GSE129734
Exonuclease Requirements for Mammalian Ribosomal RNA Biogenesis and Surveillance
Ribosomal RNAs (rRNAs) biogenesis are multistep processes requiring the activity of several nuclear and cytoplasmic exonucleases. The exact processing steps for mammalian 5.8S rRNA remains obscure. Here, using loss-of-function approaches in mouse embryonic stem cells and deep sequencing of rRNA intermediates, we investigate at nucleotide resolution the requirements of exonucleases known to be involved in 5.8S maturation, and explore the role of the Perlman syndrome-associated 3'-5' exonuclease Dis3l2 in rRNA processing. We expand the repertoire of Dis3l2 targets to three 5.8S, 5S, and 28S rRNAs. 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 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. Overall design: The 3'-end of indicated ribosomal RNAs (rRNAs) were characterized by MiSeq upon genetic perturbation of Dis3l2, Exosc3, Exosc10, or Eri1 in mouse ESCs. Occasionally, to enrich for Dis3l2 targets, FLAG-tagged mutant Dis3l2 protein was ectopically expressed in Dis3l2 knockout ESCs and Dis3l2-bound rRNAs were co-immunoprecipitated using anti-FLAG antibody, cloned and sequenced. rRNA-specific sequences and adapters were used to extract desired reads from raw data.