Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets

Jesse W. Rowley, Stephane Chappaz, Aurelie Corduan, Mark M. W. Chong, Robert Campbell, Amanda Khoury, Bhanu Kanth Manne, Jeremy G. T. Wurtzel, James V. Michael, Lawrence E. Goldfinger, Michele M. Mumaw, Marvin T. Nieman, Benjamin T. Kile, Patrick Provost, Andrew S. Weyrich

Research output: Contribution to journalArticleResearchpeer-review

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 αIIb and β3 mRNAs 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 αIIb and β3 mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion of Dicer1 resulted in increased surface expression of integrins αIIb and 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. Combined Pf4-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.
Original languageEnglish
Pages (from-to)1743-1751
Number of pages9
JournalBlood
Volume127
Issue number14
DOIs
Publication statusPublished - 7 Apr 2016
Externally publishedYes

Cite this

Rowley, J. W., Chappaz, S., Corduan, A., Chong, M. M. W., Campbell, R., Khoury, A., ... Weyrich, A. S. (2016). Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets. Blood, 127(14), 1743-1751. https://doi.org/10.1182/blood-2015-07-661371
Rowley, Jesse W. ; Chappaz, Stephane ; Corduan, Aurelie ; Chong, Mark M. W. ; Campbell, Robert ; Khoury, Amanda ; Manne, Bhanu Kanth ; Wurtzel, Jeremy G. T. ; Michael, James V. ; Goldfinger, Lawrence E. ; Mumaw, Michele M. ; Nieman, Marvin T. ; Kile, Benjamin T. ; Provost, Patrick ; Weyrich, Andrew S. . / Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets. In: Blood. 2016 ; Vol. 127, No. 14. pp. 1743-1751.
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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 αIIb and β3 mRNAs 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 αIIb and β3 mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion of Dicer1 resulted in increased surface expression of integrins αIIb and 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. Combined Pf4-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.",
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Rowley, JW, Chappaz, S, Corduan, A, Chong, MMW, Campbell, R, Khoury, A, Manne, BK, Wurtzel, JGT, Michael, JV, Goldfinger, LE, Mumaw, MM, Nieman, MT, Kile, BT, Provost, P & Weyrich, AS 2016, 'Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets' Blood, vol. 127, no. 14, pp. 1743-1751. https://doi.org/10.1182/blood-2015-07-661371

Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets. / Rowley, Jesse W.; Chappaz, Stephane; Corduan, Aurelie ; Chong, Mark M. W. ; Campbell, Robert ; Khoury, Amanda; Manne, Bhanu Kanth ; Wurtzel, Jeremy G. T. ; Michael, James V.; Goldfinger, Lawrence E.; Mumaw, Michele M. ; Nieman, Marvin T. ; Kile, Benjamin T.; Provost, Patrick; Weyrich, Andrew S. .

In: Blood, Vol. 127, No. 14, 07.04.2016, p. 1743-1751.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Dicer1-mediated miRNA processing shapes the mRNA profile and function of murine platelets

AU - Rowley, Jesse W.

AU - Chappaz, Stephane

AU - Corduan, Aurelie

AU - Chong, Mark M. W.

AU - Campbell, Robert

AU - Khoury, Amanda

AU - Manne, Bhanu Kanth

AU - Wurtzel, Jeremy G. T.

AU - Michael, James V.

AU - Goldfinger, Lawrence E.

AU - Mumaw, Michele M.

AU - Nieman, Marvin T.

AU - Kile, Benjamin T.

AU - Provost, Patrick

AU - Weyrich, Andrew S.

PY - 2016/4/7

Y1 - 2016/4/7

N2 - 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 αIIb and β3 mRNAs 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 αIIb and β3 mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion of Dicer1 resulted in increased surface expression of integrins αIIb and 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. Combined Pf4-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.

AB - 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 αIIb and β3 mRNAs 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 αIIb and β3 mRNAs can be regulated by miRNAs miR-326, miR-128, miR-331, and miR-500. Consistent with these molecular changes, the deletion of Dicer1 resulted in increased surface expression of integrins αIIb and 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. Combined Pf4-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.

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