title: Expression of muscle-specific proteins is necessary to regulate translation of the mRNA for a 40-kDa housekeeping polypeptide in rat L6 cells.
abstract: Translation of the mRNA for a housekeeping polypeptide of 40 kDa (P40) was found to be regulated under a variety of conditions in rat L6 cells. This mRNA was translated in the proliferating myoblasts but not in the non-proliferating myotubes. A number of chemicals, such as dimethyl sulfoxide, sodium butyrate and aphidicolin, were used to prevent expression of muscle-specific genes in mitogen-poor differentiation medium. In the absence of any detectable accumulation of muscle-specific alpha-actin mRNA, the P40 mRNA remained in the translated state. A fourth chemical, EGTA, a known inhibitor of fusion of muscle cells, blocked translation of muscle-specific actin and tropomyosin mRNAs. On the other hand, it showed no effect on the translation of P40 mRNA. Addition of Ca2+ to the EGTA-treated cultures, however, almost completely reversed the block of translation of actin tropomyosin mRNAs within four days. Concomitant to Ca2+ reversal of the translational block of muscle mRNAs, P40 mRNA entered the non-translated state. An inverse relationship, therefore, was observed between the translation of housekeeping P40 mRNA and muscle-specific mRNAs. The ability to mimic in vivo regulation of P40 mRNA translation was examined in mRNA-dependent micrococcal-nuclease-treated homologous cell-free extracts. The extracts from myoblasts and myotubes were able to translate P40 mRNA. Furthermore, ribosomes of both myoblast and myotube extracts containing endogenous mRNAs were also able to bind to P40 and actin mRNAs. Myotube extract, however, showed a lower binding ability to P40 mRNA than to the actin mRNA. The ability of ribosomes of myotube extract to bind P40 mRNA was somewhat enhanced by addition of proteins derived from washing these ribosomes with a high-ionic-strength buffer. In order to elucidate the role of interaction between mRNA and proteins in translational control of P40 mRNA, the polypeptide complements of polysomal and free P40 mRNA-protein (mRNP) complexes were also examined. Hybrid selection of polysomal and free P40 mRNP complexes followed the covalent joining of the RNA and protein moieties of mRNP complexes by ultraviolet irradiation of rat L6 cells. Analysis of buoyant densities of these complexes showed that free P40 mRNP had slightly less protein than polysomal P40 mRNP. Furthermore, analysis of the polypeptide complements of both free and polysomal P40 mRNP complexes showed that they were composed of identical polypeptides. The only detectable difference between the polypeptide complements of these complexes was that two polypeptides of 72 kDa and 55 kDa were more abundant in the polysomal P40 mRNP than free P40 mRNP.
authors: Department of Molecular Biology and Genetics, University of Guelph, Ontario, Canada.
authors: S K
authors: SK
authors: Pramanik
authors: J
authors: J
authors: Bag
mesh_terms: D000199:Actins / Q000235:genetics* / Q000502:physiology; D000818:Animals; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D008715:Methionine / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D010455:Peptides / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D011061:Poly A / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D012270:Ribosomes / Q000378:metabolism
chemical_list: D000199:Actins; D010455:Peptides; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D008715:Methionine