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. Author manuscript; available in PMC: 2026 Oct 14.
Published before final editing as: Mol Cell. 2026 Oct 9:S1097-2765(26)00665-9. doi: 10.1016/j.molcel.2026.09.022

40S Ribosome Remodeling Triggers 18S rRNA U-tailing and DIS3L2-dependent Decay

Akruti Shah 1, Aaztli R Coria 1, Jennifer T Miller 1, Emilien Orgebin 1, Wilfried Guiblet 2, Acong Yang 1, Shuo Gu 1, Colin Chih-Chien Wu 1,*
PMCID: PMC13660426  NIHMSID: NIHMS2212996  PMID: 42854688

SUMMARY

Eliminating defective ribosomes through quality control is essential for accurate protein synthesis. However, the mechanisms that commit defective ribosomal subunits to decay remain poorly defined. Here, we identify a tandem mechanism in which ubiquitin-dependent ribosome remodeling and 18S rRNA uridylation lead to 40S ribosomal subunit decay. Specifically, we use an in vitro reconstitution system to show that the atypical kinase RIOK3 remodels 40S ribosomal subunits, thereby exposing the 3’ end of 18S rRNA. Nanopore direct RNA sequencing reveals that this remodeling event promotes oligo-uridylation, generating uridylated 18S rRNA decay intermediates. Uridylated 18S rRNA is further degraded by the 3’−5’ exoribonuclease DIS3L2. Moreover, DIS3L2-mediated exoribonucleolytic cleavage triggers endoribonucleolytic decay of the 18S rRNA, amplifying turnover. Together, our findings define a stepwise mechanism in which ribosome remodeling and RNA tailing commit defective 40S subunits to elimination, establishing a mechanistic framework for ribosome surveillance in mammalian cells.

Graphical Abstract

graphic file with name nihms-2212996-f0001.webp

eTOC Blurb –

Translational perturbations trigger ubiquitination and turnover of 40S ribosomes, yet how these events are coupled remains unclear. Shah et al. reveal that RIOK3 remodels ubiquitinated 40S subunits, exposing the 3’ end of 18S rRNA for uridylation and subsequent DIS3L2-mediated degradation. These findings link ribosomal protein ubiquitination to selective rRNA turnover.

INTRODUCTION

Ribosomes are among the most abundant macromolecular complexes in the cell and play a central role in protein synthesis. Yet, they are also highly sensitive to physiological stress, functioning as regulatory hubs that sense translational dysfunction and engage quality control pathways to restore cellular homeostasis1–3. Recent studies have shown that diverse perturbations—including nonfunctional 18S rRNA, amino acid starvation, and disruption of 60S ribosome biogenesis— trigger ubiquitination of 40S ribosomal proteins and promote selective degradation of the small ribosomal subunit4–7. Despite these advances, the mechanism by which ubiquitinated 40S ribosomes are degraded remains unresolved.

One molecular pathway that mediates this response is 18S nonfunctional rRNA decay (NRD)8,9, a surveillance mechanism that selectively eliminates defective 40S subunits containing dysfunctional 18S rRNA. A single-nucleotide mutation in the decoding center of 18S rRNA stalls ribosomes at initiation sites, thereby triggering decay through ubiquitination of ribosomal proteins uS3 and uS55,10. Moreover, the atypical RIOK3 binds these ubiquitinated 40S subunits to promote 18S rRNA decay5,11,12. Importantly, similar mechanisms operate in response to other stress conditions that impair productive translation, including amino acid starvation and defects in 60S subunit biogenesis7,11,12. Additionally, structural analysis of 40S decay intermediates further indicates that degradation begins near the 3′ end of 18S rRNA, suggesting that defective 40S subunits may undergo localized remodeling to facilitate decay11. Despite diverse translational stress conditions converging on 18S rRNA degradation, the mechanism underlying decay remains unclear.

Among eleven terminal nucleotidyltransferases, TUT4 and TUT7 are the two well-established terminal uridylyltransferases (TUTases) for modifying cytoplasmic structured noncoding RNAs13. Oligo-uridine tailing can function as a degradation signal for RNA turnover in multiple surveillance pathways13–18. Indeed, U-tailing promotes recognition by the cytoplasmic 3′−5′ exoribonuclease DIS3L2, which preferentially binds and degrades uridylated RNA substrates, including tRNAs and microRNAs13–15,19–25. Additionally, DIS3L2 has also been linked to degradation of uridylated 5.8S rRNA biogenesis intermediates26.

Here, we show that RIOK3 remodels ubiquitinated 40S ribosomal subunits to increase accessibility of the 3′ terminus of 18S rRNA for uridylation. We further show that TUTases, TUT4 and TUT7, are in part responsible for this modification. DIS3L2 then initiates 3’−5’ degradation of uridylated 18S rRNA, promoting endoribonucleolytic decay. Together, our findings establish that defective mammalian 40S subunits are eliminated through a uridylation-dependent decay mechanism, revealing how ribosome remodeling, terminal RNA tailing, and exoribonucleolytic degradation are coupled during ribosome quality control.

RESULTS

RIOK3 remodels ubiquitinated 40S subunits to promote 18S rRNA decay

The molecular mechanism by which RIOK3 promotes 18S rRNA decay remains unknown. Given the proximity of eS26 (RPS26) to the 3’ terminus of 18S rRNA in the 40S ribosomal subunit27, its established role in ribosome repair28,29, and the related remodeling activity reported for RIOK1 during 40S maturation30, we hypothesized that RIOK3-mediated remodeling may involve eS26 displacement. Such remodeling could expose the 3’ end of 18S rRNA, thereby rendering it accessible to the RNA decay machinery. To test this, we reconstituted RIOK3 activity in vitro using purified components. We performed in vitro ubiquitination with purified 40S ribosomal subunits containing HA-tagged uS3 isolated from USP10/RIOK3 double-knockout (DKO) cells, thereby eliminating potential contamination by endogenous RIOK3 and USP10, a deubiquitinase that acts on ubiquitinated uS3 and uS531. After immobilization of 40S subunits on α-HA beads, uS3 and uS5 ubiquitination was carried out using recombinant ubiquitin, RNF10 (E3), UbcH5c (E2), and Ube1 (E1) (Figures S1A–S1B). Following the ubiquitination reaction, RNF10, UbcH5c, and Ube1 were removed by stringent washes. The ubiquitinated 40S subunits were then directly incubated with recombinant wild-type (WT) or mutant RIOK3 (Figure S1C), thereby reconstituting 40S turnover events (Figure 1A).

Figure 1. RIOK3 remodels ubiquitinated 40S ribosomal subunits.

Figure 1.

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(A) Schematic of the in vitro assay for RIOK3-mediated remodeling by monitoring eS26 dissociation.

(B) Representative immunoblotting of eS26 dissociation from immobilized 40S subunits (32.7±8.3% ubiquitinated) incubated with BSA, recombinant RIOK3-WT or ΔKD (lacking kinase domain). Supernatant (dissociated) and bead (immobilized) fractions were analyzed by immunoblotting.

(C) Quantification of percentage of eS26 dissociation shown in (B), (n= 3).

