Myosin Heavy Chain Messenger RNA from Myogenic Cell Cultures

Abstract

The appearance of messenger RNA for myosin heavy chains in chick-embryo myogenic cell cultures was investigated. Total polyribosomes were isolated from cultures at various times of development and were purified in sucrose step gradients. These polysomes were either extracted with phenol or were treated with puromycin. The ribonucleoprotein particles and ribosomal subunits released by puromycin were fractionated on sucrose gradients. RNA from polysomes or from puromycin-dissociated subunits was fractionated on oligo(dT)-cellulose columns, and the bound and unbound RNA was assayed for activity of myosin heavy chain messenger RNA in a rabbit reticulocyte cell-free system. RNA stimulating myosin heavy-chain synthesis was found predominantly in the unbound fractions of the oligo-(dT)-cellulose columns. After puromycin treatment of polysomes, the myosin heavy chain messenger RNA, which sediments at 18-26 S, was associated with a ribonucleoprotein particle sedimenting between 30 and 40 S. Myosin heavy chain messenger RNA was obtained from cultures containing well-developed myotubes and from cultures undergoing myogenic cell fusion. This messenger RNA was not detectable in early, unfused cultures, or in later cultures in which myogernic cell fusion had been prevented by treatment with ethyleneglycol bis(fl-aminoethyl ether)-NN'-tetraacetic acid. These experiments demonstrate that messenger RNA for myosin heavy chains becomes associated with ribosomes only after myogenic cell fusion has begun.During the differentiation of the multinucleated, skeletal muscle fiber the time of myogenic cell fusion and the formation of the embryonic myotube is correlated with the time of intensive synthesis of myosin and the other muscle-specific enzymes (1-4). Presently at issue is whether cell fusion is actually coupled to the regulatory mechanisms that control this intensified protein synthesis. Evidence from this laboratory on fusion-arrested chick myogenic cell cultures strongly suggests that such a coupling mechanism exists (2), and a recent report from another laboratory (5) suggests that in ratmuscle cell cultures there is a post-transcriptional regulatory event that is correlated with cell fusion.In this paper we report on the isolation of an mRNA fraction derived from polysomes of cultured chick-embryo myogenic cells. This RNA directs the incorporation of radioactive leucine into myosin heavy chains (MHCs) in a cellfree system.

Skeletal Muscle

“…The actin and myosin in 1-day cultures were of the cytoplasmic type occurring in chondroblasts or fibroblasts as well, but in 4-day cultures of fusion-arrested cells typical muscle proteins were present (CHI et al 1975a;HOLTZER et al 1977). PRZYBYLA and STROHMAN (1974) and DEVLIN and EMERSON (1979) did not find messenger RNA coding for any of the contractile proteins before fusion had commenced, indicating that the specific mRNA species begin to coordinately accumulate at fusion. PRZYBYLA and STROHMAN (1974) and DEVLIN and EMERSON (1979) did not find messenger RNA coding for any of the contractile proteins before fusion had commenced, indicating that the specific mRNA species begin to coordinately accumulate at fusion.…”

Effects of cytochaslasin B and colcemide on myogenic cultures.

“…The emergence of myoblasts bears on a second contention, namely, that fusion is required to initiate transcription of myosin mRNA, or to activate the translation of inactive myosin mRNA (19)(20)(21)(22)(45)(46)(47)(48). The emergence of mononucleated myoblasts with thick and thin filaments demonstrates that fusion is not a prerequisite event for coordinated expression of the myosin and actin genes.…”

Effect of myoblast fusion during muscle cell differentiation on myosin polysome accumulation and myosin synthesis

“…Coleman and Coleman (1968) and Yaffe and Dym (1972) de tected no myosin synthesis in prefusion muscle cultures, and, although Paterson and Strohman (1972) In contrast to these results, however, other workers have tried unsuccessfully to isolate from extracts of mononucleated myogenic cells a mMA that would direct synthesis of myosin heavy chains in a reticulocyte protein-synthesiz ing assay (Pryzbyla et al», 1973; Pryzbyla and Strohman, 1974;Strohman et al, 1974-),…”

“…Rather than attempting indirect studies, such as those reported by Buckingham et al (197^), and in view of the uncertainties involving ability to quantitatively initiate protein synthesis on myosin mRNA's (Heywood and Nwagwu, 1969) or to assay purified myosin mRNA in a cellfree assay as attempted by Strohman and coworkers (Pryzbyla et al, 1973;Pryzbyla and Strohman, 197^;Strohman et al, 197'+), a more direct approach was taken. If prefusion muscle cells synthesize myosin, then these cells must con tain myosin polysomes.…”

“…The polysome extraction procedure outlined in Materials and Methods of this dis sertation involves cell lysis by dissolving the outer cell membrane with 0.5^ Triton X-100, sedimentation of nuclei and mitochondria at 12,000 x for 10 min, and assay of the polysomes remaining in the supernatant for ability to incorporate radioactivity into myosin heavy chains. The polysome extraction procedure used by Pryzbyla and Strohman (1974); however, involves disrupting cell membranes by homogenization with a loosely fitting Bounce homogenizer followed by centrifugation at 10,000 x for 10 min.…”