Deubiquitinase OTUD1 Resolves Stalled Translation on polyA and Rare Codon Rich mRNAs

Abstract

OTUD1 is a deubiquitinating enzyme involved in many cellular processes including cancer and innate, immune signaling pathways. Here, we perform a proximity labeling-based interactome study that identifies OTUD1 largely present in the translation and RNA metabolism protein complexes. Biochemical analysis validates OTUD1 association with ribosome subunits, elongation factors and the E3 ubiquitin ligase ZNF598 but not with the translation initiation machinery. OTUD1 catalytic activity suppresses polyA triggered ribosome stalling through inhibition of ZNF598-mediated RPS10 ubiquitination and stimulates formation of polysomes. Finally, analysis of gene expression suggests that OTUD1 regulates the stability of rare codon rich mRNAs by antagonizing ZNF598.

Keywords: ribosome stalling; translation; ubiquitination.

FIG 1

OTUD1 Interacts with Elongating Ribosomes. (A) Schematic diagram of OTUD1-TurboID proximity labeling assay. (B) Gene ontology analysis of OTUD1 interactome identified by mass spectrometry. Enriched proteins with fold change more than 5 were selected for the analysis. Numbers in green bars correspond to the number of genes identified in the respective category. (C) Co-immunoprecipitation analysis of OTUD1 interaction with ribosomal proteins, initiation and elongation complex. The eluates were analyzed by Western blotting using indicated primary antibodies. WCL = whole cell lysate.

FIG 2

OTUD1 Suppresses polyA-induced Ribosome Stalling. (A) Schematic representation of the polyA ribosome stalling reporter system. (B) Normalized GFP:RFP fluorescence ratio of the polyA ribosome stalling reporter system coexpressed with OTUD1 WT and/or ZNF598. Significance was compared using the two-tailed Student's t test, * = P < 0.05; *** = P < 0.001. Error bars represent the mean ± SD. (C) OTUD1 structure (pdb code: 4BOP) with the catalytic cysteine (C320) marked in red. (D) Normalized GFP:RFP fluorescence ratio of the polyA ribosome stalling reporter system coexpressed with OTUD1 WT and OTUD1 C320R. Significance was compared using the two-tailed Student's t test. Error bars represent the mean ± SD. (E) The Western blot analysis of ribosomal protein S10 ubiquitination in HEK293 cell lysates with overexpression of OTUD1 WT or/and ZNF598. (F) Co-immunoprecipitation analysis of OTUD1 interaction with ZNF598. The eluates were analyzed by Western blotting using the indicated primary antibodies. (G) Amount of endogenous ZNF598 (left) or OTUD1 (right) mRNA in HEK293 cells upon overexpression of OTUD1 or ZNF598, respectively, were analyzed by RT-PCR. Significance was compared using the two-tailed Student's t test, *** = P < 0.001. Error bars represent the mean ± SD.

FIG 3

OTUD1 Promotes Formation of Polysomes. (A) Polysome analysis using Ribo Mega-SEC. Cell lysates from HEK293 cells transfected with empty vector or WT OTUD1 were separated using Agilent Bio SEC-5 2000 Å column. The retention time is indicated on the x axis and the UV absorbance at 260 nm is shown on the y axis. (B). Quantification of the area under the polysomal peak. The values represent a mean ± SD from two biological replicates. (C) The highlighted fractions from (A) were analyzed by Western blotting with the indicated primary antibodies. SPCs = small protein complexes (left panel). The relative abundance of ribosomal proteins (RPS6, RPS10 and RPL17) present in the polysomal fractions (1–4) was quantified by densitometry analysis of band intensities using the indicated formula (right panel).

FIG 4

OTUD1 and ZNF598 affect stability of rare codon rich mRNAs. (A) and (B) Gene set enrichment analysis of genes with low CAI using ZNF598 (A) and OTUD1 (B) expression as a continuous label. The y axis represents enrichment score (ES) and on the x axis are genes (vertical black lines) represented in the gene set. The green line connects points of ES and genes. ES is the maximum deviation from zero as calculated for each gene going down the ranked list and represents the degree of over-representation of a gene set at the top or the bottom of the ranked gene list. The colored band at the bottom represents the degree of correlation of genes with ZNF598 (A) and OTUD1 (B) expression. (C) and (D) Correlation of ZNF598 (C) and OTUD1 (D) and C-MYC mRNA levels. (E) Expression of 7 genes from the bottom 1% (those with the lowest CAI) which expression had the strongest negative correlation with OTUD1 (B), was analyzed by RT-PCR HEK293 cell lines. Significance was compared using the two-tailed Student's t test, * = P < 0.05. Error bars represent the mean ± SD. (F) Schematic model of OTUD1-mediated translation control. Abundance of rare codons causes translation slowdown eventually triggering mRNA decay (left). Alternatively in response to ribosome collision ZNF598 may activate a stalling pathway that leads to reversible translation pause; depending on presence of cognate tRNA, translation can be resumed or no-go decay is initiated. OTUD1 acts as an antagonist to ZNF598 and by suppressing stalling pathway facilitates rare codon-mediated decay (right).