(D) Representative polysome profiles from 10–30% sucrose gradients of untreated and HTN-treated (3 μg/mL for 6 h) HEK293T cells in the presence of bortezomib. Fractions were analyzed by immunoblotting with indicated antibodies, (n≥ 3). 20% of the free RNP fractions and 5% of each remaining fraction were loaded. Vinculin serves as a loading control for the free RNP fractions (red arrowheads). HTN induced uS3-ub in 10.5±1.3% of total uS3, (n= 8).

(E) Representative polysome profiles and immunoblotting for HTN-treated parental and RIOK3KO HEK293T cells. HTN induced uS3-ub in 10.9±2.0% of total uS3 (n= 3).

(F) Quantification of eS26 dissociation in parental (Par) and RIOK3KO cells. Percentage of dissociated eS26 in the free RNP fractions relative to total eS26 across all fractions, (n≥ 3).

Addition of RIOK3-WT promoted the dissociation of ~18% of eS26 from immobilized 40S subunits, resulting in significant enrichment in the supernatant fraction compared to the BSA control (Figures 1B and 1C). This is consistent with the finding that only ~30% of immobilized 40S subunits were ubiquitinated, suggesting that RIOK3 remodels a substantial fraction of ubiquitinated 40S subunits. In contrast, ubiquitinated uS3 remained associated with immobilized 40S subunits (Figure 1B). We also tested RIOK3-ΔKD, which is defective in 18S NRD (Figures S1D–S1E) and 40S subunit turnover under stress11,12. Importantly, RIOK3-ΔKD dissociated eS26 less efficiently than RIOK3-WT, although RIOK3-ΔKD bound to ubiquitinated 40S subunits with comparable efficiency (Figure S1F). Furthermore, RIOK3-WT was unable to promote eS26 dissociation without ATP (Figures S1G–S1H).

We next assessed RIOK3-mediated 40S remodeling in cells by monitoring eS26 dissociation as a measurable readout. Prior studies showed harringtonine (HTN) treatment induces ribosome stalling at translation initiation sites32, resulting in ubiquitination of uS3 and uS56,7,33 as well as mimicking the defect in the 18S decoding center5. We therefore utilized HTN treatment to trigger 40S degradation and monitored eS26 dissociation by polysome profiling followed by immunoblotting. Upon HTN treatment, eS26 was detected in the free RNP fraction (Figure 1D, compare lane 1 to 9) relative to the untreated control, indicating its dissociation form 40S subunits. Importantly, this eS26 dissociation was abolished in RIOK3KO cells (Figures 1E–1F). Although additional ribosomal proteins may also be displaced during this process, these results demonstrate that RIOK3 remodels ubiquitinated 40S subunits that is accompanied by eS26 dissociation.

18S rRNA decay intermediates accumulate uridylated tails

18S rRNA degradation proceeds through successive endo- and exoribonucleolytic cleavage events5,11. To dissect 18S rRNA decay intermediates, we performed Nanopore direct RNA sequencing on RIOK3-bound 18S rRNA from HTN-treated HEK293T cells and complemented this with amplicon-based Illumina sequencing (Figures 2A and S2A). Following alignment to the 18S rDNA reference sequence, untemplated nucleotides were extracted for downstream analysis (Figure 2A). Motif analysis revealed a striking and unexpected enrichment of oligo-uridine residues (Figure 2B).

Figure 2. RIOK3-bound 18S rRNA decay intermediates are uridylated at their 3’ termini.

Figure 2.

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(A) Schematic of the untemplated nucleotide analytical pipeline for 18S rRNA decay intermediates.

(B) MEME motif analysis revealed oligo-uridine motif in untemplated nucleotides at the 3’ ends of 18S rRNA decay intermediates. E represents the E-value significance; n denotes number of contributing sites out of 53,736 input sequences.

(C) Representative gene model of 18S rRNA depicting the read coverage of Nanopore direct RNA sequencing reads of RIOK3-bound 18S rRNA decay intermediates isolated from HTN-treated HEK293T cells, (n= 3). Positions of increased or decreased read coverage are indicated by red arrowheads. Bottom: Schematic representation of 18S rRNA domain organization.

(D-E) Representative 18S rRNA gene models illustrating the 3′ ends of oligo-uridylated (U≥ 2) from RIOK3-WT (D) and ΔKD (E) immunoprecipitation, (n≥ 2). Y-axis represents read density in reads per million (rpm).

(F) Cumulative percentage of uridylated 18S rRNA from RIOK3-WT and ΔKD immunoprecipitates isolated from HTN-treated HEK293T parental cells, (n≥ 2).

(G) Cumulative percentage of uridylated 18S rRNA for 18S:WT reporter (parental, black) and 18S:A1824C reporter from parental (red), GCN2KO (blue), RNF10KO (green), and RIOK3KO (purple) cells.

(H) Cumulative percentage of uridylated 18S rRNA reads from RIOK3-bound decay intermediates from HTN-treated HEK293T parental, TUT7KO, and TUT4/7DKO cells.

Read coverage across RIOK3-bound 18S rRNA revealed degradation within the 5’, central, and 3’ minor domains (Figure 2C), consistent with progressive endo- and exoribonucleolytic cleavages during decay5,11. Mapping of the 3’ ends of uridylated reads showed that they coincided well with the cleavage cites and were enriched toward the 3′ terminus (Figure 2D), suggesting that 18S rRNA decay intermediates are uridylated following ribonucleolytic cleavage.

Notably, ~4% of RIOK3-bound 18S rRNAs were uridylated, with a broad distribution extending up to 30 nucleotides, compared to input 18S rRNA (Figure S2B). Re-analysis of data from prolonged amino acid starvation11 revealed the same uridylation trend in RIOK3-bound 18S rRNA decay intermediates (Figure S2C), further supporting a role for RIOK3 in promoting 40S degradation5,11,12. We note that the fraction of uridylation is likely underestimated because RIOK3-bound 18S rRNAs are progressive decay intermediates that generate multiple sequencing reads per rRNA molecule, whereas intact input rRNAs generate fewer.

Because RIOK3 kinase function is critical for eS26 dissociation (Figures 1B and S1G), we next asked whether it is also required for uridylation by comparing RIOK3-WT and ΔKD immunoprecipitates. RIOK3-ΔKD-bound 18S rRNA exhibited markedly reduced uridylation (Figures S2D and 2E). To enable comparisons across samples while reducing the influence of differences in sequencing depth, we calculated cumulative distributions of untemplated oligo-U lengths, which again revealed a pronounced reduction in oligo-U tails in the RIOK3-ΔKD samples (Figure 2F).

Since nonfunctional 18S rRNA undergoes decay via the GCN2-RNF10-RIOK3 pathway5, we purified decoding-incompetent 40S subunits and sequenced the associated 18S rRNA decay intermediates to determine whether they are uridylated. Motif analysis of untemplated nucleotides revealed similar oligo-U tracts at the 3’end of decay intermediates (Figure S2E), with higher uridylation than 18S:WT counterpart (Figure 2G). Importantly, uridylation of 18S:A1824C rRNA was markedly reduced in GCN2KO, RNF10KO, and RIOK3KO cells (Figure 2G), consistent with the role of this cascade in 18S NRD5. Altogether, these findings indicate that RIOK3 promotes uridylation of 18S rRNA during 40S turnover.

