Oligo(uridine) tailing and exonucleolytic decay drive 40S ribosome degradation in response to stress

Key results

1. ShownRIOK3 brings TUT7 and DIS3L2 to 40S ribosomes during amino acid starvation. DIS3L2 was the only nuclease enriched with wild-type RIOK3 over both degradation-defective mutants, and TUT7 was enriched but TUT4 was not.

   IP-MS with three biological replicates, wild-type RIOK3 vs ΔUIM and ΔKD mutants; immunoblots confirmed loss of binding in ΔUIM, ΔKD and K290A/D406A. Catalytically dead DIS3L2 D383N bound RIOK3 in starvation only; RNF10 knockdown prevented recruitment. · RIOK3 recruits TUT7 and DIS3L2 to ribosomes; QUANTIFICATION AND STATISTICAL ANALYSIS; Figure legends

2. Shown18S rRNA in RIOK3-bound ribosomes carries 3′ oligo(U) tails, which are nearly absent with degradation-defective RIOK3 mutants. No oligo(A), (C) or (G) tails were seen.

   3′ end sequencing of RIOK3 IPs: tails on about 15% of reads at the 18S 3′ end with wild-type RIOK3, mean tail length 12 nt. Tails were defined as ≥80% U. · The rRNA in ribosomes bound by RIOK3 contains 3′…; Figure legends

3. ShownTUT7 is the main enzyme tailing 18S rRNA, and it does so selectively in starvation, via RNF10 and RIOK3. TUT7 loss also prevents DIS3L2 from associating with RIOK3.

   In total RNA from cells overexpressing DIS3L2 D383N, TUT7 knockout eliminated 18S oligo(U) tails. Starvation-induced tailing was almost exclusive to 18S among rRNAs; tRNAs and other ncRNAs carried shorter tails that starvation did not increase. RIOK3 or RNF10 knockout removed most 18S tails; TUT7 knockout, but not TUT4 knockout, blunted the starvation-induced fall in the 18S/28S ratio (n = 3). · TUT7 preferentially uridylates 18S rRNA in…; QUANTIFICATION AND STATISTICAL ANALYSIS; Figure legends

4. Shown18S decay proceeds through two 3′-truncated intermediates, a major one in h28 and a minor one in h44. These carry shorter oligo(U) tails (iterative uridylation) and depend on both TUT7 and DIS3L2.

   In RIOK3 IPs the h28 intermediate had 70% as many reads as the 18S 3′ end and h44 had 12%; together the intermediates had 82% as many reads as full-length 18S. Both intermediates and their tailed forms fell in ΔTUT7 and ΔDIS3L2 cells. · The rRNA in ribosomes bound by RIOK3 contains 3′…; DIS3L2 degrades uridylated 18S rRNA from its 3′…; DISCUSSION

5. ShownDIS3L2 loss raises 18S tailing and partly rescues 18S loss during starvation, suggesting that other nucleases can compensate to some degree.

   In RIOK3 IPs from ΔDIS3L2 cells, tails were more frequent and longer. Wild-type cells lost about 25% of normalized 18S rRNA after starvation; ΔDIS3L2 gave an intermediate rescue relative to ΔRIOK3 (n = 11 for WT and ΔDIS3L2, n = 5 for ΔRIOK3). · DIS3L2 degrades uridylated 18S rRNA from its 3′…; QUANTIFICATION AND STATISTICAL ANALYSIS