1-mam mutants and correlated well with growth phenotypes. We thus conclude that these specific translation defects, rather than generalized effects on translation, are responsible for the observed cellular phenotypes and likely contribute to DDX3X-mutant medulloblastoma. Examination of ATPase activity and RNA binding of recombinant mutant proteins also did not reveal a consistent defect, indicating that the translation defects are derived from multiple enzymatic deficiencies. This work suggests that future studies into medulloblastoma pathology should focus on this specific translation defect, while taking into account the wide spectrum of DDX3X mutations. Medulloblastoma is the most common pediatric brain cancer, and sequencing studies identified frequent mutations in DDX3X, a DEAD-box RNA helicase primarily implicated in translation. Forty-two different sites were identified, suggesting that the functional effects of the mutations are complex. To investigate how these mutations are affecting DDX3X cellular function, we constructed a full set of equivalent mutant alleles in DED1, the Saccharomyces cerevisiae ortholog of DDX3X, and characterized their effects in vivo and in vitro. Most of the medulloblastoma-associated mutants in DDX3X/DED1 (ded1-mam) showed substantial growth defects, indicating that functional effects are conserved in yeast. Further, while translation was affected in some mutants, translation defects affecting bulk mRNA were neither consistent nor correlated with the growth phenotypes. Likewise, increased formation of stress granules in ded1-mam mutants was common but did not correspond to the severity of the mutants’ growth defects. In contrast, defects in translating mRNAs containing secondary structure in their 5’ untranslated regions (UTRs) were found in almost all ded1-mam mutants and correlated well with growth phenotypes. We thus conclude that these specific translation defects, rather than generalized effects on translation, are responsible for the observed cellular phenotypes and likely contribute to DDX3X-mutant medulloblastoma. Examination of ATPase activity and RNA binding of recombinant mutant proteins also did not reveal a consistent defect, indicating that the translation defects are derived from multiple enzymatic deficiencies. This work suggests that future studies into medulloblastoma pathology should focus on this specific translation defect, while taking into account the wide spectrum of DDX3X mutations. Cancers of the central nervous system are the second-most prevalent in children (after leukemias), and medulloblastoma is the most common pediatric brain cancer (1Northcott P.A. Jones D.T. Kool M. Robinson G.W. Gilbertson R.J. Cho Y.J. Pomeroy S.L. Korshunov A. Lichter P. Taylor M.D. Pfister S.M. Medulloblastomics: The end of the beginning.Nat. Rev. Cancer. 2012; 12: 818-834Crossref PubMed Scopus (419) Google Scholar). Medulloblastoma is considered an embryonal tumor that originates in the posterior fossa, although it also occurs in adults in some subtypes (2Blessing M.M. Alexandrescu S. Embryonal tumors of the central nervous system: An update.Surg. Pathol. Clin. 2020; 13: 235-247Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). It has been divided into four subtypes (Wnt, Shh, group 3, and group 4), primarily on the basis of transcriptome differences (3Taylor M.D. Northcott P.A. Korshunov A. Remke M. Cho Y.J. Clifford S.C. Eberhart C.G. Parsons D.W. Rutkowski S. Gajjar A. Ellison D.W. Lichter P. Gilbertson R.J. Pomeroy S.L. Kool M. et al.Molecular subgroups of medulloblastoma: The current consensus.Acta Neuropathol. 2012; 123: 465-472Crossref PubMed Scopus (1036) Google Scholar). Medulloblastoma has a 5-year survival rate of 65 to 80% (1Northcott P.A. Jones D.T. Kool M. Robinson G.W. Gilbertson R.J. Cho Y.J. Pomeroy S.L. Korshunov A. Lichter P. Taylor M.D. Pfister S.M. Medulloblastomics: The end of the beginning.Nat. Rev. Cancer. 