TUTases mediate uridylation of 18S rRNA during decay

Given the oligo-U signature in RIOK3-bound and nonfunctional 18S rRNA decay intermediates (Figures 2B and S2E), we next tested its role in promoting 18S rRNA decay. TUT4KO, TUT7KO, and TUT4/7DKO did not affect uS3 ubiquitination upon HTN treatment (Figure S2F). We next sequenced RIOK3-bound 18S rRNA decay intermediates from TUT7 and TUT4/7DKO cells. TUT7KO caused a moderate reduction in uridylation (Figure 2H), consistent with its partial redundancy in uridylating miRNAs24,34. Notably, TUT4/7DKO resulted in a marked decrease in uridylation, particularly in reads containing more than five uridines (Figure 2H). Despite the overall reduction in uridylation, oligo-U tracts remained detectable in untemplated reads from TUT4/7DKO cells (Figure S2G). Consistently, decoding-incompetent 18S rRNA was not stabilized in TUT4/7DKO (Figures S2H–S2I). Together, these results support a model in which TUT4 and TUT7 are the primary enzymes mediating 18S rRNA uridylation, while additional factors may contribute to residual uridylation, which appears sufficient to support 18S rRNA decay.

DIS3L2 degrades uridylated 18S rRNA during 40S subunit turnover

To determine whether DIS3L2 degrades uridylated 18S rRNA, we assessed the stability of the constitutively expressed decoding-incompetent 18S:A1824C rRNA following inhibition of RNA polymerase I transcription. As reported previously5, 18S:A1824C rRNA was rapidly degraded in parental cells (half-life, 4.1±1.7 h) (Figure 3A), whereas 18S:WT rRNA remained stable for more than 24 h35. Strikingly, DIS3L2KO markedly stabilized 18S:A1824C rRNA, extending its half-life beyond 24 h (Figure 3A) and increasing its steady-state levels. This stabilization phenotype was rescued by DIS3L2-WT, but not the catalytic mutant D391N (Figures 3B–3C).

Figure 3. DIS3L2 degrades uridylated 18S rRNA.

Figure 3.

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(A) Half-life measurements of 18S:A1824C in parental and DIS3L2KO cells compared to 18S:WT in parental cells. Half-lives are presented as mean ±SD.

(B) Primer extension analysis of 18S NRD reporters in parental or DIS3L2KO cells complemented with DIS3L2-WT or D391N. Bottom: immunoblots for DIS3L2.

(C) Quantification of the primer extension assays shown in (B) (n≥ 4).

(D) Cumulative percentage of uridylated 18S rRNA reads from RIOK3-bound fractions from HTN-treated HEK293T parental, DIS3L2KO, and DIS3L2oe cells, (n≥ 2).

(E) MEME analysis from untemplated nucleotides at the 3’ ends of 18S decay intermediates isolated from DIS3L2KO cells. E represents the E-value significance; n denotes the number of contributing sites out of 22,631 input sequences.

(F) Representative 18S rRNA gene models illustrating the distribution of 3′ ends of RIOK3-bound 18S rRNA isolated from HEK293T parental, DIS3L2KO, and DIS3L2oe cells, (n≥ 2). Red arrowheads indicate enriched truncation in DIS3L2oe cells in the 3’ minor domain.

(G) Representative 18S rRNA gene model illustrating the 3′ ends of DIS3L2-D391N-crosslinked uridylated reads. Data from GEO (GSE81537)14.

We next examined RIOK3-bound 18S rRNA from HTN-treated DIS3L2KO cells (Figure S3A) by Nanopore direct RNA sequencing. DIS3L2KO caused a marked accumulation of uridylated reads compared to parental cells (Figure 3D). Motif analysis of untemplated nucleotides from DIS3L2KO cells revealed an even more striking enrichment of longer oligo-uridine tracts (Figure 3E). Conversely, DIS3L2 overexpression largely eliminated these uridylated species (Figure 3D).

Mapping the 3’ ends of Nanopore sequencing reads, which correspond to truncated 18S rRNA species, revealed distinct peaks across the 5’, central, and 3’ minor domains of 18S rRNA in parental cells (Figure 3F), consistent with progressive endo- and exoribonucleolytic decay11. In contrast, DIS3L2KO accumulated reads at the 3’ end, accompanied by a marked loss of internal cleavage signatures (Figure 3F), indicative of full-length 18S rRNA. Although uridylation was elevated in DIS3L2KO cells (Figure 3D), these uridylated reads are localized predominantly to the 3’ termini of intact 18S rRNA (Figure S3B), indicating stabilization of terminally uridylated 18S rRNA rather than increased degradation. Conversely, DIS3L2 overexpression (DIS3L2oe) enhanced decay signatures, characterized by increased peak intensity across multiple domains (Figure 3F) and generated a prominent new peak at G1758 (Figure 3F, red arrowhead), consistent with the accumulation of 18S decay intermediates.

Read coverage profiles supported these findings. Compared to parental cells, DIS3L2KO largely preserved 18S rRNA coverage, whereas DIS3L2oe resulted in a pronounced loss of coverage in the central and 3’ minor domains (Figure S3C), indicating removal of approximately half of the 3’ minor domain (Figure S3D). Together, these findings establish DIS3L2 as the exoribonuclease that degrades uridylated 18S rRNA during 40S turnover.

DIS3L2 acts at multiple steps of 18S rRNA degradation

To dissect whether DIS3L2 acts at additional steps of 18S rRNA decay, we re-analyzed DIS3L2 enhanced UV cross-linking and immunoprecipitation (eCLIP) data generated with the catalytically inactive mutant D391N, which stabilizes otherwise transient decay substrates14. DIS3L2 D391N-crosslinked 18S rRNA fragments contained uridylated reads at both the 3’ end and within the 5’ domain of 18S rRNA (Figure 3G). Notably, the enrichment of uridylated species immediately upstream of endoribonucleolytic cleavage sites in the 5’ domain suggests that TUTases modifies decay intermediates following endoribonucleolytic cleavage, priming them for further degradation. Consistently with its role in 18S rRNA decay, DIS3L2 was present in the free RNP and 40S fractions in unstressed cells and became enriched in the 40S fraction following HTN treatment (Figure S3E). Co-immunoprecipitation further revealed the association between RIOK3 and DIS3L2 was enhanced by HTN treatment (Figure S3F). Given that RIOK3 binds and remodels ubiquitinated 40S subunits to license decay, these findings support a model in which further uridylation occurs, thereby ensuring efficient clearance of 18S rRNA by DIS3L2. Thus, DIS3L2 acts on both the 3’ end of 18S rRNA and uridylated decay intermediates generated by endoribonucleolytic cleavage to complete 40S subunit turnover.

In vitro reconstitution of uridylation-dependent 18S rRNA decay

To define the biochemical basis of uridylation-dependent 18S rRNA decay, we reconstituted the pathway in vitro using purified recombinant TUT7 catalytic domain (TUT7CD) and DIS3L2 (Figure S4A). Uridylation at the 3’ end of 18S rRNA was quantified using the amplicon-based sequencing approach (Figure S2A). Incubation of ubiquitinated 40S subunits with TUT7CD yielded short uridine extensions relative to ubiquitinated 40S alone (Figure 4A). In contrast, addition of RIOK3 drastically increased both the length and abundance of uridylated reads. Importantly, this stimulation required RIOK3 kinase function, as RIOK3-ΔKD failed to promote robust uridylation (Figure 4B).

