RNF10 and RIOK3 facilitate 40S ribosomal subunit degradation upon 60S biogenesis disruption or amino acid starvation

Abstract

The initiation-specific ribosome-associated quality control pathway (iRQC) is activated when translation initiation complexes fail to transition to elongation-competent 80S ribosomes. Upon iRQC activation, RNF10 ubiquitylates the 40S proteins uS3 and uS5, which leads to 40S decay. How iRQC is activated in the absence of pharmacological translation inhibitors and what mechanisms govern iRQC capacity and activity remain unanswered questions. Here, we demonstrate that altering 60S:40S stoichiometry by disrupting 60S biogenesis triggers iRQC activation and 40S decay. Depleting the critical scanning helicase eIF4A1 impairs 40S ubiquitylation and degradation, indicating mRNA engagement is required for iRQC. We show that amino acid starvation conditions also stimulate iRQC-dependent 40S decay. We identify RIOK3 as a crucial iRQC factor that interacts with ubiquitylated 40S subunits to mediate degradation. Both RNF10 and RIOK3 protein levels increase upon iRQC pathway activation, establishing a feedforward mechanism that regulates iRQC capacity and subsequent 40S decay.

Keywords: 40S degradation; 60S biogenesis; CP: Molecular biology; RIOK3; RNF10; amino acid starvation; iRQC; ribosomal subunit imbalance; ribosomal ubiquitylation.

Figure 1.. Impairing 60S biogenesis stimulates ribosome ubiquitylation

(A) 293T cells transfected with non-targeting siRNA (siCon) or three independent siRNA oligonucleotides targeting the indicated translation factor were either untreated or treated with harringtonine (HTN) for 2 h. Whole-cell extracts were analyzed by SDS-PAGE followed by immunoblotting with antibodies against uS3 and uS5. The ubiquitin-modified uS3 and uS5 are indicated by the arrows. S, short exposure; L, long exposure. (B) 293T cells transfected with control siRNA or siRNAs targeting EIF4A1 or EIF6 were either untreated or treated with the indicated inhibitors for 2 h. Whole-cell extracts were analyzed by SDS-PAGE followed by immunoblotting with antibodies against uS3. The ubiquitin-modified uS3 is indicated by the arrows. S, short exposure; L, long exposure. (C) Schematic representation of ribosome biogenesis pathways with targeted 60S biogenesis factors indicated. (D) Whole-cell extracts from 293 Flp-In cells transfected with non-targeting or 60S-biogenesis-factor-targeting siRNAs were fractionated on sucrose gradients.40S, 60S, and 80S peaks are as indicated. (Top) Absorbance at 260 nm indicating ribosomal abundance across the gradient. (Bottom) The 60S:40S and 80S:40S ratio upon knockdown of the indicated 60S biogenesis factor as calculated from the sucrose gradient absorbance profile. (E) Whole-cell extracts from 293T cells transfected with siRNAs targeting the indicated 60S biogenesis factor were immunoblotted with antibodies against uS3 and uS5. The ubiquitin-modified uS3 and uS5 are indicated by the arrows. S, short exposure; L, long exposure. (F) Ubiquitylated uS5-K58 peptide intensity quantified by mass spectrometry from 293T cells transfected with non-targeting siRNA or siRNAs targeting indicated biogenesis factors. n = 3, error bars indicate SEM; **p < 0.01 by Student’s t test.

Figure 2.. Disruption of 60S biogenesis results in a concomitant reduction in 40S levels, and excess 40S subunits are translationally active