2012; 12: 818-834Crossref PubMed Scopus (419) Google Scholar, 4Li Q. Dai Z. Cao Y. Wang L. Comparing children and adults with medulloblastoma: A SEER based analysis.Oncotarget. 2018; 9: 30189-30198Crossref PubMed Scopus (7) Google Scholar), and many survivors suffer long-term developmental and cognitive damage from current therapies (5Ris M.D. Packer R. Goldwein J. Jones-Wallace D. Boyett J.M. Intellectual outcome after reduced-dose radiation therapy plus adjuvant chemotherapy for medulloblastoma: A Children's Cancer Group study.J. Clin. Oncol. 2001; 19: 3470-3476Crossref PubMed Scopus (404) Google Scholar, 6Mabbott D.J. Penkman L. Witol A. Strother D. Bouffet E. Core neurocognitive functions in children treated for posterior fossa tumors.Neuropsychology. 2008; 22: 159-168Crossref PubMed Scopus (142) Google Scholar, 7Hanzlik E. Woodrome S.E. Abdel-Baki M. Geller T.J. Elbabaa S.K. A systematic review of neuropsychological outcomes following posterior fossa tumor surgery in children.Childs Nerv. Syst. 2015; 31: 1869-1875Crossref PubMed Scopus (28) Google Scholar). Therefore, further research into the molecular defects of medulloblastoma is needed in order to design more effective and targeted treatments that address these shortcomings. Medulloblastoma was one of the first cancers to be characterized using next-generation sequencing techniques in a series of exome- and genome-wide studies with a combined cohort of several hundred patients (8Jones D.T. Jager N. Kool M. Zichner T. Hutter B. Sultan M. Cho Y.J. Pugh T.J. Hovestadt V. Stutz A.M. Rausch T. Warnatz H.J. Ryzhova M. Bender S. Sturm D. et al.Dissecting the genomic complexity underlying medulloblastoma.Nature. 2012; 488: 100-105Crossref PubMed Scopus (576) Google Scholar, 9Kool M. Jones D.T. Jager N. Northcott P.A. Pugh T.J. Hovestadt V. Piro R.M. Esparza L.A. Markant S.L. Remke M. Milde T. Bourdeaut F. Ryzhova M. Sturm D. Pfaff E. et al.Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition.Cancer Cell. 2014; 25: 393-405Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 10Pugh T.J. Weeraratne S.D. Archer T.C. Pomeranz Krummel D.A. Auclair D. Bochicchio J. Carneiro M.O. Carter S.L. Cibulskis K. Erlich R.L. Greulich H. Lawrence M.S. Lennon N.J. McKenna A. Meldrim J. et al.Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.Nature. 2012; 488: 106-110Crossref PubMed Scopus (529) Google Scholar, 11Robinson G. Parker M. Kranenburg T.A. Lu C. Chen X. Ding L. Phoenix T.N. Hedlund E. Wei L. Zhu X. Chalhoub N. Baker S.J. Huether R. Kriwacki R. Curley N. et al.Novel mutations target distinct subgroups of medulloblastoma.Nature. 2012; 488: 43-48Crossref PubMed Scopus (539) Google Scholar). One of the most prominent findings from these studies was frequent mutations in the RNA helicase DDX3X, which had not previously been linked to medulloblastoma. In fact, in a meta-analysis, DDX3X was the second-most commonly mutated gene in medulloblastoma after the transcription factor β-catenin (CTNNB1), and the mutations were not limited to a particular subtype, although they were especially frequent in Wnt and adult Shh subtypes (1Northcott P.A. Jones D.T. Kool M. Robinson G.W. Gilbertson R.J. Cho Y.J. Pomeroy S.L. Korshunov A. Lichter P. Taylor M.D. Pfister S.M. Medulloblastomics: The end of the beginning.Nat. Rev. Cancer. 2012; 12: 818-834Crossref PubMed Scopus (419) Google Scholar, 9Kool M. Jones D.T. Jager N. Northcott P.A. Pugh T.J. Hovestadt V. Piro R.M. Esparza L.A. Markant S.L. Remke M. Milde T. Bourdeaut F. Ryzhova M. Sturm D. Pfaff E. et al.Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition.Cancer Cell. 2014; 25: 393-405Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Interestingly, the identified mutations were all single-nucleotide variants or small, in-frame indels (see Table 1), suggesting that they are not simply inactivating the gene but have a more specific effect on DDX3X function. On the other hand, the mutations were spread throughout the central “helicase domains” of DDX3X, thus complicating any prediction of their effects (Fig. 1A).Table 1Growth phenotypes of DDX3X/DED1 medulloblastoma-associated mutationsThe sites of 42 DDX3X point mutations identified in four genome-wide sequencing studies of medulloblastoma patients are organized by encoded amino acid sequence, with the orthologous change in Ded1 sequence also listed. Growth assays of the respective ded1-mam mutants were performed at 16, 25, 30, and 37 °C, demonstrating a variety of growth phenotypes: no growth or greatly inhibited growth (−, red), moderately inhibited growth (−/+, pink), and growth similar to wild-type DED1 (+, white). Lethal ded1-mam alleles (those unable to grow at any temperature) are marked in red type. Open table in a new tab The sites of 42 DDX3X point mutations identified in four genome-wide sequencing studies of medulloblastoma patients are organized by encoded amino acid sequence, with the orthologous change in Ded1 sequence also listed. Growth assays of the respective ded1-mam mutants were performed at 16, 25, 30, and 37 °C, demonstrating a variety of growth phenotypes: no growth or greatly inhibited growth (−, red), moderately inhibited growth (−/+, pink), and growth similar to wild-type DED1 (+, white). Lethal ded1-mam alleles (those unable to grow at any temperature) are marked in red type. DDX3X and its orthologs, including Ded1 in Saccharomyces cerevisiae, are members of the DEAD-box family of RNA helicases, which have critical roles in many facets of RNA biology and gene expression (for review, see (12Linder P. Jankowsky E. From unwinding to clamping - the DEAD box RNA helicase family.Nat. Rev. Mol. Cell Biol. 2011; 12: 505-516Crossref PubMed Scopus (553) Google Scholar)). While DDX3X/Ded1 has been implicated in a number of processes, it has been best characterized as a translation factor (13Sharma D. Jankowsky E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 343-360Crossref PubMed Scopus (77) Google Scholar, 14Shen L. Pelletier J. General and target-specific DExD/H RNA helicases in eukaryotic translation initiation.Int. J. Mol. Sci. 2020; 21: 4402Crossref Scopus (4) Google Scholar). Current models of eukaryotic translation initiation propose that translation factors, including the 40S ribosomal subunit, first assemble on the mRNA, and this preinitiation complex (PIC) then scans the mRNA, starting from the 5’ cap, for the start codon (15Aylett C.H. Ban N. Eukaryotic aspects of translation initiation brought into focus.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2017; 372: 20160186Crossref PubMed Scopus (18) Google Scholar). Once the start codon is recognized, conformational rearrangements commit the PIC to initiation at that site, the 60S subunit joins, and translation elongation can begin. During steady-state conditions, both Ded1 and DDX3X have been shown to stimulate initiation (13Sharma D. Jankowsky E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 343-360Crossref PubMed Scopus (77) Google Scholar, 16Chuang R.Y. Weaver P.L. Liu Z. Chang T.H. Requirement of the DEAD-box protein Ded1p for messenger RNA translation.Science. 1997; 275: 1468-1471Crossref PubMed Scopus (259) Google Scholar, 17Lee C.S. Dias A.P. Jedrychowski M. Patel A.H. Hsu J.L. Reed R. Human DDX3 functions in translation and interacts with the translation initiation factor eIF3.Nucleic Acids Res. 2008; 36: 4708-4718Crossref PubMed Scopus (119) Google Scholar, 18Lai M.C. Lee Y.H. Tarn W.Y. The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as Tip-associated protein and participates in translational control.Mol. Biol. Cell. 2008; 19: 3847-3858Crossref PubMed Scopus (150) Google Scholar). Specifically, both orthologs are proposed to unwind RNA secondary structure in the 5’UTR in order to facilitate start site scanning by the PIC (13Sharma D. Jankowsky E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 343-360Crossref PubMed Scopus (77) Google Scholar, 18Lai M.C. Lee Y.H. Tarn W.Y. The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as Tip-associated protein and participates in translational control.Mol. Biol. Cell. 2008; 19: 3847-3858Crossref PubMed Scopus (150) Google Scholar, 19Soto-Rifo R. Rubilar P.S. Limousin T. de Breyne S. Decimo D. Ohlmann T. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs.EMBO J. 2012; 31: 3745-3756Crossref PubMed Scopus (150) Google Scholar). Results from both reporter assays and ribosome profiling (Ribo-seq) studies are consistent with this proposal, showing that transcripts vary in their sensitivity to DDX3X/Ded1 depending on the extent of predicted structure in their 5’ UTRs (18Lai M.C. Lee Y.H. Tarn W.Y. The DEAD-box RNA helicase DDX3 associates with export messenger ribonucleoproteins as well as Tip-associated protein and participates in translational control.Mol. Biol. Cell. 2008; 19: 3847-3858Crossref PubMed Scopus (150) Google Scholar, 20Berthelot K. Muldoon M. Rajkowitsch L. Hughes J. McCarthy J.E. Dynamics and processivity of 40S ribosome scanning on mRNA in yeast.Mol. Microbiol. 2004; 51: 987-1001Crossref PubMed Scopus (107) Google Scholar, 21Sen N.D. Zhou F. Ingolia N.T. Hinnebusch A.G. Genome-wide analysis of translational efficiency reveals distinct but overlapping functions of yeast DEAD-box RNA helicases Ded1 and eIF4A.Genome Res. 2015; 25: 1196-1205Crossref PubMed Scopus (71) Google Scholar, 22Guenther U.P. Weinberg D.E. Zubradt M.M. Tedeschi F.A. Stawicki B.N. Zagore L.L. Brar G.A. Licatalosi D.D. Bartel D.P. Weissman J.S. Jankowsky E. The helicase Ded1p controls use of near-cognate translation initiation codons in 5' UTRs.Nature. 2018; 559: 130-134Crossref PubMed Scopus (61) Google Scholar). DDX3X/Ded1 also associates with the eIF4F translation complex, which binds to the mRNA early in PIC assembly, and it appears to have a role in this process, again in an mRNA-specific fashion (19Soto-Rifo R. Rubilar P.S. Limousin T. de Breyne S. Decimo D. Ohlmann T. DEAD-box protein DDX3 associates with eIF4F to promote translation of selected mRNAs.EMBO J. 2012; 31: 3745-3756Crossref PubMed Scopus (150) Google Scholar, 23Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.Mol. Cell. 2011; 43: 962-972Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Aryanpur P.P. Regan C.A. Collins J.M. Mittelmeier T.M. Renner D.M. Vergara A.M. Brown N.P. Bolger T.A. Gle1 regulates RNA binding of the DEAD-box helicase Ded1 in its complex role in translation initiation.Mol. Cell. Biol. 2017; 37e00139-17Crossref PubMed Scopus (7) Google Scholar, 25Gupta N. Lorsch J.R. Hinnebusch A.G. Yeast Ded1 promotes 48S translation pre-initiation complex assembly in an mRNA-specific and eIF4F-dependent manner.Elife. 2018; 7e38892Crossref PubMed Scopus (19) Google Scholar, 26Gulay S. Gupta N. Lorsch J.R. Hinnebusch A.G. Distinct interactions of eIF4A and eIF4E with RNA helicase Ded1 stimulate translation in vivo.Elife. 2020; 9e58243Crossref PubMed Scopus (3) Google Scholar). Interestingly, DDX3X/Ded1 can also act as a translation repressor (23Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.Mol. Cell. 2011; 43: 962-972Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 27Shih J.W. Tsai T.Y. Chao C.H. Wu Lee Y.H. Candidate tumor suppressor DDX3 RNA helicase specifically represses cap-dependent translation by acting as an eIF4E inhibitory protein.Oncogene. 2008; 27: 700-714Crossref PubMed Scopus (122) Google Scholar). In some cases, this may be a direct effect on translation, particularly in cellular stress, as we recently showed in conditions in which the target-of-rapamycin pathway is repressed (28Aryanpur P.P. Renner D.M. Rodela E. Mittelmeier T.M. Byrd A. Bolger T.A. The DEAD-box RNA helicase Ded1 has a role in the translational response to TORC1 inhibition.Mol. Biol. Cell. 2019; 30: 2171-2184Crossref PubMed Scopus (2) Google Scholar). In addition, however, DDX3X/Ded1 is associated with stress granules (SGs), cytoplasmic accumulations of RNAs, and RNA-binding