Figure 4. In vitro reconstitution of 18S rRNA decay.

Figure 4.

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(A) Cumulative percentage of uridylated reads (≥ 1 untemplated U) at the 3’ end of 18S rRNA following reactions containing purified ubiquitinated 40S (40Sub) alone, with TUT7CD, or with both TUT7CD and RIOK3, (n≥ 2).

(B) Cumulative percentage of uridylated reads at the 3’ end of 18S rRNA in reactions containing 40Sub, TUT7CD, and RIOK3-WT or ΔKD, (n= 3).

(C) Cumulative percentage of uridylated reads in the central domain of 18S decay intermediates in reactions containing BSA or TUT7CD, (n= 2).

(D) Cumulative percentage of uridylated reads at the 3’ end of 18S rRNA in reactions containing 40Sub, TUT7CD, and RIOK3 with DIS3L2-WT or D391N or without DIS3L2, (n≥ 2).

(E) Representative read coverage across 18S 3’ minor domain in reactions containing 40Sub, RIOK3, and DIS3L2-WT with or without TUT7CD, (n= 2).

(F) Working model for uridylation- and DIS3L2-mediated 40S ribosome turnover.

To determine whether RIOK3-mediated remodeling increases accessibility of the 3’ end of 18S rRNA, we added recombinant PNO1, which occupies the 3’ end of 18S rRNA similarly to eS2611,30,36 (Figure S4B). Indeed, addition of PNO1 reduced TUT7CD-mediated uridylation levels (Figure S4C). Collectively, our results indicate that RIOK3-mediated 40S remodeling renders the 3’ termini of 18S rRNA accessible to TUTases.

Because our Nanopore sequencing indicates that RIOK3-bound 18S rRNA was endoribonucleolytically cleaved within the 5’ and central domains in cells (Figure 2D), we isolated RIOK3-bound 18S rRNA decay intermediates from HTN-treated TUT4/7DKO cells, in which uridylation is reduced (Figure 2H) and the 18S rRNA is truncated within the 5’, central, and 3’ minor domains, and used these intermediates as substrates for uridylation. TUT7CD efficiently uridylated the 3’ ends of these decay intermediates derived from both the central (Figure 4C) and 3’ minor domains (Figure S4D), indicating that TUTases modifies 18S decay intermediates following endoribonucleolytic cleavages.

Next, we tested whether uridylated 18S rRNA could be further degraded by DIS3L2 in vitro. Recombinant DIS3L2 was added to uridylation reactions catalyzed by RIOK3 and TUT7CD. Recombinant DIS3L2-WT efficiently shortened 3’ uridine tails, whereas the catalytic mutant D391N failed to do so (Figure 4D). Additionally, DIS3L2-WT degrades 18S rRNA extending upstream into the 3’ minor domain (Figure S4E). Importantly, DIS3L2-WT failed to degrade 18S rRNA in the absence of TUT7CD (Figure 4E), indicating uridylation in the 3’ end of 18S rRNA is required for DIS3L2-depedent decay. Together, these findings define a pathway for mammalian 18S rRNA quality control in which RIOK3-mediated remodeling of ubiquitinated 40S subunits enables TUTase-dependent uridylation of 18S rRNA, which is subsequently degraded by DIS3L2. This stepwise mechanism couples ribosome remodeling with rRNA decay, establishing uridylation-dependent DIS3L2 activity as the terminal execution step of 40S subunit turnover.

DISCUSSION

Here, we define the mechanism by which ubiquitinated 40S subunits are committed to decay. We show that ubiquitinated 40S subunits are remodeled to expose the 3′ terminus of 18S rRNA, enabling its uridylation by TUTases and subsequent degradation by DIS3L2. Progressive 3’-end degradation then promotes endoribonucleolytic cleavage, generating intermediates for further uridylation and DIS3L2-mediated decay (Figure 4F). Importantly, our work identifies the remodeling step that converts ubiquitinated 40S ribosomal subunits, which are still intact ribonucleoprotein particles, into degradable RNA substrates.

Loss of DIS3L2 stabilizes the decoding-incompetent 18S rRNA and leads to the accumulation of uridylated 18S rRNA species, whereas DIS3L2 overexpression has the opposite effect (Figures 3A–3E). DIS3L2 degrades uridylated 18S rRNA in the 3’−5’ direction, generating a discrete decay intermediate with a defined 3’ terminus near G1758, particularly in DIS3L2oe cells (Figure 3F). This truncation removes two of the three critical monitoring bases37–40 (A1824 and A1825) in the decoding center (Figure S3D), effectively rendering the 40S subunit translationally inactive. Bacterial 16S rRNA quality control has been linked to RNase R-dependent pathways41,42, and DIS3L2 belongs to the same RNase II/RNB exoribonuclease family21,43,44. This commonality implies that a related decay pathway targets small ribosomal subunits even though distinct upstream signals commit ribosomal RNAs to degradation.

Moreover, our Nanopore sequencing and DIS3L2 eCLIP data14 further suggest that DIS3L2 functions at multiple stages of the 18S rRNA decay process, acting both early in initiating degradation and later in clearing terminal fragments. First, DIS3L2KO leads to the accumulation of uridylated reads at the 3′ termini of 18S rRNA together with a loss of internal decay signatures (Figure 3F). This pattern suggests that DIS3L2-mediated 3′−5′ exoribonucleolytic trimming promotes the progression of 18S rRNA decay, priming substrates for subsequent endoribonucleolytic cleavage. While RNase R rearranges bacterial 30S subunits during 16S rRNA degradation42, it is plausible that DIS3L2 similarly rearranges human 40S subunits to induce endoribonucleolytic cleavage. Second, DIS3L2-D391N eCLIP identifies uridylated 18S rRNA decay intermediates derived from the 5’ domain (Figure 3G), indicating that DIS3L2 also engages internal decay fragments after they become uridylated (Figure 4F).

Together, our findings define the downstream mechanism by which mammalian 40S ribosomal subunits are committed to destruction. 40S ribosomal particles targeted for turnover are remodeled to expose the 3′ end of 18S rRNA, marked by terminal uridylation, and subsequently eliminated by DIS3L2. This pathway reveals how ribosome remodeling, terminal RNA modification, and exoribonucleolytic decay are coupled during mammalian ribosome quality control.

Limitations of the study

Our mechanistic analyses were enabled by an engineered nonfunctional 18S rRNA substrate, which allowed dissection of the RIOK3-TUTase-DIS3L2 pathway. While our biochemical reconstitution and HTN-induced stress assays support a role for RIOK3-mediated 40S remodeling, defining the molecular basis of this remodeling event and its relevance across diverse physiological stress conditions will require future studies.

RESOURCE AVAILABILITY

Lead Contact

For further information or reagent request, please contact the lead contact, Colin Wu (colin.wu2@nih.gov).

Materials Availability

Reagents generated in this study are available from the lead contact with a completed materials transfer agreement.

