Time-Dependent Decay of mRNA and Ribosomal RNA during Platelet Aging and Its Correlation with Translation Activity

Abstract

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 reticulated-enriched 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.

Slowed decay of mRNAs enhances platelet specific translation

“…Similar variation was observed using published platelet RNA-Seq datasets from Rowley et al (supplemental Figure 3B). 23 Again, these data point to an underlying active process such as MK mRNA sorting 28 or platelet mRNA decay 11,13 shaping the platelet transcriptome and creating the broad variation in platelet enrichment we observed. A limitation of this analysis, however, is that the cultures of ex vivo-differentiated MKs used for RNA-Seq by Nürnberg et al contained 0% to 10% undifferentiated cells.…”

“…Based on these data, the typical mRNA decays with a half-life of 5.7 hours in PLPs (supplemental Figure 4A), consistent with recent measurements of bulk RNA turnover in primary platelets. 11 Histone mRNAs were somewhat shorter-lived (average half-life, 4.2 hours), whereas ribosomal protein mRNAs were slightly longer-lived than average (average half-life, 6.6 hours) (supplemental Figure 4B-C), and a few specific mRNAs seemed to be especially resistant to degradation, including MYD88 and RAP1A, which have special roles in platelets [75][76][77][78] ( Figure 4D). Taken together, these data suggest that RNA decay is likely a major driver that determines transcript abundance (and therefore to a significant extent translation) in platelets.…”

“…1,2 Newly released ("reticulated") platelets retain MK-derived RNA 3 ; this reticulated subpopulation is most evident when the rate of thrombopoiesis is elevated, which occurs after acute blood loss 4 or during thrombocytopenia. [5][6][7][8][9][10][11] Because platelets lack the ability to synthesize new messenger RNA (mRNA) transcripts or assemble new ribosomes, the abundance of MK-derived RNA in platelets gradually declines over several days. 10,12,13 This bulk decrease in RNA principally reflects the clearance of ribosomes (and the constituent ribosomal RNA [rRNA]), similar to the maturation process that occurs in reticulocytes during terminal differentiation.…”

“…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. 11,13 However, some evidence exists for translation in older The data used in this article have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus database (accession number GSE94384).…”

Ribosomes in platelets protect the messenger

“…By using RNA-Seq, they demonstrated that the half-life of mRNAs in cultured PLPs is 5.7 hours, a result that coincides with previous measurements of ex vivo mRNA decay in platelets. 5 Transgenic overexpression of PELO in PLPs significantly decreased the half-life of the majority of PLP mRNAs. Given that PELO expression is low in platelets, 7 these data suggest that megakaryocytes invest low levels of PELO into platelets as a mechanism for preserving mRNA levels in anucleate platelets that are incapable of transcribing new mRNA.…”