proteins that are believed to serve as sorting and storage sites for mRNAs during conditions of translation repression (29Buchan J.R. Parker R. Eukaryotic stress granules: The ins and outs of translation.Mol. Cell. 2009; 36: 932-941Abstract Full Text Full Text PDF PubMed Scopus (851) Google Scholar, 30Anderson P. Kedersha N. Ivanov P. Stress granules, P-bodies and cancer.Biochim. Biophys. Acta. 2015; 1849: 861-870Crossref PubMed Scopus (200) Google Scholar, 31Protter D.S.W. Parker R. Principles and properties of stress granules.Trends Cell Biol. 2016; 26: 668-679Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). DDX3X/Ded1 appears to play important roles in the formation and disassembly of SGs, though the cellular consequences of this function are not completely clear (23Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.Mol. Cell. 2011; 43: 962-972Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 32Shih J.W. Wang W.T. Tsai T.Y. Kuo C.Y. Li H.K. Wu Lee Y.H. Critical roles of RNA helicase DDX3 and its interactions with eIF4E/PABP1 in stress granule assembly and stress response.Biochem. J. 2012; 441: 119-129Crossref PubMed Scopus (101) Google Scholar). These effects appear to be primarily mediated through the N- and C-terminal regions of DDX3X/Ded1, which comprise low-complexity domains that contribute to liquid–liquid phase separations (33Hondele M. Sachdev R. Heinrich S. Wang J. Vallotton P. Fontoura B.M.A. Weis K. DEAD-box ATPases are global regulators of phase-separated organelles.Nature. 2019; 573: 144-148Crossref PubMed Scopus (93) Google Scholar, 34Iserman C. Desroches Altamirano C. Jegers C. Friedrich U. Zarin T. Fritsch A.W. Mittasch M. Domingues A. Hersemann L. Jahnel M. Richter D. Guenther U.P. Hentze M.W. Moses A.M. Hyman A.A. et al.Condensation of Ded1p promotes a translational switch from housekeeping to stress protein production.Cell. 2020; 181: 818-831.e819Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The medulloblastoma-associated mutations in DDX3X are not found in these regions, however. Several studies have examined the effects of the DDX3X mutations identified in medulloblastoma. Despite the high frequency of mutations in the Wnt subtype of medulloblastoma and a reported direct effect on Wnt signaling by DDX3X (35Cruciat C.M. Dolde C. de Groot R.E. Ohkawara B. Reinhard C. Korswagen H.C. Niehrs C. RNA helicase DDX3 is a regulatory subunit of casein kinase 1 in Wnt-beta-catenin signaling.Science. 2013; 339: 1436-1441Crossref PubMed Scopus (131) Google Scholar), only minimal effects on Wnt signaling were observed with the mutants in reporter assays (10Pugh T.J. Weeraratne S.D. Archer T.C. Pomeranz Krummel D.A. Auclair D. Bochicchio J. Carneiro M.O. Carter S.L. Cibulskis K. Erlich R.L. Greulich H. Lawrence M.S. Lennon N.J. McKenna A. Meldrim J. et al.Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.Nature. 2012; 488: 106-110Crossref PubMed Scopus (529) Google Scholar). Noting that many of the mutations are located in conserved regions of the helicase core (Fig. 1, A and B), two additional studies identified biochemical defects in a subset of mutants, focusing in particular on a few mutants with severe phenotypes in vitro and in vivo (36Epling L.B. Grace C.R. Lowe B.R. Partridge J.F. Enemark E.J. Cancer-associated mutants of RNA helicase DDX3X are defective in RNA-stimulated ATP hydrolysis.J. Mol. Biol. 2015; 427: 1779-1796Crossref PubMed Scopus (35) Google Scholar, 37Floor S.N. Condon K.J. Sharma D. Jankowsky E. Doudna J.A. Autoinhibitory interdomain interactions and subfamily-specific extensions redefine the catalytic core of the human DEAD-box protein DDX3.J. Biol. Chem. 2016; 291: 2412-2421Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Further implicating translational defects in DDX3X-driven oncogenesis, Valentin-Vega and colleagues showed both an increase in SG formation and a general decrease in translation when a subset of DDX3X mutants were overexpressed