henhaobio
Sign in

bioRxivMolecular BiologyPreprint

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

Akruti ShahiD, Aaztli R. CoriaiD, Jennifer Miller, Emilien OrgebiniD, Wilfried M. GuibletiD, Acong YangiD, Shuo GuiD, Colin Chih‐Chien WuiD

PDFDOI
In-depth full-text digest available · Jump to digest ↓

Abstract

Eliminating defective ribosomes through quality control is essential for accurate protein synthesis. However, the mechanisms that commit ribosomal subunits to decay remain poorly defined. Here, we identify a tandem mechanism in which ubiquitin-mediated 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 displaces the ribosomal protein eS26 from 40S subunits, exposing the 3 end of 18S rRNA. Nanopore direct RNA sequencing and motif analyses reveal that this remodeling event promotes oligo-uridylation, generating uridylated 18S rRNA decay intermediates. We further show that this uridylated 18S rRNA is selectively 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.

In-depth summary

Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (12 Sept 2026) · CC0 1.0

RIOK3 displaces eS26 from ubiquitinated 40S subunits, exposing the 18S rRNA 3' end for U-tailing by TUT4/TUT7 and exonucleolytic decay by DIS3L2, which commits defective subunits to turnover.

Ubiquitination marks defective 40S subunits for turnover, but the paper says how these subunits are then committed to decay was unresolved. It proposes a stepwise route in which RIOK3 remodeling, 18S rRNA uridylation and DIS3L2 degradation are coupled.

Key results

  1. ShownRIOK3 releases ribosomal protein eS26 from ubiquitinated 40S subunits in a purified system. Release needs ATP and the kinase domain, and ubiquitinated uS3 stays on the subunit. In cells, harringtonine-induced eS26 release was lost in RIOK3 knockout cells.

    eS26 was enriched in the supernatant with wild-type RIOK3 vs a BSA control (n=3). RIOK3 ΔKD released it less efficiently despite comparable binding to the 40S. Release was absent without ATP. Polysome profiling in RIOK3 KO cells (n=3). · RIOK3 releases eS26 to promote 18S rRNA decay; Figure legends

  2. ShownNanopore direct RNA sequencing of RIOK3-bound 18S rRNA from harringtonine-treated HEK293T cells shows oligo-U tails on decay intermediates. These tails depend on RIOK3 kinase function and on the GCN2-RNF10-RIOK3 axis.

    About 4% of RIOK3-bound 18S rRNAs were uridylated, with tails up to 30 nucleotides, vs input reads. Uridylation was markedly lower with RIOK3 ΔKD. Decoding-incompetent 18S:A1824C showed more tails than 18S:WT, and fewer in GCN2, RNF10 and RIOK3 KO cells. MEME motif analysis used 53,736 input sequences. · 18S rRNA decay intermediates accumulate…; Figure legends

  3. ShownTUT4 and TUT7 account for much of 18S rRNA uridylation, but not all of it. Residual tailing remains in the double knockout, and the decoding-incompetent 18S rRNA is still not stabilized there.

    TUT7 KO gave a moderate reduction in oligo-U levels. TUT4/7 DKO gave a more pronounced reduction, especially for reads with more than five uridines. Oligo-U tracts were still found in DKO cells, and 18S:A1824C was not stabilized relative to parental cells. · TUTases mediate uridylation of 18S rRNA during…

  4. ShownDIS3L2 is the exoribonuclease that degrades uridylated 18S rRNA. Its loss stabilizes the mutant rRNA and leaves terminally uridylated, full-length-like 18S. It also removes the internal cleavage signatures, which suggests 3'-5' trimming primes endonucleolytic cleavage.

