The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21

Sloan, Katherine E.; Leisegang, Matthias; Doebele, Carmen; Ramírez, Ana S.; Simm, Stefan; Safferthal, Charlotta; Kretschmer, Jens; Schorge, Tobias; Markoutsa, Stavroula; Haag, Sara; Karas, Michael; Ebersberger, Ingo; Schleiff, Enrico; Watkins, Nicholas J.; Bohnsack, Markus T.

doi:10.1093/nar/gku1291

Cited by 71 publications

(84 citation statements)

References 41 publications

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“…To identify NSUN3 target RNAs, we performed UV cross‐linking and analysis of cDNA (CRAC; Bohnsack et al , 2012; Sloan et al , 2015) experiments using the NSUN3‐HisPrcFLAG cell line; a HEK293 cell line expressing only the HisPrcFLAG tag was used as a control. In addition, cells expressing NSUN3‐HisPrcFLAG were treated with the cytidine derivative 5‐azacytidine (5‐AzaC) as a cross‐linking reagent, which is incorporated into nascent RNA and specifically traps m 5 C RNA methyltransferases on their target nucleotides in a covalent protein–RNA intermediate during the methylation reaction (Fig 2A; Khoddami & Cairns, 2013).…”

Section: Resultsmentioning

confidence: 99%

“…The catalytically inactive NSUN3 C265A mutant was generated by site‐directed mutagenesis (Haag et al , 2015b). The constructs were transfected into HEK293 Flp‐In T‐Rex cells according to the manufacturer's instructions and as described (Sloan et al , 2015). UV and 5‐AzaC cross‐linking and analysis of cDNA (CRAC) experiments were carried out as previously described (Bohnsack et al , 2012; Haag et al , 2015a; see also Appendix Supplementary Methods).…”

Section: Methodsmentioning

confidence: 99%

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NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

et al. 2016

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Mitochondrial gene expression uses a non‐universal genetic code in mammals. Besides reading the conventional AUG codon, mitochondrial (mt‐)tRNAM et mediates incorporation of methionine on AUA and AUU codons during translation initiation and on AUA codons during elongation. We show that the RNA methyltransferase NSUN3 localises to mitochondria and interacts with mt‐tRNAM et to methylate cytosine 34 (C34) at the wobble position. NSUN3 specifically recognises the anticodon stem loop (ASL) of the tRNA, explaining why a mutation that compromises ASL basepairing leads to disease. We further identify ALKBH1/ABH1 as the dioxygenase responsible for oxidising m5C34 of mt‐tRNAM et to generate an f5C34 modification. In vitro codon recognition studies with mitochondrial translation factors reveal preferential utilisation of m5C34 mt‐tRNA Met in initiation. Depletion of either NSUN3 or ABH1 strongly affects mitochondrial translation in human cells, implying that modifications generated by both enzymes are necessary for mt‐tRNAM et function. Together, our data reveal how modifications in mt‐tRNAM et are generated by the sequential action of NSUN3 and ABH1, allowing the single mitochondrial tRNAM et to recognise the different codons encoding methionine.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

et al. 2016

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“…Crosslinking and analysis of cDNA (CRAC) was carried out as previously described Sloan et al 2015). In brief, HEK293 cells expressing NSUN6-HisPrcFlag, NSUN6-C373A-HisPrcFLAG or the HisPrcFlag tag alone were induced using 1 μg/ mL tetracycline for 24 h and UV crosslinking was carried out using a Stratalinker (Stratagene).…”

Section: Crosslinking and Analysis Of Cdna (Crac)mentioning

confidence: 99%

NSUN6 is a human RNA methyltransferase that catalyzes formation of m⁵C72 in specific tRNAs

Haag

Warda

Kretschmer

et al. 2015

RNA

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Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m 5 C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m 5 C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA Cys and tRNA Thr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3 ′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3 ′ -CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.

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“…3i). In addition to transcription, DDX21 is also required for rRNA processing 5,14 through its interaction with both rRNA and small nucleolar RNAs (snoRNAs). Pol I inhibition disengaged DDX21 from both the 5′ external transcribed spacer, a site of processing in the rRNA, and from the snoRNAs (Extended Data Fig.…”

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Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders

Calo

Bowen

et al. 2018

Nature

132

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Many craniofacial disorders are caused by heterozygous mutations in general regulators of housekeeping cellular functions such as transcription or ribosome biogenesis1,2. Although it is understood that many of these malformations are a consequence of defects in cranial neural crest cells, a cell type that gives rise to most of the facial structures during embryogenesis3,4, the mechanism underlying cell-type selectivity of these defects remains largely unknown. By exploring molecular functions of DDX21, a DEAD-box RNA helicase involved in control of both RNA polymerase (Pol) I- and II-dependent transcriptional arms of ribosome biogenesis5, we uncovered a previously unappreciated mechanism linking nucleolar dysfunction, ribosomal DNA (rDNA) damage, and craniofacial malformations. Here we demonstrate that genetic perturbations associated with Treacher Collins syndrome, a craniofacial disorder caused by heterozygous mutations in components of the Pol I transcriptional machinery or its cofactor TCOF1 (ref. 1), lead to relocalization of DDX21 from the nucleolus to the nucleoplasm, its loss from the chromatin targets, as well as inhibition of rRNA processing and downregulation of ribosomal protein gene transcription. These effects are cell-type-selective, cell-autonomous, and involve activation of p53 tumour-suppressor protein. We further show that cranial neural crest cells are sensitized to p53-mediated apoptosis, but blocking DDX21 loss from the nucleolus and chromatin rescues both the susceptibility to apoptosis and the craniofacial phenotypes associated with Treacher Collins syndrome. This mechanism is not restricted to cranial neural crest cells, as blood formation is also hypersensitive to loss of DDX21 functions. Accordingly, ribosomal gene perturbations associated with Diamond-Blackfan anaemia disrupt DDX21 localization. At the molecular level, we demonstrate that impaired rRNA synthesis elicits a DNA damage response, and that rDNA damage results in tissue-selective and dosage-dependent effects on craniofacial development. Taken together, our findings illustrate how disruption in general regulators that compromise nucleolar homeostasis can result in tissue-selective malformations.

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The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21

Cited by 71 publications

References 41 publications

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

NSUN6 is a human RNA methyltransferase that catalyzes formation of m⁵C72 in specific tRNAs

Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders

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The association of late-acting snoRNPs with human pre-ribosomal complexes requires the RNA helicase DDX21

Cited by 71 publications

References 41 publications

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA Met to expand codon recognition in mitochondrial translation

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders

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Product

Resources

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NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

NSUN6 is a human RNA methyltransferase that catalyzes formation of m⁵C72 in specific tRNAs