Nanopore long-read transcriptome sequencing reveals miR-22-3p-mediated transcriptomic remodeling and alternative...

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.