DDX3 interacts with USP9X and participates in deubiquitination of the anti‐apoptotic protein MCL1

Abstract

Here, we describe a novel interaction between the RNA helicase DDX3 and the deubiquitinase ubiquitin-specific peptidase 9 X-linked (USP9X) in human cells. Domain mapping studies reveal that the C-terminal region of DDX3 interacted with the N terminus of USP9X. USP9X was predominantly localized in the cytoplasm where the interaction between DDX3 and USP9X occurred. USP9X was not visibly enriched in cytoplasmic stress granules (SGs) under oxidative stress conditions, whereas overexpression of GFP-DDX3 induced SG formation and recruited USP9X to SGs in HeLa cells. Luciferase reporter assays showed that depletion of USP9X had no significant effect on DDX3-mediated translation. Given that DDX3 is not ubiquitinated upon ubiquitin overexpression, it is unlikely that DDX3 serves as a substrate of USP9X. Importantly, we found that ubiquitinated MCL1 was accumulated upon depletion of USP9X and/or DDX3 in MG132-treated cells, suggesting that USP9X and DDX3 play a role in regulating MCL1 protein stability and anti-apoptotic function. This study indicates that DDX3 exerts anti-apoptotic effects probably by coordinating with USP9X in promoting MCL1 deubiquitination.

The human DEAD-box helicase DDX3X as a regulator of mRNA translation

“…In a similar fashion to many other SG proteins, overexpression of DDX3X can trigger spontaneous SG formation ( Shih et al, 2012 ; Lai et al, 2021 ). Several disease-associated DDX3X mutations are more prone to triggering spontaneous SGs than wild-type DDX3X, and it is thought that this contributes to disease pathophysiology in medulloblastoma and neurodevelopmental delay (DDX3X syndrome) ( Valentin-Vega et al, 2016 ; Lennox et al, 2020 ).…”

“…Conflicting reports also come from studies using 5′UTR luciferase reporter assays as read-outs of DDX3X-mediated translation regulation. Several studies found that DDX3X selectively regulated transcripts with highly structured 5′UTRs and did not affect translation efficiency of reporter mRNAs containing various unstructured 5′UTRs ( Soto-Rifo et al, 2012 ; Çelik et al, 2015 ; Lai et al, 2021 ). However, Geissler et al (2012) observed negative effects on translation of both structured and unstructured 5′UTR mRNA reporters upon DDX3X knockdown.…”

“…Given that it binds many different mRNAs and translation machinery components (see section 2 and Section 3 ), it is unsurprising that DDX3X is detectable in SGs in response to a variety of stresses ( Lai et al, 2008 ; Shih et al, 2012 ; Ayalew et al, 2016 ; Adjibade et al, 2017 ; Lai et al, 2021 ). DDX3X incorporation into SGs has been suggested to be dependent on its interaction with eIF4E (see Section 3.1 ) ( Shih et al, 2012 ).…”

TGFBR2 is a novel substrate and indirect transcription target of deubiquitylase USP9X in granulosa cells

“…Increasing evidence from functional assays has demonstrated that USP9X acts as a vital modulator of cell apoptosis via direct or indirect regulation of the cell death‐related pathways including the extrinsic death receptor pathway and intrinsic mitochondrial pathway (Chen et al, 2021; Vucic et al, 2011). Multiple components such as c‐FLIP and ASK1 in the death receptor pathway, and MCL‐1 of the mitochondrial pathway are direct substrates for DUB USP9X (Kim et al, 2019; Lai et al, 2021; Nagai et al, 2009; Schwickart et al, 2010). Additionally, USP9X has an indirect impact on cell apoptosis via the cell death signaling pathways: for example, USP9X induces apoptosis in cholangiocarcinoma cells by increasing the expression of cleaved‐caspase3, a common effector of both the death receptor and the mitochondrial pathways through deubiquitination and preventing the degradation of EGLN3 (Chen et al, 2021).…”

Roles of USP9X in cellular functions and tumorigenesis (Review)

“…USP9X stabilizes MCL-1 through its interaction with the protein and the removal of the Lys48-linked polyubiquitin chains that mark a protein for proteasomal degradation ( 53 ). A recent study reported that the RNA helicase Asp-Glu-Ala-Asp-box polypeptide 3 interacted with the N-terminus of USP9X and participated in deubiquitination of MCL-1 ( 61 ). Therefore, human tumors with a high level of MCL-1 expression may be accompanied by the overexpression of USP9X ( 53 , 62 ).…”