Medulloblastoma-associated DDX3 variant selectively alters the translational response to stress.

Seajin Oh ,Ryan D Leib,Michael D Taylor,Stephen N Floor,Marco A Marra,Yisu Li,Paul A Northcott,Howard Y Chang,Dedeepya Vaka,Ryan A Flynn,James Purzner,Peter Lichter,Stefan M Pfister,Lance Martin,Jennifer A Doudna,Marcel Kool,Simone Schubert,Volker Hovestadt,Christopher M Adams,David Malkin,David Jones ,Hans Jörg Warnatz ,Marcus R Breese ,T Brian ,A Sorana Morrissy ,Thomas S Risch ,Marie Laure Yaspo ,Yoon Jae Cho

doi:10.18632/oncotarget.8612

Abstract

DDX3X encodes a DEAD-box family RNA helicase (DDX3) commonly mutated in medulloblastoma, a highly aggressive cerebellar tumor affecting both children and adults. Despite being implicated in several facets of RNA metabolism, the nature and scope of DDX3′s interactions with RNA remain unclear. Here, we show DDX3 collaborates extensively with the translation initiation machinery through direct binding to 5′UTRs of nearly all coding RNAs, specific sites on the 18S rRNA, and multiple components of the translation initiation complex. Impairment of translation initiation is also evident in primary medulloblastomas harboring mutations in DDX3X, further highlighting DDX3′s role in this process. Arsenite-induced stress shifts DDX3 binding from the 5′UTR into the coding region of mRNAs concomitant with a general reduction of translation, and both the shift of DDX3 on mRNA and decreased translation are blunted by expression of a catalytically-impaired, medulloblastoma-associated DDX3R534H variant. Furthermore, despite the global repression of translation induced by arsenite, translation is preserved on select genes involved in chromatin organization in DDX3R534H-expressing cells. Thus, DDX3 interacts extensively with RNA and ribosomal machinery to help remodel the translation landscape in response to stress, while cancer-related DDX3 variants adapt this response to selectively preserve translation.

Highlights

The DEAD-box RNA helicases are a large family of multifaceted proteins that bind and remodel RNA and RNA–protein complexes in an ATP-dependent manner
To identify the RNA targets of wild-type DDX3X encodes a DEAD-box family RNA helicase (DDX3) and the impact of a catalytically defective DDX3 variant harboring a recurrent substitution at arginine 534 with histidine (DDX3R534H) [5] on RNA binding, we performed individual-nucleotide crosslinking and immunoprecipitation (FAST-iCLIP) [17] in HEK293 cell line, where a large volume of data for RNA-protein global interaction and translation landscape are available for direct comparison with our study
We did not identify a specific consensus DDX3 binding motif, we found DDX3 binds to sites high in GC content (Supplementary Figure S1E), supporting its proposed role in unwinding of secondary structures in messenger RNA (mRNA) 5′untranslated regions (UTR), and sequences 3′ to DDX3 binding sites were high in guanidine (G) content (Supplementary Figure S1E)

Summary

Introduction

The DEAD-box RNA helicases are a large family of multifaceted proteins that bind and remodel RNA and RNA–protein complexes in an ATP-dependent manner. Medulloblastoma-associated DDX3X mutations occur exclusively the helicase domains [3, 4] and have been shown (or are predicted) to result in catalytically impaired proteins with limited ATPase and helicase activity [4,5,6]. Though these mutants potentiate Wnt pathway activity when co-expressed with stabilized β-catenin [4], how the impairment of DDX3’s catalytic activity contributes to oncogenesis remains unclear given we know very little about the extent and nature of DDX3’s binding to RNA normally

Methods

Results

Conclusion