in mammalian cell lines (38Valentin-Vega Y.A. Wang Y.D. Parker M. Patmore D.M. Kanagaraj A. Moore J. Rusch M. Finkelstein D. Ellison D.W. Gilbertson R.J. Zhang J. Kim H.J. Taylor J.P. Cancer-associated DDX3X mutations drive stress granule assembly and impair global translation.Sci. Rep. 2016; 6: 25996Crossref PubMed Scopus (57) Google Scholar). Finally, hinting that the translational effects of the DDX3X mutations may be complex, ribosome profiling of cells expressing a medulloblastoma-associated DDX3X mutant showed some mRNA-specific effects, particularly on stress-related genes (39Oh S. Flynn R.A. Floor S.N. Purzner J. Martin L. Do B.T. Schubert S. Vaka D. Morrissy S. Li Y. Kool M. Hovestadt V. Jones D.T. Northcott P.A. Risch T. et al.Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress.Oncotarget. 2016; 7: 28169-28182Crossref PubMed Scopus (35) Google Scholar). This previous work has contributed greatly to our understanding of the role of DDX3X/Ded1 in medulloblastoma and cancer generally. A limitation of these studies, however, is that they have generally focused on a small subset of the 42 distinct mutation sites that have been identified, largely for technical reasons. Therefore, it is unclear to what extent the results are representative or broadly applicable to the identified DDX3X mutations as a whole. Here, we have begun to address this question by taking advantage of the genetic tractability of budding yeast to construct a full set of mutations in DDX3X/DED1 at all 42 identified sites (Fig. 1A and Table 1). The S. cerevisiae model system does not lend itself to detailed pathological analysis; however, since DDX3X and Ded1 are highly conserved in both sequence and function (13Sharma D. Jankowsky E. The Ded1/DDX3 subfamily of DEAD-box RNA helicases.Crit. Rev. Biochem. Mol. Biol. 2014; 49: 343-360Crossref PubMed Scopus (77) Google Scholar, 40Sharma D. Putnam A.A. Jankowsky E. Biochemical differences and similarities between the DEAD-box helicase orthologs DDX3X and Ded1p.J. Mol. Biol. 2017; 429: 3730-3742Crossref PubMed Scopus (18) Google Scholar), we conducted a comparison of molecular and cellular defects in the mutants. After an initial screening of the full set of mutants, we further characterized the translational and biochemical defects in a large, representative subset that reflects the spectrum of effects produced by the mutations. This analysis revealed that while some mutants had significant defects in bulk translation and particular biochemical properties, the most common and well-correlated defects in medulloblastoma-associated mutants of DDX3X/DED1 were in specific translation processes, suggesting that these should be a focus in future studies of DDX3X-driven oncogenesis. To characterize the impact of the medulloblastoma-associated mutations on cellular function, we began our study by mutating yeast DED1 at the 42 conserved sites identified in medulloblastoma patient sequencing studies (Fig. 1, A and B and Table 1) (8Jones D.T. Jager N. Kool M. Zichner T. Hutter B. Sultan M. Cho Y.J. Pugh T.J. Hovestadt V. Stutz A.M. Rausch T. Warnatz H.J. Ryzhova M. Bender S. Sturm D. et al.Dissecting the genomic complexity underlying medulloblastoma.Nature. 2012; 488: 100-105Crossref PubMed Scopus (576) Google Scholar, 9Kool M. Jones D.T. Jager N. Northcott P.A. Pugh T.J. Hovestadt V. Piro R.M. Esparza L.A. Markant S.L. Remke M. Milde T. Bourdeaut F. Ryzhova M. Sturm D. Pfaff E. et al.Genome sequencing of SHH medulloblastoma predicts genotype-related response to smoothened inhibition.Cancer Cell. 2014; 25: 393-405Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 10Pugh T.J. Weeraratne S.D. Archer T.C. Pomeranz Krummel D.A. Auclair D. Bochicchio J. Carneiro M.O. Carter S.L. Cibulskis K. Erlich R.L. Greulich H. Lawrence M.S. Lennon N.J. McKenna A. Meldrim J. et al.Medulloblastoma exome sequencing uncovers subtype-specific somatic mutations.Nature. 