A comprehensive long RNA landscape of multi-regional porcine lung-derived small extracellular vesicles

Tissue-derived small extracellular vesicles (sEVs) are vital mediators of intercellular communication; however, the molecular landscape of their long RNA cargo, including full-length mRNAs and long noncoding RNAs, and the mechanisms controlling specific RNA sorting into sEVs are still poorly understood. Here, we successfully isolated sEVs from five anatomical regions of healthy porcine lung and performed polyadenylated transcriptome profiling using short- and long-read sequencing.

We identified a diverse repertoire of long RNAs in lung-derived sEVs, with high concordance in transcript quantification between sequencing platforms and substantial functional conservation across anatomical regions. Notably, approximately 30% of sEV-associated long RNAs were full-length transcripts, comprising 8,374 unique isoforms. Comparative transcriptomic analyses revealed pronounced differences between lung sEVs and their parental tissues. Specifically, sEV-enriched long RNAs (557) were characterized by low-GC and repetitive motifs, in contrast to the GC-rich motifs predominantly observed in sEV-depleted transcripts (1,076). We identified that KHDRBS1 is a candidate RNA-binding protein (RBP) associated with sEV mRNA enrichment. KHDRBS1 knockdown specifically impaired the sorting of 21 sEV-enriched mRNAs that contain predicted KHDRBS1 binding sites. Additionally, epithelial and immune cells were established as the predominant contributors to lung sEVs.

This study provides a comprehensive transcriptional landscape of porcine lung sEVs. Our results present evidence that KHDRBS1 represents a critical candidate RNA-binding protein (RBP) associated with sEV mRNA enrichment of porcine lung.