Structure-function Analysis of Yeast RNA Debranching Enzyme (Dbr1), a Manganese-Dependent Phosphodiesterase

Nucleic Acids Res. 2005 Nov 7;33(19):6349-60. doi: 10.1093/nar/gki934. Print 2005.

Abstract

Saccharomyces cerevisiae Dbr1 is a 405-amino acid RNA debranching enzyme that cleaves the 2'-5' phosphodiester bonds of the lariat introns formed during pre-mRNA splicing. Debranching appears to be a rate-limiting step for the turnover of intronic RNA, insofar as the steady-state levels of lariat introns are greatly increased in a Deltadbr1 strain. To gain insight to the requirements for yeast Dbr1 function, we performed a mutational analysis of 28 amino acids that are conserved in Dbr1 homologs from other organisms. We identified 13 residues (His13, Asp40, Arg45, Asp49, Tyr68, Tyr69, Asn85, His86, Glu87, His179, Asp180, His231 and His233) at which alanine substitutions resulted in lariat intron accumulation in vivo. Conservative replacements at these positions were introduced to illuminate structure-activity relationships. Residues important for Dbr1 function include putative counterparts of the amino acids that comprise the active site of the metallophosphoesterase superfamily, exemplified by the DNA phosphodiesterase Mre11. Using natural lariat RNAs and synthetic branched RNAs as substrates, we found that mutation of Asp40, Asn85, His86, His179, His231 or His233 to alanine abolishes or greatly diminishes debranching activity in vitro. Dbr1 sediments as a monomer and requires manganese as the metal cofactor for debranching.

Publication types

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

MeSH terms

  • Alanine / genetics
  • Amino Acid Sequence
  • DNA Mutational Analysis
  • Introns
  • Manganese / chemistry*
  • Molecular Sequence Data
  • Phosphoric Diester Hydrolases / chemistry*
  • Phosphoric Diester Hydrolases / genetics
  • Phosphoric Diester Hydrolases / metabolism*
  • RNA Nucleotidyltransferases / chemistry*
  • RNA Nucleotidyltransferases / genetics
  • RNA Nucleotidyltransferases / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Alignment
  • Structure-Activity Relationship

Substances

  • Saccharomyces cerevisiae Proteins
  • Manganese
  • RNA Nucleotidyltransferases
  • RNA debranching enzyme Dbr1, S cerevisiae
  • mRNA debranching enzyme
  • Phosphoric Diester Hydrolases
  • Alanine