RNA transcription in porcine skeletal muscle nuclei during postnatal development

Postnatal developmental pretranslational regulation of skeletal muscle alpha-actin gene expression was investigated. Northern blot analysis of skeletal muscle alpha-actin and beta-tubulin mRNA from 1- and 28-day-old pigs indicated that there are developmental increases in alpha-actin mRNA abundance (P < 0.03) and no significant changes in beta-tubulin mRNA (P > 0.1). A system for isolation of nuclei from porcine skeletal muscle and for transcriptional "run-on" analysis was established in order to investigate the regulatory mechanism of developmental changes in porcine skeletal muscle protein. Skeletal muscle nuclei were isolated from longissimus dorsi (LD) muscle of 1- and 28-day-old pigs by adapting a method to isolate nuclei from cardiac muscle. Results from a [3H]-UTP incorporation assay indicate that these nuclei preparations have the capacity to synthesize RNA and attain maximum incorporation after 40-45 min at 26 degrees C. Messenger RNA syntheses from skeletal muscle nuclei from 1- and 28-day-old pigs were not significantly different (P > 0.25). All nascent tRNA, rRNA, and mRNA in the nuclei were elongated since [3H]-UTP incorporation was reduced after addition of 0.05 micrograms/ml alpha-amanitin to the transcription mixture. Transcription "run-on" assay results indicated that more (P < 0.02) skeletal muscle alpha-actin pre-mRNA was synthesized in the 28-day-old pig skeletal muscle nuclei than in the 1-day-old pig skeletal muscle nuclei. These results indicate that the relative increase in skeletal muscle alpha-actin mRNA observed in the older animals was due, at least in part, to an increase in the transcriptional activity of the skeletal muscle alpha-actin gene.