title: A human 3' miR-499 mutation alters cardiac mRNA targeting and function.
abstract: BACKGROUND
  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.
  
  
  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.
authors.affiliation: Center for Pharmacogenomics, Department of Internal Medicine and Center for Pharmacogenomics, 660 S. Euclid Ave., Campus Box 8220, St. Louis, MO 63110, USA. gdorn@dom.wustl.edu
authors.forename: Gerald W
authors.initials: GW
authors.lastname: Dorn
authors.forename: Scot J
authors.initials: SJ
authors.lastname: Matkovich
authors.forename: William H
authors.initials: WH
authors.lastname: Eschenbacher
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001483:Base Sequence; D004195:Disease Models, Animal; D020869:Gene Expression Profiling; D006321:Heart / Q000503:physiopathology*; D006333:Heart Failure / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009206:Myocardium / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: C548683:MIRN499 microRNA, human; C573190:MIRN499 microRNA, mouse; D035683:MicroRNAs; D012333:RNA, Messenger; D009218:Myosins