DDX3X and DDX3Y are redundant in protein synthesis

Abstract

DDX3 is a DEAD-box RNA helicase that regulates translation and is encoded by the X- and Y-linked paralogs DDX3X and DDX3Y While DDX3X is ubiquitously expressed in human tissues and essential for viability, DDX3Y is male-specific and shows lower and more variable expression than DDX3X in somatic tissues. Heterozygous genetic lesions in DDX3X mediate a class of developmental disorders called DDX3X syndrome, while loss of DDX3Y is implicated in male infertility. One possible explanation for female-bias in DDX3X syndrome is that DDX3Y encodes a polypeptide with different biochemical activity. In this study, we use ribosome profiling and in vitro translation to demonstrate that the X- and Y-linked paralogs of DDX3 play functionally redundant roles in translation. We find that transcripts that are sensitive to DDX3X depletion or mutation are rescued by complementation with DDX3Y. Our data indicate that DDX3X and DDX3Y proteins can functionally complement each other in the context of mRNA translation in human cells. DDX3Y is not expressed in a large fraction of the central nervous system. These findings suggest that expression differences, not differences in paralog-dependent protein synthesis, underlie the sex-bias of DDX3X-associated diseases.

Keywords: DEAD-box proteins; RNA; sex differences; translational control.

FIGURE 1.

(A) Graphic indicating the locations of DDX3X and DDX3Y on their respective chromosomes. Pseudoautosomal regions (PAR) are indicated. (B) Alignment of human DDX3X and DDX3Y demonstrating ∼92% sequence identity. Domain architecture of DDX3X is indicated. (C) Phylogenetic tree indicating distances between the sequences of DDX3X and DDX3Y in mammals (where sequence is available, only selected species indicated, see Supplemental Figure S1 for tree of all available mammalian sequences). The cluster of mammalian DDX3Y orthologs is indicated with red branches. Human orthologs are highlighted in yellow.

FIGURE 2.

(A) Western blot for degradation of endogenous DDX3X tagged with mini-AID (mAID) upon treatment with an auxin (indole-3-acetic acid) in HCT-116 human colorectal carcinoma cell line expressing OsTIR1 under the control of a CMV promoter (DDX3^degr). (B) Experimental schematic for ribosomal profiling after replacement of endogenous DDX3X with exogenous DDX3X or 3Y. (C) Western blot for expression of exogenous FLAG-tagged DDX3X and DDX3Y in HCT-116 cells after degradation of endogenous DDX3X. (D, left) Full and (right) zoomed in plots of differential expression analysis of RNA and ribosome profiling changes upon complementation of DDX3X with either DDX3Y or DDX3X. Point size indicates P-value of differential translation (two biological replicates, each condition). (E) Fold-change in TE between DDX3Y and DDX3X expression in mRNAs classified based on DDX3 sensitivity (as in Supplemental Fig. S2A). (F) The fold-change of the riboskew, or ratio in ribosome occupancy in the 5′ UTR versus the coding sequence under DDX3 depletion (left) or the ratio between complementation with DDX3Y or DDX3X (right). Effect size (Cliff's delta) between the “Not_significant” and “TE_down” groups is indicated.

FIGURE 3.

(A) Selected DDX3-sensitive transcripts are indicated by the effect of DDX3 on their translation (top) and the half-lives of their protein products (bottom). (B) Experimental schematic for Methionine-chase assay. (C) Western blot for protein products of DDX3-sensitive or control transcripts upon DDX3X depletion and complementation with DDX3X or DDX3Y.

FIGURE 4.

(A) Experimental schematic for in vitro translation after replacement of endogenous DDX3X with exogenous DDX3X or 3Y. (B) Western blot for degradation of endogenous DDX3X tagged with mini-AID (mAID) and expression of exogenous FLAG-tagged DDX3X and DDX3Y in HCT-116 cells after degradation of endogenous DDX3X in in vitro translation extracts. (C) Translation of in vitro transcribed reporter RNAs in lysates from control (DMSO) or DDX3X-degraded (auxin treated) HCT-116 cells as well as either FLAG-tagged DDX3X or DDX3Y on top of depletion of endogenous DDX3X (three biological replicates, each condition).

FIGURE 5.

(A) Western blots of endogenous and transfected DDX3 paralogs (DDX3X and DDX3Y) for 48 h after treatment with cycloheximide (100 µg/mL). Myc is used as a control for rapid degradation. (B, left) Scaled TPM expression of DDX3X and DDX3Y across a number of human tissues (data from GTeX). (Right) Quartile-normalized expression of DDX3X and DDX3Y across a number of human tissues (data from GTeX). DDX3Y expresson is particularly prominent in male-specific tissue types such as prostate and seminal vesicles, in the testes, and notably deplete in numerous tissues of the central nervous system.