2012; 488: 106-110Crossref PubMed Scopus (529) Google Scholar, 11Robinson G. Parker M. Kranenburg T.A. Lu C. Chen X. Ding L. Phoenix T.N. Hedlund E. Wei L. Zhu X. Chalhoub N. Baker S.J. Huether R. Kriwacki R. Curley N. et al.Novel mutations target distinct subgroups of medulloblastoma.Nature. 2012; 488: 43-48Crossref PubMed Scopus (539) Google Scholar). Because DED1 is an essential gene in yeast, these ded1-mam (medulloblastoma-associated mutation) alleles were introduced into ded1-null yeast on single-copy CEN plasmids via plasmid shuffle, replacing wild-type DED1 as the only copy in cells. Following successful replacement, 9 of the 42 ded1-mam mutations resulted in lethality (Table 1). The lethal mutations were located across the helicase core and in different conserved motifs. This result is consistent with the lethality of several other known ded1 point mutations in the conserved DEAD-box motifs (16Chuang R.Y. Weaver P.L. Liu Z. Chang T.H. Requirement of the DEAD-box protein Ded1p for messenger RNA translation.Science. 1997; 275: 1468-1471Crossref PubMed Scopus (259) Google Scholar, 23Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.Mol. Cell. 2011; 43: 962-972Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Aryanpur P.P. Regan C.A. Collins J.M. Mittelmeier T.M. Renner D.M. Vergara A.M. Brown N.P. Bolger T.A. Gle1 regulates RNA binding of the DEAD-box helicase Ded1 in its complex role in translation initiation.Mol. Cell. Biol. 2017; 37e00139-17Crossref PubMed Scopus (7) Google Scholar). Furthermore, two of the lethal mutants (G261V and G284E) were previously shown to be incapable of complementing a temperature-sensitive ded1 mutant in Saccharomyces pombe and had severe defects in enzymatic activity (36Epling L.B. Grace C.R. Lowe B.R. Partridge J.F. Enemark E.J. Cancer-associated mutants of RNA helicase DDX3X are defective in RNA-stimulated ATP hydrolysis.J. Mol. Biol. 2015; 427: 1779-1796Crossref PubMed Scopus (35) Google Scholar). The lethality of these ded1-mam mutants suggests an acute loss of DDX3X/Ded1 function for a subset of the medulloblastoma-associated mutations (see Discussion). The viable ded1-mam mutants were further studied to determine if other growth defects were present. These mutants were screened for changes in growth on nutrient-rich agar at 16, 25, 30, and 37 °C (Fig. 1C and Table 1). Many of the medulloblastoma-associated mutants displayed temperature-dependent growth inhibition, while some mutants had no noticeable defect. Of the 33 viable ded1-mam mutants, 19 showed temperature-sensitive growth defects, including 16 cold-sensitive and 3 heat-sensitive (Table 1). Overall, growth inhibition or lethality occurred in two-thirds (28 out of 42) of the medulloblastoma-associated DDX3X/ded1 mutants. To further examine the effects of the ded1-mam mutations on DDX3X/Ded1 function, we took advantage of the ability of wild-type DED1 to inhibit growth when highly overexpressed with a galactose-inducible promoter (23Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution of an eIF4F-mRNA complex.Mol. Cell. 2011; 43: 962-972Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Aryanpur P.P. Regan C.A. Collins J.M. Mittelmeier T.M. Renner D.M. Vergara A.M. Brown N.P. Bolger T.A. Gle1 regulates RNA binding of the DEAD-box helicase Ded1 in its complex role in translation initiation.Mol. Cell. Biol. 2017; 37e00139-17Crossref PubMed Scopus (7) Google Scholar, 41Beckham C. Hilliker A. Cziko A.M. Noueiry A. Ramaswami M. Parker R. The DEAD-box RNA helicase Ded1p affects and accumulates in Saccharomyces cerevisiae P-bodies.Mol. Biol. Cell. 2008; 19: 984-993Crossref PubMed Scopus (73) Google Scholar). Previously, the ability of ded1 mutations to suppress this effect by rescuing growth correlated with effects on Ded1 function in translation and SG dynamics (23Hilliker A. Gao Z. Jankowsky E. Parker R. The DEAD-box protein Ded1 modulates translation by the formation and resolution
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