Data and Code Availability

  • Sequencing data generated in this study are available in the SRA (PRJNA1451181). Original gel images are available in Mendeley Data (DOI: 10.17632/2k3dykry2k.1).

  • This study does not report original code.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

STAR METHODS

EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

Cell lines and culture conditions

HEK293T and Flp-In T-REx HeLa cells were grown in Dulbecco’s modified medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine. HEK293T parental, TUT4KO, TUT7KO, TUT4/7DKO, and DIS3L2KO cells were generated previously24,34. Flp-In T-REx HeLa RIOK3KO cells were generated previously5. Cells were maintained in a 5% CO2 humidified incubator and passaged every 2–3 days.

Generation of knockout cell lines

sgRNAs targeting human 3’−5’ exoribonuclease DIS3L2 and USP10 were annealed and cloned into lentiCRISPRv2-puro vector (Addgene, #98290) and lentiviral particles were generated as previously described5. In brief, 1 mL of lentiCRISPRv2 viral particles were incubated with 2 × 105 Flp-In T-REx HeLa cells and polybrene transduction reagent (10 μg/mL) at 37°C for 30 minutes. This mixture was then plated onto a 6-well plate. 24 hours after transduction, media was changed to DMEM supplemented with 10% FBS and 2 mM L-glutamine. 48 hours post transduction, cells were selected with puromycin (1 μg/mL). Monoclonal DIS3L2KO and USP10/RIOK3 DKO cell lines were generated using a limiting dilution method. Loss of protein of interest was validated by immunoblotting and Sanger sequencing.

Generation of 18S rRNA reporter cell lines

2 × 105 HeLa Flp-In T-REx DIS3L2KO cells were seeded in a 6-well plate a day prior to transfection with 1 μg of 18S:A1824C plasmid and 1 μg of PiggyBac transposase as previously described5. 4 days after transfection, cells were trypsinized for single cell sorting. tagBFP-positive cells were sorted into three 96-well plates. Expression of 18S:A1824C rRNA was subsequently confirmed by primer extension analysis.

METHOD DETAILS

In vitro ubiquitination assay

For Figure S1A–S1B, ribosomes were isolated from whole-cell extracts by layering the extract onto a 35% sucrose cushion followed by centrifugation at 100,000 × g for 1 h. The ribosome pellet was resuspended in ribosome resuspension buffer (50 mM Tris-HCl [pH 8.0], 25 mM KCl, and 5 mM MgCl2). 20 U of ribosomes were ubiquitinated in vitro using a ubiquitination kit (Enzo Life Sciences) according to the manufacturer’s protocol. Briefly, the reaction contained 1× ubiquitination buffer, IPP (100 U/mL), 1 mM DTT, 5 mM Mg-ATP, 100 nM E1 enzyme, 500 nM E2 enzyme, 450 nM RNF10, 2.5 μM biotinylated ubiquitin, and 2 U SUPERaseIn RNase inhibitor. Reactions were carried out at 37°C for 3 h.

In vitro eS26 displacement assay

Preparation of 40S ribosomal subunits:

To purify human 40S ribosomal subunits, we adapted a previously published protocol45. Frozen cell pellets collected from four 15-cm dishes of Flp-In T-REx HeLa USP10/RIOK3 DKO uS3-HA cells were lysed in 1 mL lysis buffer per plate (30 mM HEPES [pH 7.5], 50 mM KCl, 10 mM MgCl2, 220 mM sucrose, 2 mM DTT, 0.5 mM EDTA, 0.5% Igepal, and 0.5 mg/mL heparin, 1× cOmplete Mini Protease Inhibitor Cocktail, 20 U SUPERaseIn). Lysates were incubated on ice for 10 min, centrifuged at 10,000 × g for 5 min at 4°C, and the supernatants were transferred to fresh tubes. The lysates were further clarified by centrifugation at 21,000 × g for 30 min at 4°C. PEG 20,000 was added to the clarified lysate to a final concentration of 1.35%, followed by incubation on ice for 15 min. After centrifugation at 20,000 × g for 12 min at 4°C, the supernatant was collected, adjusted to 130 mM KCl, and incubated for 5 min. PEG 20,000 was then increased to a final concentration of 4.5%, and samples were incubated on ice for an additional 10 min. Ribosomal particles were collected by centrifugation at 17,500 × g for 10 min at 4°C, and the resulting pellet was resuspended in buffer R (30 mM HEPES [pH 7.5], 125 mM KCl, 7.5 mM MgCl2, 2 mM DTT, and 0.5 mg/mL heparin, 1× cOmplete Mini Protease Inhibitor Cocktail). Resuspended ribosomes were loaded onto 10–30% sucrose gradients prepared in high-salt gradient buffer (25 mM HEPES [pH 7.5], 550 mM KCl, 5 mM MgCl2, and 2 mM DTT) and centrifuged at 39,000 rpm for 3 h at 4°C in an SW40 rotor (Beckman Coulter). Gradients were fractionated into 1 mL fractions, and fractions corresponding to the 40S peak were pooled. Pooled 40S fractions were then incubated overnight with washed anti-HA beads to capture uS3-HA-containing 40S ribosomes.

In vitro ubiquitination of immunoprecipitated uS3-HA-containing 40S ribosomes:

For Figures 1B and S1G, anti-HA beads bound to uS3-HA-containing 40S ribosomes were washed once with IP lysis buffer (50 mM HEPES [pH 7.5], 5 mM MgCl2, 150 mM NaCl, 1% Triton X-100, and 1× cOmplete Mini Protease Inhibitor Cocktail) and once with ribosome resuspension buffer (50 mM Tris-HCl [pH 8.0], 25 mM KCl, and 5 mM MgCl2). The bead-bound 40S ribosomes were then subjected to an in vitro ubiquitination reaction using a ubiquitination kit (Enzo Life Sciences) according to the manufacturer’s protocol. Briefly, each reaction contained 1× ubiquitination buffer, IPP (100 U/mL), 1 mM DTT, 5 mM Mg-ATP, 100 nM E1 enzyme, 500 nM E2 enzyme, 450 nM RNF10, 2.5 μM biotinylated ubiquitin, and 2 U SUPERaseIn RNase inhibitor. Reactions were performed at 37°C for 3 h, after which the beads were washed once with ribosome resuspension buffer and resuspended in the same buffer for subsequent reactions. ubiquitination of uS3 was quantified as the ratio of ubiquitinated uS3 to total uS3 signal.

RIOK3-mediated displacement assay:

Ubiquitinated uS3-HA 40S ribosomes (23 nM) immobilized on anti-HA beads were incubated with 800 nM recombinant RIOK3 WT, RIOK3-ΔKD, or BSA control, in reactions containing 20 mM Tris-HCl [pH 8.0], 150 mM KCl, 20 mM MgCl2, 1 mM ATP, and 20 U SUPERaseIn. Reactions were incubated for 1 h at 37°C. Following incubation, supernatants were collected and mixed with 1× SDS sample buffer. Beads were washed once with ribosome resuspension buffer, and bead-associated material was recovered in 1× SDS sample buffer. eS26 dissociation in the supernatant fraction was quantified as the ratio of eS26 signal in the supernatant to the total eS26 signal in the supernatant and bead fractions by ImageJ.