    The 18S:A1824C half-life was about 4.1 ± 1.7 h in parental cells and above 24 h in DIS3L2 KO, vs more than 24 h for 18S:WT. Stabilization was rescued by wild-type DIS3L2 but not the D391N catalytic mutant. DIS3L2 overexpression gave a new 3' peak at G1758. Re-analysed eCLIP of the D391N mutant also showed U-tailed reads at the 3' terminus and within the 5' domain. · DIS3L2 degrades uridylated 18S rRNA during 40S…; DIS3L2 acts at multiple steps of 18S rRNA…; DISCUSSION

  5. ShownA reconstitution with recombinant proteins reproduces the sequence of steps. RIOK3 kinase activity stimulates TUT7 catalytic-domain uridylation of the 18S 3' end. PNO1 reduces it, and DIS3L2 shortens the tails and degrades upstream only when uridylation has occurred.

    RIOK3 added to ubiquitinated 40S plus TUT7CD increased the length and abundance of uridylated reads, whereas RIOK3 ΔKD did not. DIS3L2 wild type shortened tails but D391N did not. DIS3L2 did not degrade 18S rRNA without TUT7CD (n≥2 or n=2 per panel). · In vitro reconstitution of uridylation-dependent…; Figure legends

Methods, data and reproducibility

In vitro reconstitution with purified ubiquitinated 40S subunits and recombinant RIOK3, TUT7 catalytic domain and DIS3L2 was combined with Nanopore direct RNA sequencing and amplicon-based Illumina sequencing of untemplated 3' nucleotides. Knockout and overexpression cell lines and a reporter-rRNA pulse-chase gave the cellular data.

  • Nanopore and Illumina amplicon sequencing of RIOK3-bound 18S rRNA from harringtonine-treated cells (parental, ΔKD, TUT7 KO, TUT4/7 DKO, DIS3L2 KO/oe) and in vitro reaction libraries · human (HEK293T and HeLa cell lines) · n = n≥2 or n=3 per condition · PRJNA1451181
  • Re-analysis of 18S:WT and 18S:A1824C reporter rRNA decay intermediates in parental, GCN2 KO, RNF10 KO and RIOK3 KO cells · human · PRJNA1195343
  • Re-analysis of DIS3L2 D391N eCLIP data · human · GSE81537
Methods & tools
Nanopore direct RNA sequencing (SQK-RNA004, GridION), Illumina NextSeq 1000/2000, minimap2, STAR, skewer, MEME, MinKNOW, CRISPR knockout cell lines, polysome profiling, primer extension, CX-5461 pulse-chase
Reproducibility gaps
Analysis used custom Python 3 and R scripts, but no code repository or URL is given. The sequencing data are deposited in SRA under accession PRJNA1451181, which is printed without a URL.
Limitations (4)
  • The frequency of 18S rRNA uridylation is likely underestimated, because decay intermediates yield multiple reads per molecule while input rRNAs are largely intact. · stated by the authors
  • Oligo-U tracts persist in TUT4/7 DKO cells, so additional factors probably contribute to the residual uridylation. · stated by the authors
  • How DIS3L2 recognizes and degrades uridylated 18S rRNA within remodeled 40S subunits, including any remodeling of the subunit, is untested and left to future structural and biophysical studies. · stated by the authors
  • The cell experiments use harringtonine treatment or a mutant 18S reporter in HEK293T and HeLa lines, with only two to three replicates for the sequencing comparisons. The reconstitution uses a truncated TUT7 catalytic domain, so physiological stoichiometry and TUT4 are not tested in vitro. · noted in this summary

Read this if you study ribosome quality control, rRNA decay or RNA tailing, or want Nanopore-based analysis of untemplated 3' uridines on rRNA decay intermediates.

Citations
1

Reproducibility ledger

Nobody has reported trying to reproduce this paper yet. Did you replicate an experiment or run the code? Your report saves the next lab a week.

Sign in to file a reproducibility report.

Related papers

Similarity Map
SelectedSame fieldCross-field
THISDiehl '26Buzovetsky '25Heuvel '21Plassart '21Parker '24Blottnitz '26
83% semantic matchMolecular Biology
Frances F. Diehl, Allen R. Buskirk, Rachel Green·Molecular Cell·Oct 1, 2026· 2 citations