title: NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs.
abstract: Autosomal-recessive loss of the NSUN2 gene has been identified as a causative link to intellectual disability disorders in humans. NSun2 is an RNA methyltransferase modifying cytosine-5 in transfer RNAs (tRNAs), yet the identification of cytosine methylation in other RNA species has been hampered by the lack of sensitive and reliable molecular techniques. Here, we describe miCLIP as an additional approach for identifying RNA methylation sites in transcriptomes. miCLIP is a customized version of the individual-nucleotide-resolution crosslinking and immunoprecipitation (iCLIP) method. We confirm site-specific methylation in tRNAs and additional messenger and noncoding RNAs (ncRNAs). Among these, vault ncRNAs contained six NSun2-methylated cytosines, three of which were confirmed by RNA bisulfite sequencing. Using patient cells lacking the NSun2 protein, we further show that loss of cytosine-5 methylation in vault RNAs causes aberrant processing into Argonaute-associated small RNA fragments that can function as microRNAs. Thus, impaired processing of vault ncRNA may contribute to the etiology of NSun2-deficiency human disorders.
authors.affiliation: Wellcome Trust - Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK.
authors.forename: Shobbir
authors.initials: S
authors.lastname: Hussain
authors.forename: Abdulrahim A
authors.initials: AA
authors.lastname: Sajini
authors.forename: Sandra
authors.initials: S
authors.lastname: Blanco
authors.forename: Sabine
authors.initials: S
authors.lastname: Dietmann
authors.forename: Patrick
authors.initials: P
authors.lastname: Lombard
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Sugimoto
authors.forename: Maike
authors.initials: M
authors.lastname: Paramor
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Gleeson
authors.forename: Duncan T
authors.initials: DT
authors.lastname: Odom
authors.forename: Jernej
authors.initials: J
authors.lastname: Ule
authors.forename: Michaela
authors.initials: M
authors.lastname: Frye
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003596:Cytosine / Q000378:metabolism*; D019175:DNA Methylation; D057809:HEK293 Cells; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D022661:RNA, Untranslated / Q000235:genetics / Q000378:metabolism*; D059467:Transcriptome; D014162:Transfection
chemical_list: D022661:RNA, Untranslated; D012313:RNA; D003596:Cytosine; D008780:Methyltransferases; C515610:NSUN2 protein, human