Medulloblastoma-associated mutations in the DEAD-box RNA helicase DDX3X/DED1 cause specific defects in translation.
Medulloblastoma-associated mutations in the DEAD-box RNA helicase DDX3X/DED1 cause specific defects in translation.
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DOI：
10.1016/j.jbc.2021.100296
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发表时间：
2021-01
期刊：
影响因子：
--
通讯作者：
Bolger TA
中科院分区：
文献类型：
--
作者：
Brown NP;Vergara AM;Whelan AB;Guerra P;Bolger TA
髓母细胞瘤是最常见的儿科脑癌，测序研究发现DDX 3X（一种主要参与翻译的DEAD盒RNA解旋酶）中的频繁突变。42个不同的网站被确定，这表明突变的功能影响是复杂的。为了研究这些突变如何影响DDX 3X细胞功能，我们在DDX 3X的酿酒酵母直系同源物DED 1中构建了一整套等同的突变等位基因，并在体内和体外表征了它们的作用。DDX 3X/DED 1（ded 1-mam）中的大多数髓母细胞瘤相关突变体显示出显著的生长缺陷，表明功能效应在酵母中是保守的。此外，虽然在一些突变体中翻译受到影响，但影响批量mRNA的翻译缺陷既不一致也不与生长表型相关。同样，ded 1-mam突变体中胁迫颗粒的形成增加是常见的，但与突变体生长缺陷的严重程度不相对应。相反，在几乎所有的ded 1-mam突变体中发现了5'非翻译区（UTR）含有二级结构的mRNA翻译缺陷，并且与生长表型相关。因此，我们得出结论，这些特定的翻译缺陷，而不是对翻译的一般性影响，是负责观察到的细胞表型，并可能有助于DDX 3X突变型髓母细胞瘤。重组突变蛋白的ATP酶活性和RNA结合的检查也没有发现一致的缺陷，表明翻译缺陷来自多种酶的缺陷。这项工作表明，未来对髓母细胞瘤病理学的研究应该集中在这种特定的翻译缺陷上，同时考虑到DDX 3X突变的广泛性。
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.
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