In vitro uridylation assay

40S ribosomal subunits were prepared as described above from Flp-In T-REx HeLa USP10/RIOK3 DKO uS3-HA cells treated with HTN (3 μg/mL) for 30 min before harvest. Briefly, fractions from 10–30% sucrose gradients prepared in high-salt gradient buffer (25 mM HEPES [pH 7.5], 550 mM KCl, 5 mM MgCl2, and 2 mM DTT) and centrifuged at 39,000 rpm for 3 h at 4°C in an SW40 rotor (Beckman Coulter) were pooled. PEG 20,000 was then added to a final concentration of 4.5% and incubate on ice for 10 min. The resulting solution was spun at 100,000 rpm in a TLA100.3 rotor for 2 h at 4°C. The final 40S pellet was resuspended in buffer S (10 mM HEPES [pH 7.5], 100 mM KCl, 7.55 mM MgCl2, 1 mM DTT). Aliquots were flash frozen in liquid nitrogen and stored at −80°C.

Reactions were carried out in 20 μL reaction buffer (20 mM Tris-HCl [pH 8], 150 mM KCl, 20 mM MgCl2, 0.5 mM DTT, 1 mM ATP, 0.25 mM UTP, and 20 U SUPERaseIn). When indicated, purified 40S ribosomal subunits (46 nM), recombinant RIOK3-WT or ΔKD (960 nM), and recombinant TUT7CD (560 nM) were included, and reactions were incubated at 37°°C for 2 h (Figures 4 and S4). For DIS3L2 in vitro assays (Figures 4D, 4E and S4E), recombinant DIS3L2-WT or D391N (205 nM) was added to the above reactions and reactions were incubated at 37°C for additional 30 min. In experiments examining the effect of PNO1 (Figure S4C), purified 40S subunits (46 nM) were incubated with RIOK3-WT (960 nM) in 20 μL reaction buffer (20 mM Tris-HCl [pH 8], 150 mM KCl, 20 mM MgCl2, 0.5 mM DTT, 1 mM ATP, and 20 U SUPERaseIn) for 1 h. Then BSA or recombinant PNO1 was added to 1.7 μM and reactions were incubated for additional 30 min. To uridylate 18S rRNA, TUT7CD (800 nM) and UTP (0.25 mM) were added, and reactions were incubated for an additional 1 h. RNA was isolated by phenol-chloroform extraction followed by sodium acetate/isopropanol precipitation.

Library preparation and data analysis for In vitro uridylation assays

Recovered RNA was resuspended in 10 mM Tris-HCl [pH 8.0], heated to 80°C for 2 min, chilled on ice, and subjected to 3′ dephosphorylation using T4 polynucleotide kinase for 1 h at 37°C. A pre-adenylated 3′ linker (oCW001, 5’-Phos/App-NNNNNNCACTCGGGCACCAAGGA/−3’ddC) was then ligated using T4 RNA ligase 2 truncated and incubating for 3 h at 37°C, followed by overnight sodium acetate precipitation at −80°C. The ligated RNA was resuspended in water and reverse transcribed using an RT primer (anti-oBZ407, TCCTTGGTGCCCGAGTG) and SuperScript III at 55°C for 30 min. RNA templates were hydrolyzed with NaOH, and cDNA was recovered by sodium acetate/isopropanol precipitation. cDNA was amplified by an initial PCR using NEBNext Ultra II Q5 Master Mix, reverse primer (5’-TCCTTGGTGCCCGAGTG-3’) and forward primer (PCR1_Fwd2, 5’-CCCTACACGACGCTCTTCCGATCTNNNNNTTTCCGTAGGTGAACC-3’) for the 3’ minor domain or (PCR1_central, 5’-CCCTACACGACGCTCTTCCGATCTNNNNNCTCCAATAGCGTATATTAAAG-3’) for the central domain, purified with AMPure XP beads, and subjected to a second indexing PCR with barcoded primers. Barcoded libraries were resolved on 8% native PAGE, extracted using sodium acetate/isopropanol precipitation, and on an Illumina NextSeq 1000/2000.

3’ adapter (NNNNNNCACTCGGGCACCAAGGA) was trimmed off using skewer46. Trimmed reads were aligned to human 18S rRNA using STAR47. For sequence motif analyses, untemplated reads starting with ‘ACG’ were removed to exclude biogenesis intermediates11,48, and the resulting fasta files were analyzed by MEME49. For counting untemplated U reads, only untemplated sequences beginning with “T” were considered. Thymidines were counted, while non-T nucleotides (A, C, G) were treated as mismatches and tolerated up to 3 mismatches. Mismatches immediately followed by a thymidine were permitted. Scanning terminated when the mismatch threshold was exceeded. Cumulative fractions were calculated as the proportion of reads containing at least one terminal untemplated T.

RIOK3 immunoprecipitation

For analysis of RIOK3-associated 18S rRNA, cells with indicated genetic background grown in a 15-cm dish were transfected with 20 μg of pcDNA-RIOK3-FLAG (WT or ΔKD) plasmid. 48 h post transfection, cells were treated with 3 μg/mL HTN for 30 mins and lysed in IP lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM MgCl2, 0.5% Triton X-100, 1× cOmplete Mini Protease Inhibitor Cocktail, 200 U SUPERaseIn RNase inhibitor, and 400 U murine RNase inhibitor). Lysates were clarified and incubated with 50 μL anti-DYKDDDDK magnetic agarose beads with rotation at 4 °C for 16 h. Beads were subsequently washed three times with IP wash buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 0.5 mM DTT). FLAG-tagged RIOK3 complexes were eluted using DYKDDDDK peptide (0.5 mg/mL in 100 μL PBS supplemented with 60 U SUPERaseIn RNase inhibitor) for 30 min at 4 °C. RNA was then isolated from the eluate by phenol-chloroform extraction and resuspended in 10 μL RNase/DNase-free water.

Library preparation and data analysis for Nanopore sequencing

For nanopore sequencing library preparation, purified RIOK3-associated RNA was polyadenylated using E. coli poly(A) polymerase according to Oxford Nanopore protocols. The poly(A)-tailed RNA was then used to generate sequencing libraries using a Direct RNA Sequencing Kit following the manufacturer’s recommendations. Libraries were loaded onto SpotON flow cells and sequenced on a GridION instrument.

Basecalling was performed by the MinKNOW software. Reads were mapped to 18S rRNA reference sequence using minimap250 with the following parameters: “–secondary=no”. Aligned reads were analyzed using custom scripts written in Python 3 and R51. For counting untemplated U reads, only untemplated sequences beginning with “TT” were considered. Thymidines were counted, while non-T nucleotides (A, C, G) were treated as mismatches and tolerated up to 3 mismatches. Mismatches immediately followed by a thymidine were permitted. Scanning terminated when the mismatch threshold was exceeded. Cumulative fractions were calculated as the proportion of reads containing at least two terminal untemplated T.

