N6-methyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs). The discovery of FTO as the first m6A mRNA demethylase established the concept of reversible RNA modification. Here, we present a comprehensive transcriptome-wide analysis of RNA demethylation and uncover FTO as a potent regulator of nuclear mRNA processing events such as alternative splicing and 3΄ end mRNA processing. We show that FTO binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced (AS) exons and poly(A) sites. FTO knockout (KO) results in substantial changes in pre-mRNA splicing with prevalence of exon skipping events. The alternative splicing effects of FTO KO anti-correlate with METTL3 knockdown suggesting the involvement of m6A. Besides, deletion of intronic region that contains m6A-linked DRACH motifs partially rescues the FTO KO phenotype in a reporter system. All together, we demonstrate that the splicing effects of FTO are dependent on the catalytic activity in vivo and are mediated by m6A. Our results reveal for the first time the dynamic connection between FTO RNA binding and demethylation activity that influences several mRNA processing events.

• MeSH
• 3' nepřekládaná oblast genetika   MeSH
• adenosin analogy a deriváty   metabolismus   MeSH
• alternativní sestřih * MeSH
• exony genetika   MeSH
• gen pro FTO genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• introny genetika   MeSH
• lidé MeSH
• methyltransferasy genetika   metabolismus   MeSH
• mutace MeSH
• mutageneze cílená MeSH
• poly A genetika   MeSH
• prekurzory RNA genetika   metabolismus   MeSH
• stanovení celkové genové exprese metody   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs.

• MeSH
• aminokyselinové motivy MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• oligoribonukleotidy metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající RNA analýza   chemie   metabolismus   MeSH
• RNA malá jaderná chemie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• uracilnukleotidy metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Recruitment of appropriate RNA processing factors to the site of transcription is controlled by post-translational modifications of the C-terminal domain (CTD) of RNA polymerase II (RNAP II). Here, we report the solution structure of the Ser5 phosphorylated (pSer5) CTD bound to Nrd1. The structure reveals a direct recognition of pSer5 by Nrd1 that requires the cis conformation of the upstream pSer5-Pro6 peptidyl-prolyl bond of the CTD. Mutations at the complex interface diminish binding affinity and impair processing or degradation of noncoding RNAs. These findings underpin the interplay between covalent and noncovalent changes in the CTD structure that constitute the CTD code.

• MeSH
• fosforylace MeSH
• molekulární modely MeSH
• nekódující RNA metabolismus   MeSH
• prolin metabolismus   MeSH
• proteiny vázající RNA chemie   metabolismus   MeSH
• RNA-polymerasa II metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   metabolismus   MeSH
• Saccharomyces cerevisiae cytologie   enzymologie   genetika   metabolismus   MeSH
• serin metabolismus   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů MeSH
• viabilita buněk MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Non-coding RNA polymerase II transcripts are processed by the poly(A)-independent termination pathway that requires the Nrd1 complex. The Nrd1 complex includes two RNA-binding proteins, the nuclear polyadenylated RNA-binding (Nab) 3 and the nuclear pre-mRNA down-regulation (Nrd) 1 that bind their specific termination elements. Here we report the solution structure of the RNA-recognition motif (RRM) of Nab3 in complex with a UCUU oligonucleotide, representing the Nab3 termination element. The structure shows that the first three nucleotides of UCUU are accommodated on the β-sheet surface of Nab3 RRM, but reveals a sequence-specific recognition only for the central cytidine and uridine. The specific contacts we identified are important for binding affinity in vitro as well as for yeast viability. Furthermore, we show that both RNA-binding motifs of Nab3 and Nrd1 alone bind their termination elements with a weak affinity. Interestingly, when Nab3 and Nrd1 form a heterodimer, the affinity to RNA is significantly increased due to the cooperative binding. These findings are in accordance with the model of their function in the poly(A) independent termination, in which binding to the combined and/or repetitive termination elements elicits efficient termination.

• MeSH
• genetická transkripce MeSH
• jaderné proteiny chemie   genetika   metabolismus   MeSH
• konformace proteinů MeSH
• magnetická rezonanční spektroskopie MeSH
• multimerizace proteinu MeSH
• oligonukleotidy chemie   metabolismus   MeSH
• proteiny vázající RNA chemie   genetika   metabolismus   MeSH
• roztoky MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence nukleotidů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH