RESEARCH ARTICLE
Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity Catherine Angénieux1,2,3,4, Blandine Maître1,2,3,4, Anita Eckly1,2,3,4, François Lanza1,2,3,4, Christian Gachet1,2,3,4, Henri de la Salle1,2,3,4* 1 UMR_S949, INSERM, Strasbourg, France, 2 Etablissement Français du Sang-Alsace (EFS-Alsace), Strasbourg, France, 3 Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France, 4 Université de Strasbourg, Strasbourg, France * [email protected]
Abstract
OPEN ACCESS Citation: Angénieux C, Maître B, Eckly A, Lanza F, Gachet C, de la Salle H (2016) Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity. PLoS ONE 11(1): e0148064. doi:10.1371/journal. pone.0148064 Editor: Christian Schulz, Medical Faculty, Ludwig Maximilians University Munich, GERMANY Received: May 6, 2015 Accepted: January 12, 2016 Published: January 25, 2016 Copyright: © 2016 Angénieux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Association de Recherche et Développement en Médecine et Santé Publique supports laboratory UMR-S949. This funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Previous investigations have indicated that RNAs are mostly present in the minor population of the youngest platelets, whereas translation in platelets could be biologically important. To attempt to solve this paradox, we studied changes in the RNA content of reticulated platelets, i.e., young cells brightly stained by thiazole orange (TObright), a fluorescent probe for RNAs. We provoked in mice strong thrombocytopenia followed by dramatic thrombocytosis characterized by a short period with a vast majority of reticulated platelets. During thrombocytosis, the TObright platelet count rapidly reached a maximum, after which TOdim platelets accumulated, suggesting that most of the former were converted into the latter within 12 h. Experiments on platelets, freshly isolated or incubated ex vivo at 37°C, indicated that their “RNA content”, here corresponding to the amounts of extracted RNA, and the percentage of TObright platelets were positively correlated. The “RNA Content” normalized to the number of platelets could be 20 to 40 fold higher when 80–90% of the cells were reticulated (20–40 fg/platelet), than when only 5–10% of control cells were TObright (less than 1fg/ platelet). TObright platelets, incubated ex vivo at 37°C or transfused into mice, became TOdim within 24 h. Ex vivo at 37°C, platelets lost about half of their ribosomal and beta actin RNAs within 6 hours, and more than 98% of them after 24 hours. Accordingly, fluorescence in situ hybridization techniques confirmed the presence of beta actin mRNAs in most reticulatedenriched platelets, but detected them in only a minor subset of control platelets. In vitro, constitutive translation decreased considerably within less than 6 hours, questioning how protein synthesis in platelets, especially in non-reticulated ones, could have a biological function in vivo. Nevertheless, constitutive transient translation in young platelets under pathological conditions characterized by a dramatic increase in circulating reticulated platelets could deserve to be investigated.
Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0148064 January 25, 2016
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Translation Only Transiently Occurs in Young Platelets.
Introduction The major primary hemostatic functions of blood platelets (PLTs) imply a series of rapid signal transduction events generally completed within a few seconds or minutes [1]. PLTs can also participate in vascular inflammation, through mechanisms which are less well characterized in terms of their kinetics, especially in vivo [2]. The idea that PLTs might possess a functional translation system is attractive since according to this view, these cells would play a role in longer term cellular and immunological responses [3]. Translation in PLTs has been documented for nearly half a century [4]. However, its reality has been questioned on the grounds of the possible presence of contaminating leukocytes in PLT preparations [5]. Moreover, human PLTs contain rather small amounts of RNA [6], which would question its biological function. Staining of PLTs with new methylene blue, a rough endoplasmic reticulum (RER) dye, allows the identification of a minor RER-rich population known as reticulated PLTs (retPLTs) [7]. This population can also be distinguished by flow cytometry (FC) after staining with thiazole orange (TO), a fluorescent probe for double stranded RNA molecules [7]. Animal studies have shown that TObright PLTs are relatively young, being less than 36 h old [8,9], whereas the PLT life span is 5 or 10 days in mouse and human, respectively; so, they represent a minor population, between less than 10% and a few percent of all PLTs, respectively. Increased TObright PLT counts have been associated with acute blood loss or reactive thrombopoiesis, in particular in cases of idiopathic thrombocytopenic purpura [10,11]. In this latter pathological context, transmission electron microscopy (TEM) confirmed that retPLTs contained RER, while metabolic labeling indicated that protein synthesis occurred in these cells [12]. Up until now, the precise characterization of the RNA content of PLTs has been neglected because PLTs were predominantly considered to be vestigial remnants of megakaryocytes (MKs). The experimental evidence that translation can occur in PLTs nevertheless challenged this view and incites one to better characterize their RNA content. In this objective, we studied the time-dependent changes of the “RNA content” of PLTs (defined as the RNA molecules extracted from PLTs), the integrity of their rRNA, the life span of beta actin mRNA in platelets placed in ex vivo conditions and, the constitutive biosynthetic activity in PLTs. Steady state conditions were compared with experimental conditions characterized by the transient presence of a vast majority of retPLTs. Our results were then correlated with previously published genetic studies of PLTs to assess the biological relevance of the deduced relative amounts of specific mRNAs in PLTs. Our experimental system also provides a unique means of investigating in vivo the functional characteristics of retPLTs.
Results Ablation of megakaryocytes (MKs) and experimental non-immune thrombocytopenia Mice transgenic for Cre-inducible diphtheria toxin receptor (DTR) [13] and Cre recombinase under the control of the MK-specific PF4 promoter [14] received DT for 4 days. Profound thrombocytopenia developed during the following days. In general, PLT counts reached a nadir three days later, i.e. on day 7 (22 +/- 17 103 PLTs/μL, n = 20) and rebounded to 250–800 103 cells/μL on day 8 (Fig 1A). In control animals receiving saline, PLT counts were stable (1110 +/- 163 103 PLTs/μL, n = 5). Since the life span of mouse PLTs is about 5 days [15], this time course indicated that DT killed maturing MKs, while PLT rebound occurred after the restoration of full MK differentiation. This hypothesis was confirmed by TEM analysis of bone marrow from saline- or DTtreated animals, one day after the last dose of DT, i.e on day 5. DT treatment resulted in a two-
PLOS ONE | DOI:10.1371/journal.pone.0148064 January 25, 2016
2 / 24
Translation Only Transiently Occurs in Young Platelets.
Fig 1. Enhanced megakaryopoiesis and thrombopoieisis after DT-mediated MK ablation. F1 iDTR x PF4-Cre mice received 100 ng/day DT or saline for 4 days (first injection on day 1). PLT counts decreased during the following days and PLT rebound occurred between days 7 and 8. (A) PLT counts of salineand DT-treated mice (n = 5 and 20, respectively) were measured on day 7 and 8. Horizontal bars indicate the mean values of the counts. Comparing the different conditions, a two way ANOVA analysis followed by a Bonferroni post-test indicated a p value of
Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity Catherine Angénieux1,2,3,4, Blandine Maître1,2,3,4, Anita Eckly1,2,3,4, François Lanza1,2,3,4, Christian Gachet1,2,3,4, Henri de la Salle1,2,3,4* 1 UMR_S949, INSERM, Strasbourg, France, 2 Etablissement Français du Sang-Alsace (EFS-Alsace), Strasbourg, France, 3 Fédération de Médecine Translationnelle de Strasbourg (FMTS), Strasbourg, France, 4 Université de Strasbourg, Strasbourg, France * [email protected]
Abstract
OPEN ACCESS Citation: Angénieux C, Maître B, Eckly A, Lanza F, Gachet C, de la Salle H (2016) Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity. PLoS ONE 11(1): e0148064. doi:10.1371/journal. pone.0148064 Editor: Christian Schulz, Medical Faculty, Ludwig Maximilians University Munich, GERMANY Received: May 6, 2015 Accepted: January 12, 2016 Published: January 25, 2016 Copyright: © 2016 Angénieux et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: Association de Recherche et Développement en Médecine et Santé Publique supports laboratory UMR-S949. This funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Previous investigations have indicated that RNAs are mostly present in the minor population of the youngest platelets, whereas translation in platelets could be biologically important. To attempt to solve this paradox, we studied changes in the RNA content of reticulated platelets, i.e., young cells brightly stained by thiazole orange (TObright), a fluorescent probe for RNAs. We provoked in mice strong thrombocytopenia followed by dramatic thrombocytosis characterized by a short period with a vast majority of reticulated platelets. During thrombocytosis, the TObright platelet count rapidly reached a maximum, after which TOdim platelets accumulated, suggesting that most of the former were converted into the latter within 12 h. Experiments on platelets, freshly isolated or incubated ex vivo at 37°C, indicated that their “RNA content”, here corresponding to the amounts of extracted RNA, and the percentage of TObright platelets were positively correlated. The “RNA Content” normalized to the number of platelets could be 20 to 40 fold higher when 80–90% of the cells were reticulated (20–40 fg/platelet), than when only 5–10% of control cells were TObright (less than 1fg/ platelet). TObright platelets, incubated ex vivo at 37°C or transfused into mice, became TOdim within 24 h. Ex vivo at 37°C, platelets lost about half of their ribosomal and beta actin RNAs within 6 hours, and more than 98% of them after 24 hours. Accordingly, fluorescence in situ hybridization techniques confirmed the presence of beta actin mRNAs in most reticulatedenriched platelets, but detected them in only a minor subset of control platelets. In vitro, constitutive translation decreased considerably within less than 6 hours, questioning how protein synthesis in platelets, especially in non-reticulated ones, could have a biological function in vivo. Nevertheless, constitutive transient translation in young platelets under pathological conditions characterized by a dramatic increase in circulating reticulated platelets could deserve to be investigated.