Co-immunoprecipitation analysis

Cells were transfected with either pcDNA-RIOK3-FLAG or pCDNA-RIOK3 ΔKD-FLAG plasmid for 48 h. 48 h post-transfection, cells were treated with 3 μg/mL HTN for 30 mins and lysed in IP lysis buffer (50 mM HEPES [pH 7.5], 15 mM MgCl2, 100 mM KCl, 0.25% NP-40, 5% glycerol, 1× cOmplete Mini Protease Inhibitor Cocktail). Lysates were incubated on ice for 10 min followed by centrifugation at 10,000 × g for 10 min. Clarified extracts were incubated with 20 μL of anti-FLAG beads for 2 h, followed by five washes with IP wash buffer (50 mM HEPES [pH 7.5], 5 mM MgCl2, 150 mM NaCl, and 1% Triton X-100). Bound proteins were eluted in 30 μL of 2× SDS sample buffer and immunoblotted.

Polysome profiling

When indicated, cells were treated with harringtonine (HTN) at 3 μg/mL for 6 h in the presence of 50 nM bortezomib prior to lysis in footprint lysis buffer (20 mM Tris-HCl [pH 8.0], 150 mM KCl, 15 mM MgCl2, 1% Triton X-100, 1 mM DTT, and 0.1 mg/mL cycloheximide). Lysates were clarified by centrifugation at 21,000 × g for 10 min and the clarified lysate containing 500 μg of RNA was loaded onto a 10–30% sucrose gradient. Gradients were centrifuged in an SW40Ti rotor at 39,000 rpm for 3 h at 4°C and fractionated using a Biocomp fractionator. Proteins in each fraction were precipitated with 8% trichloroacetic acid (TCA) and analyzed by immunoblotting. eS26 dissociation was quantified as the percentage of eS26 in the free RNP fraction relative to the total eS26 signal across all sucrose gradient fractions.

Purification of recombinant DIS3L2 proteins

6His-SUMO-DIS3L2 in pET32a was a gift from Dr. Sandy Wolin (NCI/NIH). Mutant D391N was created by Gibson assembly and verified by whole plasmid sequencing. The proteins were expressed in Rosetta (DE3) cells (Novagen) induced with 1 mM IPTG and grown overnight at 16 °C. Cells were lysed by sonication in 50 mM HEPES [pH 7.5], 150 mM NaCl and the clarified supernatant was incubated with NiNTA-agarose resin (Qiagen) equilibrated in 50 mM HEPES [pH 7.5], 150 mM NaCl at 4 °C with gentle rotation for 1 h. Resin was first washed with 10 column volumes 50 mM HEPES [pH 7.5], 150 mM NaCl, 30 mM Imidazole and then 10 column volumes 50 mM HEPES [pH 7.5], 500 mM NaCl, 30 mM Imidazole. Protein was eluted with 3 volumes of 50 mM HEPES [pH 7.5], 150 mM NaCl and 500 mM Imidazole. Fractions containing the protein were pooled and concentrated using an Amicon Ultra-15 10KDa MWCO (Sigma-Aldrich, UFC901008D). The SUMO tag was cleaved by adding 1/10 volume of 10X SUMO salt free buffer (500mM Tris-HCl [pH 8], 2% Igepal, 1.5M NaCl, 10mM DTT) and 6 units of SUMO protease (UlpI) per microgram of full-length tagged 6HSUMODIS3L2 for 16 h at 4°C. The reaction was diluted to 30mM imidazole (1:17) with 50mM HEPES [pH 7.5] and re-applied to NiNTA-agarose in 50 mM HEPES [pH 7.5], 150 mM NaCl. The flow through containing the cleaved DIS3L2 species was applied to a 1mL Hitrap heparin HP column (Cytiva) equilibrated with 10 CV in 25 mM HEPES [pH 7.5], 50 mM NaCl, 5 mM DTT, followed by a 10 CV wash in 25 mM HEPES [pH 7.5], 50 mM NaCl, 5 mM DTT, and a 20 CV linear gradient from 50mM to 1M NaCl. Appropriate fractions were pooled and concentrated to 5 mL and loaded onto a size exclusion column (16/600 Superdex200pg, Cytiva) equilibrated with 50 mM HEPES [pH 7.5], 150 mM NaCl, 5 mM DTT. Fractions containing pure protein were pooled, concentrated, dialyzed overnight at 4°C against 20mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM DTT and 20% glycerol, and stored at −80°C. The identities of the recombinant proteins were confirmed by mass spectrometry.

Purification of recombinant TUT7CD proteins

The plasmid p6His-SUMO-TUT7CD-HA was created by Gibson assembly between the backbone of pSumo-His6-SUMO-AtTPR1(1–209) (Addgene #177858) and a truncated human TUT7 gene encoding amino acids 951 to 1495 with a C-terminal HA tag. The protein was expressed in Rosetta2 (DE3) cells with 1 mM IPTG, cells were lysed and purified by IMAC as above for DIS3L2. Concentrated TUT7CD fractions were applied to a Superdex 200 size exclusion column equilibrated with 50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM DTT. Fractions containing pure protein were pooled, concentrated, dialyzed overnight at 4 °C against 20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT and 20% glycerol, and stored at −80°C.

Purification of recombinant RIOK3 proteins

The plasmid p6His-SUMO-RIOK3 was created by Gibson assembly between the backbone of pSumo-His6-SUMO-AtTPR1(1–209) and either the full-length human RIOK3 gene or a deletion of the kinase domain (residues 350 to 618). The proteins were expressed in Rosetta2 (DE3) cells with 1 mM IPTG, cells were lysed and purified by IMAC as above for DIS3L2. Concentrated fractions were applied to a Superdex 200 size exclusion column equilibrated with 50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM DTT. Fractions containing pure protein were pooled, concentrated, dialyzed overnight at 4°C against 20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM DTT and 20% glycerol, and stored at −80°C.

The plasmid pMAL-c2G-RIOK3-MBP-6His was created by Gibson assembly between the backbone of pMAL-c2G (Addgene #75290) and either the full-length human RIOK3 gene or a deletion of the kinase domain (residues 350 to 618) with a C-terminal 6X His tag. The proteins were expressed in Rosetta2 (DE3) cells with 1mM IPTG, cells were lysed, purified by IMAC followed by Superdex 200 size exclusion, concentrated and dialyzed as described above for p6His-SUMO-RIOK3.

Purification of recombinant RNF10 protein

The plasmid pMAL-c2G-RNF10–6His was created by Gibson assembly between the backbone of pMAL-c2G and the human RNF10 gene with a C-terminal 6X His tag, replacing the MBP fusion protein. The protein was expressed in Rosetta2 (DE3) cells overnight at 18°C after induction with 1mM IPTG. Cells were lysed and the clarified supernatant was purified by IMAC as above for DIS3L2.

Primer extension assay

Primer extension analysis was performed essentially as previous described5. Total RNA was isolated from indicated cells using a Quick-RNA Miniprep Kit (Zymo, R1055) and quantified by NanoDrop 2000. Purified 1 μg RNA was incubated with 0.67 mM dNTPs and 0.67 mM Cy5-labeled oligonucleotide at 65 °C for 5 min and then placed on ice. Reverse transcription was performed with 5× First Strand Buffer and 100 U Maxima RT at 53 °C for 30 min, followed by RNA hydrolysis with 0.1 N NaOH at 95 °C for 15 min. cDNA was precipitated, resolved on a 10% Criterion TBE-Urea polyacrylamide gel (Bio-Rad, 3450088), and imaged using an Amersham Typhoon scanner. Band intensities for endogenous 18S and 18S-H rRNA were quantified in ImageJ. Relative 18S-H levels were calculated as the ratio of 18S-H to endogenous 18S signal, and values from at least three biological replicates were used to determine the mean ± SD.

