title: Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets.
abstract: Human platelets contain microRNAs (miRNAs) and miRNA processing machinery, but their contribution to platelet function remains incompletely understood. Here, we show that murine megakaryocyte (MK)-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduces the level of the majority of miRNAs in platelets. This leads to altered platelet messenger RNA (mRNA) expression profiles and mild thrombocytopenia. Fibrinogen receptor subunits Itga2b (αIIb) and Itgb3 (β3) mRNAs were among the differentially expressed transcripts that are increased in platelets lacking Dicer1. Argonaute 2 (Ago2), a member of the miRNA silencing complex, co-immunoprecipitated with αIIband β3mRNAs in wild-type platelets. Furthermore, co-immunoprecipitation experiments suggested reduced αIIb/β3/Ago2 complexes in miRNA-deficient platelets. These results suggested that miRNAs regulate both integrin subunits. Subsequent 3' untranslated region luciferase reporter assays confirmed that the translation of both αIIband β3mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion ofDicer1resulted in increased surface expression of integrins αIIband β3, and enhanced platelet binding to fibrinogen in vivo and in vitro. Heightened platelet reactivity, shortened tail-bleeding time, and reduced survival following collagen/epinephrine-induced pulmonary embolism were also observed in Dicer1-deficient animals. CombinedPf4-cre-mediated deletion of Drosha and Dicer1 did not significantly exacerbate phenotypes observed in single Dicer1 knockout mice. In summary, these findings indicate that Dicer1-dependent generation of mature miRNAs in late-stage MKs and platelets modulates the expression of target mRNAs important for the hemostatic and thrombotic function of platelets.
authors.affiliation: The Molecular Medicine Program and Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT;
authors.forename: Jesse W
authors.initials: JW
authors.lastname: Rowley
authors.affiliation: Australian Cancer Research Foundation Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia;
authors.forename: Stéphane
authors.initials: S
authors.lastname: Chappaz
authors.affiliation: Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Université Laval, Québec, QC, Canada;
authors.forename: Aurélie
authors.initials: A
authors.lastname: Corduan
authors.affiliation: Australian Cancer Research Foundation Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia;
authors.forename: Mark M W
authors.initials: MM
authors.lastname: Chong
authors.affiliation: The Molecular Medicine Program and Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT;
authors.forename: Robert
authors.initials: R
authors.lastname: Campbell
authors.affiliation: The Molecular Medicine Program and.
authors.forename: Amanda
authors.initials: A
authors.lastname: Khoury
authors.affiliation: The Molecular Medicine Program and.
authors.forename: Bhanu Kanth
authors.initials: BK
authors.lastname: Manne
authors.affiliation: Department of Anatomy and Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA; Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA; and.
authors.forename: Jeremy G T
authors.initials: JG
authors.lastname: Wurtzel
authors.affiliation: Department of Anatomy and Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA; Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA; and.
authors.forename: James V
authors.initials: JV
authors.lastname: Michael
authors.affiliation: Department of Anatomy and Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA; Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA; and.
authors.forename: Lawrence E
authors.initials: LE
authors.lastname: Goldfinger
authors.affiliation: Deparment of Pharmacology, Case Western Reserve University, Cleveland, OH.
authors.forename: Michele M
authors.initials: MM
authors.lastname: Mumaw
authors.affiliation: Deparment of Pharmacology, Case Western Reserve University, Cleveland, OH.
authors.forename: Marvin T
authors.initials: MT
authors.lastname: Nieman
authors.affiliation: Australian Cancer Research Foundation Chemical Biology Division, The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia;
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Kile
authors.affiliation: Centre Hospitalier Universitaire de Québec Research Center and Faculty of Medicine, Université Laval, Québec, QC, Canada;
authors.forename: Patrick
authors.identifier: http://orcid.org/0000-0002-6099-6562
authors.initials: P
authors.lastname: Provost
authors.affiliation: The Molecular Medicine Program and Department of Internal Medicine, University of Utah School of Medicine, Salt Lake City, UT;
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Weyrich
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D039421:Integrin alpha2 / Q000096:biosynthesis / Q000235:genetics; D039661:Integrin beta3 / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D011655:Pulmonary Embolism / Q000139:chemically induced / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*
chemical_list: C526735:Ago2 protein, mouse; D060565:Argonaute Proteins; D039421:Integrin alpha2; D039661:Integrin beta3; D035683:MicroRNAs; D012333:RNA, Messenger; C509781:Dicer1 protein, mouse; C513400:Drosha protein, mouse; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases