Identification of novel transcripts from the porcine MYL1 gene and initial characterization of its promoters

Abstract

The fast skeletal alkali myosin light polypeptide 1 (MYL1) gene is one of three mammalian alkali MLC genes and encodes two isoforms, 1f and 3f, which play a vital role in embryonic, fetal, and adult skeletal muscle development. We isolated the MYL1 gene from a pig BAC library with the goal of characterizing its promoter and identifying its transcripts. Genes and isoforms were identified by reverse transcriptase-PCR, northern blot and RACE (Rapid Amplification of cDNA Ends). Potential MYL1 gene promoters were characterized using a luciferase reporter assay and electrophoretic mobility shift assays (EMSA). MLC1f, MLC3f, and three additional isoforms of porcine MYL1, MLC5f-A, -B, and -C were identified. Up to now, the three novel isoforms had not been reported in human or mouse. Northern blot analysis indicated that MLC1f, MLC3f, and MLC5fs were expressed only in longissimus dorsi muscles. Two transcription initiation and termination sites were identified by RACE. Promoter analysis and EMSA demonstrated the presence of a MEF3 (skeletal muscle-specific transcriptional enhancer) binding site (+384 to +481), which might be essential for porcine MYL1 transcription. Our results suggested that five transcript variants were generated using alternative promoters, two transcription start sites, and polyA sites, as well as variable splicing of the pig MYL1 exon 5. The identification of alternative promoters and splice variants, the expression of the splice variants in different muscle tissues, and the definition of regulatory elements provide important molecular genetic knowledge concerning the MYL1 gene.