Pulse-chase analysis

Pulse-chase analysis of 18S rRNA was performed essentially as described previously5. Cells expressing 18S:WT or 18S:A1824C rRNA in parental or DIS3L2KO backgrounds were seeded in 6-well plates 1 day before treatment. CX-5461 was added at 10 μM to inhibit RNA Polymerase I transcription, and cells were harvested at the indicated time points for total RNA isolation using a Quick-RNA Miniprep Kit. Samples were analyzed by primer extension, and relative tagged 18S rRNA levels were normalized to the 0 h time point, which was set to 100%.

Quantification and Statistical Analysis

Significance was calculated using unpaired Student’s t-test (*: p < 0.05, **: p < 0.01, and ***: p < 0.001, ns: not significant) as described in figure legends. Error bars denote the standard deviations (SD) unless otherwise specified.

Supplementary Material

1

Key resources table

REAGENT or RESOURCE SOURCE IDENTIFIER
Antibodies
FLAG Sigma-Aldrich A8592; RRID: AB_439702
HA Sigma-Aldrich 12013819001; RRID: AB_390917
uS3 Bethyl Laboratories Inc. A303–840A; RRID: AB_2620191
uS5 Bethyl Laboratories Inc. A303–794A; RRID: AB_11218192
β-actin Cell Signaling Technology 51255; RRID: AB_1903890
RIOK3 Proteintech 13593–1-AP; RRID: AB_2178105
eS26 Proteintech 14909–1-AP; RRID: AB_2180361
uL5 Bethyl Laboratories Inc. A303–931A; RRID: AB_2620280
TUT4 (ZCCHC11) Proteintech 18980–1-AP; RRID: AB_10598327
TUT7 (ZCCHC6) Proteintech 25196–1-AP; RRID: AB_2879953
DIS3L2 Sigma-Aldrich HPA035796; RRID: AB_2674786
vinculin Santa Cruz Biotechnologies sc-73614; RRID: AB_1131294
Chemicals, peptides, and recombinant proteins
Ambion DNase I (RNase-free) Invitrogen AM2222
T4 Polynucleotide Kinase New England Biosciences M0201L
SUPERaseIn Thermo Fischer Scientific AM2694
T4 RNA Ligase 2, Truncated New England Biosciences M0242L
Recombinant PNO1 MedChemExpress LLC HY-P702824
Superscript III Thermo Fischer Scientific 56575
GlycoBlue Thermo Fischer Scientific AM9515
BSA Thermo Fischer Scientific 15561020
UlpI ScienCell MB9018
Maxima Reverse Transcriptase Thermo Fischer Scientific EP0743
NEBNext Ultra II Q5 Master Mix New England Biolabs M0544X
Murine RNase inhibitor New England Biolabs M0314
E. coli poly(A) polymerase New England Biolabs M0276S
PolyJet DNA transfection reagent SignaGen Laboratories SL100688
Puromycin Sigma-Aldrich P7255
Polybrene Sigma-Aldrich TR-1003
Hygromycin B Thermo Fisher Scientific 10687010
DYKDDDDK peptide Genscript USA Inc RP10586
cOmplete mini protease inhibitor cocktail Roche 11836170001
Harringtonine Cayman 15361
CX-5461 Sigma-Aldrich 5092650001
Direct RNA Sequencing Kit Oxford Nanopore SQK-RNA004
SuperSignal™ West Pico PLUS Chemiluminescent Substrate Thermo Fisher Scientific 34580
SuperSignal™ West Femto Maximum Sensitivity Substrate Thermo Fisher Scientific 34095
Critical commercial assays
CellTox Green Cytotoxicity Promega G8741
ubiquitination kit Enzo Life Sciences BML-UW9920
Deposited data
Sequencing data This study, SRA PRJNA1451181
Raw data of gels and blots This study, Mendeley Data DOI: 10.17632/2k3dykry2k.1
Experimental models: Cell lines
Flp-In T-REx HeLa Wu et al.52 N/A
Flp-In T-REx HeLa RIOK3KO Coria et al.5 N/A
Flp-In T-REx HeLa USP10/RIOK3DKO This study N/A
Flp-In T-REx HeLa 18S:WT Coria et al.5 N/A
Flp-In T-REx HeLa 18S:A1824C Coria et al.5 N/A
Flp-In T-REx HeLa DIS3L2KO 18S:A1824C This study N/A
HEK293T Yang et al.34 N/A
HEK293T TUT4KO Yang et al.34 N/A
HEK293T TUT7KO Yang et al.34 N/A
HEK293T TUT4/7DKO Yang et al.34 N/A
HEK293T DIS3L2KO Yang et al.34 N/A
HEK293T RIOK3KO This study N/A
Oligonucleotides
See Table S1 for oligo sequences This study N/A
Recombinant DNA
pcDNA-RIOK3WT-FLAG This study N/A
pcDNA-RIOK3ΔKD-FLAG This study N/A
PB-hPGK-DIS3L2WT This study N/A
PB-hPGK-DIS3L2D391N This study N/A
p6His-SUMO-RIOK3WT This study N/A
p6His-SUMO-RIOK3ΔKD This study N/A
pMAL-c2G-RIOK3WT-MBP-6His This study N/A
pMAL-c2G-RIOK3ΔKD-MBP-6His This study N/A
pMAL-c2G-RNF10–6His This study N/A
6His-SUMO-DIS3L2WT-pET32a This study N/A
6His-SUMO-DIS3L2D391N-pET32a This study N/A
p6His-SUMO-TUT7CD-HA This study N/A
Software and algorithms
skewer Jiang et al.46 github.com/relipmoc/skewer
STAR Dobin et al.47 github.com/alexdobin/STAR
seqtk GitHub github.com/lh3/seqtk
GraphPadPrism GraphPad software lnc www.graphpad.com
Image J National Institutes of Health imagej.net/
Other
Biocomp Gradient Fractionator Biocomp 100–001
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Highlights –

  • RIOK3 remodels ubiquitinated 40S ribosomes to expose the 3’ end of 18S rRNA

  • TUT4 and TUT7 contribute to 18S rRNA U-tailing

  • DIS3L2 degrades U-tailed 18S rRNA and promotes further decay

ACKNOWLEDGMENTS

We would like to thank Dr. Sandy Wolin (NCI/NIH) for sharing DIS3L2 plasmids. This work was supported by the Intramural Research Program of the National Institutes of Health (NIH) (1ZIABC012037 to C.C.C.W and 1ZIABC011566 to S.G.). The contributions of the NIH authors are considered works of the United States Government. However, the findings and conclusions presented in this paper are those of the authors and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

DISCLOSURE OF INTERESTS

The authors declare no competing interests.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS2212996-supplement-1.pdf (6.3MB, pdf)

Data Availability Statement

  • Sequencing data generated in this study are available in the SRA (PRJNA1451181). Original gel images are available in Mendeley Data (DOI: 10.17632/2k3dykry2k.1).

  • This study does not report original code.

  • Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

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