2'-O-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO

PLoS One. 2018 Mar 30;13(3):e0193804. doi: 10.1371/journal.pone.0193804. eCollection 2018.

Abstract

The 5' RNA cap structure (m7GpppRNA) is a key feature of eukaryotic mRNAs with important roles in stability, splicing, polyadenylation, mRNA export, and translation. Higher eukaryotes can further modify this minimal cap structure with the addition of a methyl group on the ribose 2'-O position of the first transcribed nucleotide (m7GpppNmpRNA) and sometimes on the adjoining nucleotide (m7GpppNmpNmpRNA). In higher eukaryotes, the DXO protein was previously shown to be responsible for both decapping and degradation of RNA transcripts harboring aberrant 5' ends such as pRNA, pppRNA, GpppRNA, and surprisingly, m7GpppRNA. It was proposed that the interaction of the cap binding complex with the methylated cap would prevent degradation of m7GpppRNAs by DXO. However, the critical role of the 2'-O-methylation found in higher eukaryotic cap structures was not previously addressed. In the present study, we demonstrate that DXO possesses both decapping and exoribonuclease activities toward incompletely capped RNAs, only sparing RNAs with a 2'-O-methylated cap structure. Fluorescence spectroscopy assays also revealed that the presence of the 2'-O-methylation on the cap structure drastically reduces the affinity of DXO for RNA. Moreover, immunofluorescence and structure-function assays also revealed that a nuclear localisation signal is located in the amino-terminus region of DXO. Overall, these results are consistent with a quality control mechanism in which DXO degrades incompletely capped RNAs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endoribonucleases / genetics
  • Endoribonucleases / metabolism*
  • Escherichia coli
  • Exoribonucleases
  • Fluorescent Antibody Technique
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Methylation
  • Mutagenesis, Site-Directed
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • RNA Caps / metabolism*
  • RNA Stability*
  • RNA, Messenger / metabolism*
  • Recombinant Proteins / metabolism
  • Spectrometry, Fluorescence
  • Structure-Activity Relationship
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*

Substances

  • Nuclear Proteins
  • RNA Caps
  • RNA, Messenger
  • Recombinant Proteins
  • Trans-Activators
  • DXO protein, human
  • Endoribonucleases
  • Exoribonucleases
  • DCP1A protein, human

Grants and funding

This work was supported by grants from the Canadian Institutes of Health Research (CIHR)​ and the Natural Sciences and Engineering Research Council of Canada (NSERC) (MB)​. M.B. is a Chercheur-boursier Sénior from the Fonds de recherche du Québec-Santé (FRQ-S). C.B. holds a graduate training award from the Faculté de médecine et des sciences de la santé de l’Université de Sherbrooke. A.A. holds a graduate training award from the CIHR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.