(A) Relative median intensity of all 60S and 40S proteins as quantified by mass spectrometry in 293T cells transfected with the indicated siRNA. (B) Relative ratio of the summed intensity of 60S proteins to 40S proteins as quantified by mass spectrometry in 293T cells transfected with the indicated siRNA. (C) Normalized intensity of individual 40S and 60S proteins in 293T cells transfected with the indicated siRNA. The median protein intensity is indicated by the black bar. (D) Enhanced view of disome-containing sucrose gradient fractions from 293 Flp-In cells after knockdown of the indicated genes. Absorbance curves were aligned based on the local maximum of the disome peak. Fraction numbers indicate collections from siControl. Disome-associated half-mer peak is indicated by the arrow. (E) Representative 260 nm absorbance from selected sucrose gradient fractions for whole-cell extracts from 293 Flp-In cells transfected with non-targeting or GTPBP4-targeting siRNA. Half-mer peaks are indicated by arrows. (F) Relative ratio of the median 40S to 60S protein intensity as quantified by mass spectrometry in the indicated sucrose gradient fractions. For (A)–(F), n = 3, error bars indicate SEM; *p < 0.05 and **p < 0.01 by Student’s t test.

Figure 3.. RNF10 is required for 40S decay and is post-transcriptionally upregulated in response to 60S biogenesis disruption

(A) Volcano plot showing differentially expressed proteins between 293T cells transfected with GTPBP4- or control-targeting siRNA. Proteins with an absolute log₂ fold change >0.6 and a negative log₁₀ adjusted p > 1.3 are colored as indicated. (B) Relative mean intensity of RNF10 protein as quantified by mass spectrometry in 293T cells transfected with non-targeting (Con) or 60S-biogenesis-factor-targeting siRNA as indicated. (C) Relative median intensity of 60S and 40S proteins, normalized to siCon quantified by mass spectrometry in 293 Flp-In parental or RNF10 KO cells transfected with the indicated siRNA. (D) Schematic of the RNF10 5′ UTR with ribosome occupancy from RiboSeq data. Five putative uORFs are outlined by boxes; color indicates the reading frame of each uORF. uORF start locations and their sequence context are annotated below the corresponding tracks. (E) Top, schematic of the dual luciferase reporter. Bottom, Rluc:Fluc ratio relative to the WT RNF10 5′ UTR reporter of cells transfected with the indicated RNF10 5′ UTR reporters. (F) Rluc:Fluc ratio relative to siCon for cells co-transfected with either the WT RNF10 5′ UTR reporter or the 5′ UTR reporter lacking all five uORF start codons and GTPBP4-targeting siRNA. For (A)–(F), n = 3, error bars indicate SEM; NS, not significant; *p < 0.05 and **p < 0.01 by Student’s t test. Red lines or asterisks indicate a comparison to knockdown of 60S biogenesis alone.

Figure 4.. RNF10-mediated ribosomal ubiquitylation and 40S decay requires EIF4A1

(A) Relative median intensity of 60S and 40S proteins, normalized to siCon, as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting either GTPBP4 or EIF4A1 or both GTPBP4 and eIF4A1 as indicated. (B) Whole-cell extracts from 293 Flp-In cells transfected with siRNA targeting GTPBP4 and/or EIF4A1 as indicated were immunoblotted with antibodies against uS3 and uS5. The ubiquitin-modified uS3 and uS5 are indicated by the arrows. S, short exposure; L, long exposure. * indicates a non-specific band. (C) Relative mean intensity of RNF10 protein as quantified by mass spectrometry in 293T cells transfected with non-targeting siRNAs or siRNAs targeting either GTPBP4 or EIF4A1 or both GTPBP4 and EIF4A1 as indicated. (D) Relative median intensity of 60S and 40S proteins, normalized to siCon as quantified by mass spectrometry in 293 Flp-In cells transfected with non-targeting siRNA (siCon) or EIF4A1-targeting siRNA and with or without induction of RNF10 overexpression as indicated. For (A)–(D), n = 3, error bars indicate SEM; *p < 0.05 and **p < 0.01 by Student’s t test. Red lines, text, or asterisks indicate a comparison to siGTPBP4 alone.

Figure 5.. 60S:40S subunit imbalance activates iRQC

(A) Relative ratio of the summed intensity of 60S proteins to 40S proteins as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 and NFX1 or NFXL1 as indicated. (B) Relative median intensity of 60S and 40S proteins, normalized to non-targeting siRNA, as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 and the indicated RQC factors. (C) Relative median intensity of 60S and 40S proteins, normalized to siCon, as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting the indicated ribosomal proteins. (D) Whole-cell extracts from 293T cells transfected with siRNA targeting GTPBP4 and either non-targeting (siCon) siRNA or siRNAs targeting the indicated 40S biogenesis factor or 40S protein were immunoblotted with antibodies against uS3 and uS5. The ubiquitin-modified uS3 and uS5 are indicated by the arrows. S, short exposure; L, long exposure. (E) Ubiquitylated uS5-K58 and uS3-K214 peptide intensity quantified by mass spectrometry from 293 Flp-In cells transfected with siCon or siRNAs targeting the indicated 40S biogenesis factor or 40S protein with or without GTPBP4-targeting siRNA. (F) Relative mean intensity of RNF10 protein as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting the indicated factor. For (A)–(F), n = 3, error bars indicate SEM; NS, not significant; *p < 0.05 and **p < 0.01 by Student’s t test. Red lines, text, or asterisks indicate a comparison to siGTPBP4 alone.

Figure 6.. RIOK3 is required for RNF10-dependent 40S decay

(A) Relative mean intensity of RIOK3 protein as quantified by mass spectrometry in 293T cells transfected with non-targeting (Con) or 60S-biogenesis-factor-targeting siRNA as indicated. (B) Relative median intensity of 60S and 40S proteins, normalized to non-targeting siRNA, as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 or RIOK3. (C) Ubiquitylated uS5-K58 and uS3-K214 peptide intensity quantified by mass spectrometry from 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 or RIOK3 as indicated. (D) Relative mean intensity of indicated proteins as quantified by mass spectrometry in 293 Flp-In cells transfected with siCon or siRNAs targeting GTPBP4 or RIOK3 as indicated. For (A)–(D), n = 3, error bars indicate SEM; NS, not significant; *p < 0.05 and **p < 0.01 by Student’s t test. Red lines, text, or asterisks indicate a comparison to siGTPBP4 alone.

Figure 7.. RIOK3 ubiquitin binding is necessary to facilitate 40S degradation upon iRQC pathway activation

(A) AlphaFold 3 model with RIOK3 MIU-2 domain (gray) and ubiquitin (orange). MIU domain residues (gray text) and ubiquitin residues (orange text) at the putative interaction surface are indicated. (B) Schematics of RIOK3 WT, ΔN, and KM constructs. (C) FLAG-hemagglutinin (HA)-tagged RIOK3 was immunoprecipitated from cell lines with stable expression of wild-type or mutant RIOK3. Whole-cell extracts and HA immunoprecipitates were immunoblotted with antibodies as indicated. (D) Relative median intensity of 60S and 40S proteins, normalized to non-targeting siRNA, as quantified by mass spectrometry from parental or RIOK3 stably expressing lines transfected with siRNAs targeting GTPBP4 or RIOK3 as indicated. RIOK3 transgenes are resistant to RIOK3 siRNA. n = 3, error bars indicate SEM; NS, not significant; *p < 0.05 and **p < 0.01 by Student’s t test. (E) Whole-cell extracts from parental or RIOK3 variant stably expressing lines transfected with siRNAs targeting GTPBP4 or RIOK3 or not targeting (siCon), as indicated, were immunoblotted with antibodies against uS3 and uS5. The ubiquitin-modified uS3 and uS5 are indicated by the arrows. S, short exposure; L, long exposure. (F) Relative 60:40 summed protein abundance ratio as quantified by mass spectrometry from parental 293 Flp-In cells (black line/circles), RNF10-KO cells(red line/boxes), or RIOK3-KO cells (orange line/triangles) cultured in lysine- and arginine-free medium for the indicated time. n = 3, error bars indicate SEM; **p < 0.01 by Tukey’s HSD test comparing parental to both RNF10-KO and RIOK3-KO cell lines. ††p < 0.01 by Dunnett’s test comparing the zero time point for the cell line indicated by color. (G) Model for RNF10- and RIOK3-mediated 40S decay.