Many categories of sRNA, including miRNA, piwi-interacting RNA, transfer RNA (tRNA)/tRNA fragments (tRFs), and Y RNAs, play important roles in the regulation of gene expression. The profile of these RNAs in the bloodstream and other biofluids holds promise as an approach for diagnostic monitoring of human disease. Extracellular RNAs with more than a fleeting half-life are contained within EVs, a vesicle population that includes low-density EVs released by budding from the plasma membrane and higher-density EVs released upon fusion of cytoplasmic multivesicular bodies with the plasma membrane.
To improve community knowledge of EV sRNA inventories, University of California, Berkeley researchers generated and sequenced ordered two-template relay (OTTR) cDNA libraries from EV populations. From the breast cancer–derived MDA-MB-231 cell line, EVs were sampled as crude EV preparations from conditioned medium and as highly purified vesicles floated in a sucrose density step gradient (Floated EVs) and treated with micrococcal nuclease before detergent lysis to remove nucleic acids not enclosed within the vesicles. The length profile of total cellular RNA included major peaks for 18S and 28S rRNAs and tRNAs, whereas bulk or highly purified EV RNAs had lengths predominantly of tRNA size or smaller. For sequencing comparison the researchers used filtration to enrich total cellular sRNA of less than 200 nt prior to library generation. They also used a different human cell line, HEK 293T, to generate similarly size-enriched total cellular sRNA and floated vesicles for comparison
Isolated RNA pools were used directly, without gel purification, for OTTR cDNA library synthesis and sequencing. Unsurprisingly, total cellular sRNA and EV library reads were dominated by tRNAs or tRFs and rRNA fragments, as evident from pie charts comparing RNA species across all mapped reads and EV-enriched populations
OTTR RNA-seq inventorying of EV sRNA
(A) Schematic of EV purification. (B) Agilent Bioanalyzer RNA traces for cellular RNA (purple), the 100,000 × g pellet (blue), and Floated EVs (peach). Peaks corresponding to tRNA and 18S and 28S rRNA are indicated. (C) Pie charts of mapped read assignments of MDA-MB-231 sRNA libraries from two biological replicates. tRAX and miRDeep2 were used to map tRNA and miRNA reads, respectively. rRNA, ncRNA, and protein-coding reads aligned to annotated transcripts or genomic ncRNA loci. Intronic, intergenic, and mitochondrial (mt) DNA reads mapped in corresponding locations. Among ncRNA reads, vt is vault and miscRNA includes all ncRNA not split out into other pie slices. (D) EV miRNA enrichment in MDA-MB-231 based on DESeq2 log2 fold change estimates between, as pairwise combinations, 100,000 × g pellet and Total cell, Floated EVs and Total cell, and Floated EVs and 100,000 × g pellet. The 25 miRBase sRNAs included in the panel are the most abundant in read count in MDA-MB-231 Floated EVs, with most abundant of the 25 at top as schematized by the thicker end of the gray wedge. The sequences not considered bona fide miRNA based on miRGeneDB are in red italic. The sequence of miRBase hsa-mir-142 corresponds to miRGeneDB hsa-mir-142-v3.
Overall, we suggest that NGS libraries that oblige defined end-to-end sequence capture of entire sRNA pools will be useful for appreciating differences in the precision of RNA processing and for EV RNA diagnostics.