Circular RNA enrichment in platelets is a signature of transcriptome degradation

Abstract

Key Points• Circular RNAs are hugely enriched in platelets compared with nucleated cell types.• Lack of enrichment in megakaryocte progenitors implicates degradation of platelet linear RNAs.In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17-to 188-fold relative to nucleated tissues and 14-to 26-fold relative to samples digested with RNAse R to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched on average 12.7 times in platelets relative to nucleated tissues and identify 3162 genes significantly enriched for circRNAs, including some where all RNAseq reads appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in cultured megakaryocytes, and demonstrate that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost >90% of their progenitor mRNAs and that translation in platelets occurs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts previously classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artifacts. (Blood. 2016;127(9):e1-e11)

Selective release of circRNAs in platelet‐derived extracellular vesicles

“…Strikingly, in platelets, a large set of circular isoforms (70% in resting, 66% in activated platelets) are predominantly expressed, and 95% of these abundant circRNAs are identical in resting and activated platelets. These results are consistent with the previous study by Alhasan et al [20], which showed that circRNAs are highly enriched in platelets and, in addition, that for some genes only the circular isoform was detected; especially the enrichment of back-splice junctions detected is clearly in line with our finding, when comparing the circular/linear-junction reads in the different hematopoietic cell types (see Figure 1(a)).…”

“…Moreover, the ratio between the linear spliced mRNA and the circular isoform is very often shifted towards the circular form, which is more stable than linear RNA [20]. Therefore, Alhasan et al had explained this phenomenon by differential degradation of cellular platelet RNA [20].…”

Rat BodyMap transcriptomes reveal unique circular RNA features across tissue types and developmental stages

“…, testes (Liang et al 2017b), peripheral whole blood 62 (Memczak et al 2015), peripheral blood mononucleotide cells (Qian et al 2018), platelets 63 (Alhasan et al 2016), and exosomes (Li et al 2015a). CircRNAs have also been related to the 64 development of the fetal human brain (Szabo et al 2015), mouse brain (You et al 2015), fetal 65 porcine brain (Veno et al 2015), and Drosophila neural systems (Westholm et al 2014).…”

Slowed decay of mRNAs enhances platelet specific translation

“…[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. [14][15][16] The mechanisms of mRNA and ribosome decay in platelets are not known.…”

“…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).…”