63 Identifying extracellular vesicles coupled miRNA sequence motifs and their regulatory RNA binding proteins in bovine reproductive cells

Reproductive cell-derived extracellular vesicles (EVs), whether obtained in vivo or in vitro from cells under controlled environments, transport diverse bioactive compounds, notably microRNAs (miRNAs), to the target cells functionally modulating their stress response. The intricate process of miRNA packaging into EVs is facilitated in part by the involvement of multiple RNA binding proteins (RBPs), which selectively recognise particular sequence motifs present amid miRNA molecules. The primary objective of our investigation was to explore the potential interactions between sequence motifs and their respective RBPs in the packaging and release of particular miRNAs through EVs in bovine oviducal organoids and follicular cells under heat stress (HS) conditions. To achieve this, we conducted a comparative small RNA sequencing analysis of EVs obtained from in vitro-cultured oviducal organoids, granulosa cells (GCs), and in vivo-collected follicular fluid under conditions of HS. This analysis aimed to identify the miRNAs that are commonly released via EVs during HS. Sequence motif analysis was performed using Multiple Em for Motif Elicitation (MEME) suite software (https://meme-suite.org/) using a zero or 1 occurrence per sequence model with a motif between 4 to 8 nucleotides long. To identify associating RBPs, the resultant motif was then submitted to the motif comparison tool TOMTOM within the same software. Compared to thermoneutral conditions, we identified specific miRNA sequence motifs present in the HS-associated EV-miRNAs. Our findings revealed that miR-10b, miR-181a, and miR-150 were consistently present among the HS EV-miRNAs across different experiments. Through in silico sequence motif analysis, we observed common miRNA motifs, such as CUGU and GGAG on the HS-associated EV-miRNAs, which are predicted to be targeted by RBPs including LIN28A, HuR, RBM6, RBM42, SRSF1, YBX1, and YBX2. Subsequently, we further examined the transcriptional levels of these predicted RBPs in GCs cultured under HS (42°C) compared to thermoneutral (38.5°C) conditions using qRT–PCR. All tested RBP genes exhibited significantly higher expression levels (>2-fold change, P < 0.05) in the HS-GCs, except for RBM42. Notably, the expression of the LIN28A gene was found to be more than 8-fold higher in the HS-GCs. Certain RBPs, such as YBX and LIN28A, are already known for their involvement in miRNA sorting into EVs and the regulation of cellular stress. The identification and characterisation of these miRNA sequence motifs and their interaction with RBPs will present a unique opportunity to investigate the underlying mechanisms of miRNA-mediated intercellular communication during conditions of HS. Our future research will focus on a mechanistic analysis of the identified motifs and RBPs, exploring their roles in the packaging and release of specific miRNAs via EVs and assessing the subsequent impact of this interplay on cell survival and ovarian response under thermal stress conditions.