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A profile of small non-coding RNAs from porcine follicular fluid exosomes using a Pandora sequencing method

agricultural-science
developmental-biology
veterinary-medicine

Abstract

Follicular fluid exosomes are critical mediators of intercellular communication in the ovarian follicle, regulating oocyte maturation and follicular development via bioactive cargo such as small non-coding RNAs (SncRNAs). However, traditional small RNA sequencing suffers from systematic biases due to RNA modifications (such as m¹A and m¹G) that impede adapter ligation and reverse transcription, leading to underrepresentation of modified SncRNAs (piRNAs, tRNA, and snoRNAs ). To comprehensively profile the SncRNA landscape in porcine follicular fluid (PFF) exosomes (EXs) using Pandora-sequence, which can overcome RNA modification-induced biases and elucidate the functional SncRNA classes (piRNAs, miRNAs, snoRNAs, rRNAs, tRNAs). The results show that isolated PFF EXs exhibited a peak particle size of 69 nm, average size of 103 nm, and concentration of 2.65×10¹¹ particles/mL, Pandora-seq revealed piRNAs as the dominant SncRNA class (46.32% of total SncRNA s), followed by rRNAs (32.99%), tRNAs (20.26%), snoRNAs (0.36%), and miRNAs (0.06%), which challeng e the traditional paradigm of miRNA dominance in exosomes. Among rRNAs, 18S rRNA accounted for 77.36% of the total rRNAs. For tRNAs, the most abundant were those corresponding to glycine, valine, and histidine, while serine, cysteine, and tyrosine tRNAs were the least abundant. GO enrichment analysis showed: (1) piRNA target genes enriched in actin cytoskeleton organization and small GTPase-mediated signal transduction; (2) miRNA target genes enriched in positive regulation of cell differentiation and in utero embryonic development; (3) snoRNA target genes enriched in autophagy and intracellular transport. These distinct functional profiles of SncRNA classes, piRNAs in cytoskeletal dynamics, miRNAs in cell differentiation, and snoRNAs in autophagy provide a layered regulatory network for follicular homeostasis. These findings offer potential biomarkers for oocyte quality assessment (glycine/valine tRNA levels) and could inform strategies to improve in vitro maturation efficiency in assisted reproductive technologies.
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