Ribosomal RNA (rRNA) biogenesis is a multistep process requiring several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remain obscure. Here, using loss-of-function approaches in mouse embryonic stem cells (mESCs) and deep sequencing of rRNA intermediates, we investigate the requirements of exonucleases known to be involved in 5.8S maturation at nucleotide resolution and explore the role of the Perlman syndrome–associated 3′–5′ exonuclease Dis3l2 in rRNA processing. We uncover a novel cytoplasmic intermediate that we name ‘7SB’ rRNA that is generated through sequential processing by distinct exosome complexes. 7SB rRNA can be oligoadenylated by an unknown enzyme and/or oligouridylated by TUT4/7 and subsequently processed by Dis3l2 and Eri1. Moreover, exosome depletion triggers Dis3l2-mediated decay (DMD) as a surveillance pathway for rRNAs. Our data identify previously unknown 5.8S rRNA processing steps and provide nucleotide-level insight into the exonuclease requirements for mammalian rRNA processing.
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This work was supported by grants to R.I.G. from the US National Institutes of Health (R01GM086386; R01CA211328) and the March of Dimes Foundation (FY15-3339). M.P. was supported by a research fellowship from the Manton Center for Orphan Disease Research.
Integrated supplementary information
(a) Western blot analysis of Dis3l2 expression in wild type (WT), heterozygous (Het), and knockout (KO) mESC lines used in this study. (b) Probing 7SB rRNA in Dis3l2 stable knockdown V6.5 mESC line. (c) qRT-PCR analysis of relative uridylation for indicated mature or intermediate rRNA species in mESCs stably transduced with shLacZ (as control) and shDis3l2 shRNAs. qRT-PCR (d) and northern blot (e) analyses of FLAG-mutant Dis3l2-bound 5.8S rRNA with indicated primer sets or probe, respectively. Western blot (f) and qRT-PCR (g) indicate that 7SB uridylation can be rescued by FLAG-wild type Dis3l2 re-expression. Bars represent mean ± SEM; *p ≤ 0.05,**p ≤ 0.01, ***p ≤ 0.001, Student’s t-test (n ≥ 3); ns, not significant. Uncropped blot/gel images are shown in Supplementary Data Set 1.
(a) Schematic representation of circular RACE (cRACE) protocol to study 7SB rRNA tails. Total RNAs from mutant FLAG-Dis3l2 RIP and input samples were isolated, and treated with T4 RNA ligase. After DNase I treatment, reverse transcription was performed using reverse primer (overlapping 5.8S rRNA and ITS2) to enrich for 7SB species. PCR with divergent forward and reverse primers generated products that were purified and sequenced by MiSeq (see methods section for details). (b) Pie chart representation of various tails in extended 5.8S in RIP samples showed that tails contain mostly U or both A and then U (purple). Only a small set of the extended reads have A-tail but no U-tail (blue). (c) Size distribution of extensions (Ext.), A- and U-tails in RIP samples. (d) qRT-PCR analysis of 5.8S rRNA levels in Dis3l2 depleted mESCs. (e) Left panel: human DIS3L2 mRNA expression in stable knockdown human cell lines. Relative uridylation levels of human 7SB rRNA (middle panel) and pre-let-7a1 miRNA (right panel as a positive control) are measured by qRT-PCR. Bars represent mean ± SEM; **p ≤ 0.01, ***p ≤ 0.001, Student’s t-test (n ≥ 3); ns, not significant.
Supplementary Figure 3 Dis3l2 re-expression attenuates uridylation of pre-rRNAs stabilized by Exosc3- and Exosc10-depletion.
(a) Left panel: qRT-PCR analysis shows specific knockdown of Exosc3 and Exosc10 using specific siRNAs in Dis3l2 knockout ESCs. Right panel: qRT-PCR analysis revealing the rescue effect of Dis3l2 on uridylation of rRNAs. 7SK uridylation was unchanged, whereas Rmrp uridylation was only rescued by Dis3l2 re-expression but not induced upon Exosc3- or Exosc10-depletion.