Transcript Expression, Intron Retention, and RNA Editing Are Conserved in Human Platelets over a 4 Year Period

The use of circulating, anucleate platelet RNA signatures has been proposed as a new "liquid biopsy platform" for precision diagnostics. Changes within the platelet transcriptome during disease have also been used to identify the molecular basis of altered platelet phenotypes. Nevertheless, the durability of the platelet transcriptome in health is unknown. To determine the inter- and intra-individual durability of the human platelet transcriptome, we performed next-generation RNA-sequencing (RNA-seq) on purified platelets isolated from the same healthy individuals repeatedly over time (up to 4 years). We analyzed individual transcript expression (e.g. mRNAs) as well as the conservation of Adenosine to Inosine (A to I) RNA editing, and intron retention within and among individuals over time.

We initially examined the platelet transcriptome in 31 healthy individuals at visit 1 (time 0) and again 4 months later. Intra-individual durability over this 4 month period among both protein coding and non-coding transcripts was very high (Spearman correlation coefficient [rho] > 0.975), while comparisons between subjects (inter-individual) was significantly lower. The percentage of transcripts with low variability (CV<0.20) over 4 months was significantly higher within subjects (e.g. self-pairs) than among subjects (e.g. random-pairs) (78% vs. 20%, p<0.05). To determine the long-term durability of the platelet transcriptome, we performed RNA-seq in healthy subjects (n=7) studied repeatedly over 4 years. Consistent with short-term findings, we found that intra-individual durability of the human platelet transcriptome remained remarkably high over the 4 year study period. Differentially expressed transcripts specific for gender and age were also highly correlated over 4 years.

The protein end products of RNA expression are dictated by RNA features beyond absolute transcript expression. For example, intron retention alters the product and quantity of synthesized protein. Therefore, we assessed the extent and durability of intron retention in human platelets. The extent of intron retention, as measured by individual intron/total exon ratios, was highly durable within the same individual over time. We focused on the mitochondrial superoxide dismutase gene (SOD2), which mediates ROS generation and programmed cell death. Interestingly, retention of intron 5 of SOD2 was common in platelets (35% to 100% retention) and the extent of SOD2 intron retention was highly conserved over time within the same individual (rho 0.88, Figure 1A and 1B). A to I RNA editing also exhibited high durability among self-pairs. Post-transcriptional A to I RNA editing regulates host inflammatory responses to dsRNA, and can alter protein coding regions (I is read as G by polymerases and ribosomes). We examined A to I RNA editing in a protein coding region (Q->R coding change) of Filamin A (FLNA), a cytoskeletal regulator known to undergo RNA editing. FLNA was variably edited among individuals, (18-50% of transcripts) but remarkably durable within the same individuals over time (rho=0.82, Figure 1C and 1D).

This is the first prospective, longitudinal study to demonstrate that in health, the human platelet transcriptome is highly durable over a 4-year period. Both protein-coding and non-coding mRNAs exhibited remarkable durability and precise signatures related to gender and age. We also identified for the first time that intron retention and RNA-editing, features of RNA expression that may modulate the platelet proteome, are remarkably conserved in the same individuals over time. These findings support the concept of the human platelet transcriptome as a diagnostic tool for precision medicine. We also suggest the intriguing possibility of using platelet RNA-seq as a reliable window to understand the architecture of human gene expression.

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