A human 3' miR-499 mutation alters cardiac mRNA targeting and function

Circ Res. 2012 Mar 30;110(7):958-67. doi: 10.1161/CIRCRESAHA.111.260752. Epub 2012 Feb 28.

Abstract

Rationale: MyomiRs miR-499, miR-208a and miR-208b direct cardiac myosin gene expression. Sequence complementarity between miRs and their mRNA targets determines miR effects, but the functional consequences of human myomiR sequence variants are unknown.

Objective: To identify and investigate mutations in human myomiRs in order to better understand how and to what extent naturally-occurring sequence variation can impact miR-mRNA targeting and end-organ function.

Methods and results: Screening of ≈2,600 individual DNAs for myomiR sequence variants identified a rare mutation of miR-499, u17c in the 3' end, well outside the seed region thought to determine target recognition. In vitro luciferase reporter analysis showed that the 3' miR-499 mutation altered suppression of a subset of artificial and natural mRNA targets. Cardiac-specific transgenic expression was used to compare consequences of wild-type and mutant miR-499. Both wild-type and mutant miR-499 induced heart failure in mice, but miR-499 c17 misdirected recruitment of a subset of miR-499 target mRNAs to cardiomyocyte RNA-induced silencing complexes, altering steady-state cardiac mRNA and protein make-up and favorably impacting cardiac function. In vitro analysis of miR-499 target site mutations and modeling of binding energies revealed abnormal miR-mRNA duplex configurations induced by the c17 mutation.

Conclusions: A naturally occurring miR-499 mutation outside the critical seed sequence modifies mRNA targeting and end-organ function. This first description of in vivo effects from a natural human miR mutation outside the seed sequence supports comprehensive studies of individual phenotypes or disease-modification conferred by miR mutations.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Disease Models, Animal
  • Gene Expression Profiling
  • Heart / physiopathology*
  • Heart Failure / genetics
  • Heart Failure / metabolism
  • Humans
  • In Vitro Techniques
  • Mice
  • Mice, Transgenic
  • MicroRNAs / genetics*
  • Molecular Sequence Data
  • Mutation / genetics*
  • Myocardium / metabolism*
  • Myosins / genetics
  • Myosins / metabolism
  • Proteomics
  • RNA, Messenger / metabolism*

Substances

  • MIRN499 microRNA, human
  • MIRN499 microRNA, mouse
  • MicroRNAs
  • RNA, Messenger
  • Myosins