Nanopore long-read transcriptome sequencing reveals miR-22-3p-mediated transcriptomic remodeling and alternative splicing in chicken pectoral muscle

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ABSTRACT The full-length transcriptomic architecture mediated by gga-miR-22-3p in poultry muscle development remains largely unexplored. To address this, a lentiviral knockdown vector targeting miR-22-3p was constructed, and an in vivo miR-22-3p knockdown model was established in the pectoral muscle of 14-day-old Qingyuan partridge chickens through subalar injection. Oxford Nanopore Technologies (ONT) long-read RNA sequencing was employed to profile the full-length transcriptome and its remodeling under miR-22-3p knockdown. In total, 24,727 unannotated transcripts were identified. Differential expression analysis revealed 1,448 differentially expressed transcripts (DETs) and 535 differentially expressed genes (DEGs) that were significantly responsive to miR-22-3p knockdown. Gene set enrichment analysis (GSEA) showed that the downregulation of miR-22-3p was associated with significant suppression of muscle development-related pathways, including extracellular matrix (ECM)-receptor interaction, focal adhesion, and collagen metabolic processes, accompanied by the reduced expression of core genes such as COL1A2, COL6A1 , and EGF . Moreover, alternative splicing analysis demonstrated that miR-22-3p knockdown induced extensive remodeling of splicing patterns and transcript isoform usage. This included splicing switches in key functional domains of ENSGALG00000003955 and altered isoform ratios of ESD and LOC101747587 , despite their stable gene-level expression. Taken together, these findings demonstrate that miR-22-3p may regulate the transcriptomic homeostasis of ECM and focal adhesion-related genes in chicken pectoral muscle by coordinating both gene expression levels and isoform-specific splicing. This study provides a full-length transcriptomic framework for understanding miR-22-3p-mediated post-transcriptional regulation during avian muscle development, offering new insights into the molecular regulation of poultry meat quality.

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