Existence of a microRNA pathway in anucleate platelets

Abstract

Platelets have a crucial role in the maintenance of hemostasis as well as in thrombosis and vessel occlusion, which underlie stroke and acute coronary syndromes. Anucleate platelets contain mRNAs and are capable of protein synthesis, raising the issue of how these mRNAs are regulated. Here we show that human platelets harbor an abundant and diverse array of microRNAs (miRNAs), which are known as key regulators of mRNA translation in other cell types. Further analyses revealed that platelets contain the Dicer and Argonaute 2 (Ago2) complexes, which function in the processing of exogenous miRNA precursors and the control of specific reporter transcripts, respectively. Detection of the receptor P2Y(12) mRNA in Ago2 immunoprecipitates suggests that P2Y(12) expression may be subjected to miRNA control in human platelets. Our study lends an additional level of complexity to the control of gene expression in these anucleate elements of the cardiovascular system.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. Human platelets contain an abundant and diverse array of miRNAs

(a,b) Characterization of the purified platelet preparations from starting platelet-rich plasma (PRP) by reverse transcriptase-polymerase chain reaction (RT-PCR) (a) and hemocytometer counting (b). (c) Bioanalyzer assessment of RNA samples prepared from purified platelets (left) or megakaryocytes (right). (d) Platelet miRNA profiling analysis. MiRNA probes with signals above the detection threshold are shown in order of increasing relative fluorescence unit (RFI). (e) Northern blot validation of 4 selected miRNAs shown in order of decreasing micro-array RFI. A 10-nt RNA ladder was used as a size marker. cDNA, complementary DNA; GP, glycoprotein; Leuk., leukocyte; nt, nucleotide.

Figure 2. Platelets can synthesize miRNAs from pre-miRNAs

(a) Detection of the protein components of the miRNA-guided RNA silencing machinery in megakaryocytes (left) or platelets (right) by Western blot. β-Actin is used as a control. (b) Assessment of Dicer processing activity in protein extracts and Dicer immunoprecipitates (IP) prepared from purified platelets upon incubation with 32P-labeled human let-7a-3 pre-miRNA. (c) Extracts from purified platelets were separated by gel filtration on a Superose 6 column and the fractions analyzed by Western blot using anti-Dicer antibody, as described,. Selected (odd) fractions were tested for Dicer activity as in b. (d) Immunoblot analysis of TRBP2 IP derived from platelet extracts by using an anti-Dicer antibody. (e) Dicer processing activity assays on IP derived from platelet extracts using anti-TRBP2, control IgG or anti-Dicer antibodies. S10, 10,000 g supernatant fraction.

Figure 3. Platelets harbor Ago2·miRNA effector complexes functionally competent in gene silencing

(a, b)In vitro RNA-induced silencing complex (RISC) activity assays. (a) Protein extracts were incubated in the presence of a 32P-labeled RNA sensor bearing a binding site complementary to miR-223 without or with proteinase K (Prot. K; 1 mg ml−1) (center-right panel) or EDTA (5 mM) (right panel). (b) Same as in a (left panel), but using sensor RNAs bearing a binding site complementary to let-7c (left panel), miR-19a (center panel) or miR-199a-3p (right panel). (c) Same as in Figure 2c, except using anti-Ago2 antibody for immunoblotting. (d) Pooled fractions were probed for miR-223 (upper panel) and let-7c (lower panel) content by Northern blot. (e) Selected (even) fractions were tested for RISC activity as in a. (f) Ago2 immunoprecipitates (IP) were analyzed by Northern blot for the presence of miR-223. −, indicates IP using control IgG. (g) Ago2 IP were analyzed for RISC activity. (h,i) Same as in a, except in the presence of an antisense RNA to miR-223 (+) or Rluc (−) (h), or upon disrupting pairing of either the miR-223 cleavage site (lane 2) or seed region (lane 3) on the sensor transcript (i). * Indicates the expected cleavage products (38–40 nt). Temp., temperature.

Figure 4. Ago2·miR-223 complexes may regulate P2Y12 mRNA expression in platelets

(a) Base pairing of the miR-223 seed region to its predicted binding site, wild-type (WT) or mutated (Mut), in the 3′ untranslated region (3′UTR) of P2Y12 messenger RNA (mRNA). (b) Detection of P2Y12 protein (left) and mRNA (right) in extracts of platelets and megakaryocytes by Western blot and RT-PCR, respectively. (c) Functional validation of the predicted binding site for miR-223 present in P2Y12 mRNA 3′UTR by reporter gene assays performed in HEK293 cells (left and center panels) or megakaryocytes (right panel). Results are expressed as mean ± standard error of the mean (s.e.m.) (n = 5 to 7 experiments, in duplicate). * p<0.05; **p<0.001; *** p<0.0001 (two-tailed, unpaired Student’s t-test). (d) Detection of P2Y12 mRNA in Ago2 immunoprecipitates (IP) derived from either megakaryocyte or platelet extracts by RT-PCR. (e) Proposed model for platelet inheritance of a functional miRNA pathway, devoid of its initiation step, from megakaryocyte precursor cells. BS, binding site; miRNP, miRNA-containing ribonucleoprotein complex; RISC, RNA-induced silencing complex.