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.

Slowed decay of mRNAs enhances platelet specific translation

“…29,38,[84][85][86] Through ribosome profiling, we show that much of this transcriptional complexity indeed shapes the platelet proteome through protein synthesis by platelet ribosomes. We combine our high-throughput RNA-Seq and ribosome profiling data with published mass spectrometry studies to present the first integrated view of platelet gene expression.…”

“…Numerous specific examples of proteins synthesized by platelets have been described, [32][33][34][35][36] and an active microRNA pathway has been studied 26,27,29,37,38 and implicated in regulating mRNA decay and translation in platelets. 29 Because it is technically difficult to study platelet populations of different ages in vivo, 39 RNA-Seq studies have reported on an "ensemble" average of platelet transcripts that are influenced by the underlying age distribution of circulating platelets at the time of sample collection. Given the documented clearance of RNA by platelets during the first several days of circulation, 3,10,12 it seems likely that platelet protein synthesis primarily occurs within a few days of release from the MK.…”

“…29 As with mRNAs in other tissues, platelet mRNAs are marked by 59 m 7 G-caps 30 and 39polyA-tails, 25,31 which are important features recognized by the eukaryotic translational machinery. Numerous specific examples of proteins synthesized by platelets have been described, [32][33][34][35][36] and an active microRNA pathway has been studied 26,27,29,37,38 and implicated in regulating mRNA decay and translation in platelets. 29 Because it is technically difficult to study platelet populations of different ages in vivo, 39 RNA-Seq studies have reported on an "ensemble" average of platelet transcripts that are influenced by the underlying age distribution of circulating platelets at the time of sample collection.…”

The clinical significance of platelet microparticle-associated microRNAs

“…A more recent study by Rowley et al [32], however, showed that murine megakaryocyte-specific knockdown of Dicer1, the ribonuclease that cleaves miRNA precursors into mature miRNAs, reduced the level of the majority of microRNAs in platelets, altered platelet mRNA expression profiles and induced mild thrombocytopenia. Dicer1 deletion resulted in increased surface expression of integrins alphaIIb and beta3, and enhanced platelet binding to fibrinogen in vivo and in vitro.…”

Genomics and transcriptomics of megakaryocytes and platelets: Implications for health and disease

“…A critical transcription factor for megakaryocyte development and thrombopoiesis is RUNX1, which has been shown to repress the erythroid gene expression program, thus facilitating megakaryocyte differentiation 22. Furthermore, RUNX1 regulates cytoskeletal genes such as MYH9 and MYH10 and plays a role in megakaryocyte ploidy and proplatelet formation 23, 24, 25. ETV6, a member of the ETS transcription factor family, is required for late megakaryopoiesis and hematopoietic stem cell survival 26.…”