A novel 5'-hydroxyl dinucleotide hydrolase activity for the DXO/Rai1 family of enzymes

Nucleic Acids Res. 2020 Jan 10;48(1):349-358. doi: 10.1093/nar/gkz1107.

Abstract

Modifications at the 5'-end of RNAs play a pivotal role in determining their fate. In eukaryotes, the DXO/Rai1 family of enzymes removes numerous 5'-end RNA modifications, thereby regulating RNA turnover. Mouse DXO catalyzes the elimination of incomplete 5'-end caps (including pyrophosphate) and the non-canonical NAD+ cap on mRNAs, and possesses distributive 5'-3' exoribonuclease activity toward 5'-monophosphate (5'-PO4) RNA. Here, we demonstrate that DXO also catalyzes the hydrolysis of RNAs bearing a 5'-hydroxyl group (5'-OH RNA). The crystal structure of DXO in complex with a 5'-OH RNA substrate mimic at 2.0 Å resolution provides elegant insight into the molecular mechanism of this activity. More importantly, the structure predicts that DXO first removes a dinucleotide from 5'-OH RNA. Our nuclease assays confirm this prediction and demonstrate that this 5'-hydroxyl dinucleotide hydrolase (HDH) activity for DXO is higher than the subsequent 5'-3' exoribonuclease activity for selected substrates. Fission yeast Rai1 also has HDH activity although it does not have 5'-3' exonuclease activity, and the Rat1-Rai1 complex can completely degrade 5'-OH RNA. An Arabidopsis DXO1 variant is active toward 5'-OH RNA but prefers 5'-PO4 RNA. Collectively, these studies demonstrate the diverse activities of DXO/Rai1 and expands the collection of RNA substrates that can undergo 5'-3' mediated decay.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Arabidopsis / enzymology
  • Arabidopsis / genetics
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Binding Sites
  • Chloroplast Proteins / chemistry
  • Chloroplast Proteins / genetics
  • Chloroplast Proteins / metabolism*
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Exoribonucleases / chemistry
  • Exoribonucleases / genetics
  • Exoribonucleases / metabolism*
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Mice
  • Models, Molecular
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • RNA, Messenger / chemistry
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Substrate Specificity
  • Trans-Activators / chemistry
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*

Substances

  • Arabidopsis Proteins
  • Chloroplast Proteins
  • Dom3Z protein, mouse
  • Nuclear Proteins
  • RNA, Messenger
  • RNA-Binding Proteins
  • Rai1 protein, S cerevisiae
  • Rai1 protein, mouse
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Trans-Activators
  • RAT1 protein, S cerevisiae
  • AT4G17620 protein, Arabidopsis
  • Exoribonucleases