Competing Interests: The authors have declared that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0148064 January 25, 2016
1 / 24
Translation Only Transiently Occurs in Young Platelets.
Introduction The major primary hemostatic functions of blood platelets (PLTs) imply a series of rapid signal transduction events generally completed within a few seconds or minutes [1]. PLTs can also participate in vascular inflammation, through mechanisms which are less well characterized in terms of their kinetics, especially in vivo [2]. The idea that PLTs might possess a functional translation system is attractive since according to this view, these cells would play a role in longer term cellular and immunological responses [3]. Translation in PLTs has been documented for nearly half a century [4]. However, its reality has been questioned on the grounds of the possible presence of contaminating leukocytes in PLT preparations [5]. Moreover, human PLTs contain rather small amounts of RNA [6], which would question its biological function. Staining of PLTs with new methylene blue, a rough endoplasmic reticulum (RER) dye, allows the identification of a minor RER-rich population known as reticulated PLTs (retPLTs) [7]. This population can also be distinguished by flow cytometry (FC) after staining with thiazole orange (TO), a fluorescent probe for double stranded RNA molecules [7]. Animal studies have shown that TObright PLTs are relatively young, being less than 36 h old [8,9], whereas the PLT life span is 5 or 10 days in mouse and human, respectively; so, they represent a minor population, between less than 10% and a few percent of all PLTs, respectively. Increased TObright PLT counts have been associated with acute blood loss or reactive thrombopoiesis, in particular in cases of idiopathic thrombocytopenic purpura [10,11]. In this latter pathological context, transmission electron microscopy (TEM) confirmed that retPLTs contained RER, while metabolic labeling indicated that protein synthesis occurred in these cells [12]. Up until now, the precise characterization of the RNA content of PLTs has been neglected because PLTs were predominantly considered to be vestigial remnants of megakaryocytes (MKs). The experimental evidence that translation can occur in PLTs nevertheless challenged this view and incites one to better characterize their RNA content. In this objective, we studied the time-dependent changes of the “RNA content” of PLTs (defined as the RNA molecules extracted from PLTs), the integrity of their rRNA, the life span of beta actin mRNA in platelets placed in ex vivo conditions and, the constitutive biosynthetic activity in PLTs. Steady state conditions were compared with experimental conditions characterized by the transient presence of a vast majority of retPLTs. Our results were then correlated with previously published genetic studies of PLTs to assess the biological relevance of the deduced relative amounts of specific mRNAs in PLTs. Our experimental system also provides a unique means of investigating in vivo the functional characteristics of retPLTs.
Results Ablation of megakaryocytes (MKs) and experimental non-immune thrombocytopenia Mice transgenic for Cre-inducible diphtheria toxin receptor (DTR) [13] and Cre recombinase under the control of the MK-specific PF4 promoter [14] received DT for 4 days. Profound thrombocytopenia developed during the following days. In general, PLT counts reached a nadir three days later, i.e. on day 7 (22 +/- 17 103 PLTs/μL, n = 20) and rebounded to 250–800 103 cells/μL on day 8 (Fig 1A). In control animals receiving saline, PLT counts were stable (1110 +/- 163 103 PLTs/μL, n = 5). Since the life span of mouse PLTs is about 5 days [15], this time course indicated that DT killed maturing MKs, while PLT rebound occurred after the restoration of full MK differentiation. This hypothesis was confirmed by TEM analysis of bone marrow from saline- or DTtreated animals, one day after the last dose of DT, i.e on day 5. DT treatment resulted in a two-
PLOS ONE | DOI:10.1371/journal.pone.0148064 January 25, 2016
2 / 24
Translation Only Transiently Occurs in Young Platelets.
Fig 1. Enhanced megakaryopoiesis and thrombopoieisis after DT-mediated MK ablation. F1 iDTR x PF4-Cre mice received 100 ng/day DT or saline for 4 days (first injection on day 1). PLT counts decreased during the following days and PLT rebound occurred between days 7 and 8. (A) PLT counts of salineand DT-treated mice (n = 5 and 20, respectively) were measured on day 7 and 8. Horizontal bars indicate the mean values of the counts. Comparing the different conditions, a two way ANOVA analysis followed by a Bonferroni post-test indicated a p value of
