Satyapriya Sarkar

Changes in tropomyosin subunits and myosin light chains during development of chicken and rabbit straited muscles

Abstract We have selected tropomyosin subunits and myosin light chains as representative markers of the myofibrillar proteins of the thin and thick filaments and have studied changes in the type of proteins present during development in chicken and rabbit striated muscles. The β subunit of tropomyosin is the major species found in all embryonic skeletal muscles studied. During development the proportion of the α subunit of tropomyosin gradually increases so that in adult skeletal muscles the α subunit is either the only or the major species present. In contrast, cardiac muscles of both chicken and rabbit contain only the α subunit which remains invariant with development. Two subspecies of the α subunit of tropomyosin which differ in charge only were found in adult and embryonic chicken skeletal muscles. Only one of these subspecies seems to be common to chicken cardiac tropomyosin. With respect to myosin light chains, embryonic skeletal fast muscle myosin of both species resembles the adult fast muscle myosin except that the LC 3 light chain characteristic of the adult skeletal fast muscle is present in smaller amounts. The significance of these isozymic changes in the two myofibrillar proteins is discussed in terms of a model of differential gene expression during development of chicken and rabbit skeletal muscles.

Thermal chromatography of eukaryotic messenger ribonucleoprotein particles on oligo(dT)-cellulose: Evidence for common mRNA-associated proteins in various cell types

Eukaryotrc mRNAs exist m the cytoplasm as protein-bound complexes (messenger rrbonucleoprotein or mRNP) m polysomes (PmRNP) and m several instances as free nonpolysomal (CmRNP) particles [l-3] . The orgamzation and brologrcal functron of the proteins m mRNP are not well understood, and the published reports on their varratrons and drversrty remam a matter of considerable dispute [2] . PmRNP derived from a large number of cells contains two maJor protems, iWr SO-52 X lo3 (P52) and 74-78 X lo3 (P78), and a number of additional polypeptrdes, usually 2-13, m the 15-150 X lo3 mol. wt range [2,3,5-161 The P78 protein and a protein of srmrlar size have been reported to be specrfically bound to the 3’-poly(A) tracts of PmRNP [5,6,9,12,15] and heterogeneous nuclear RNP (hnRNP) [ 171. Several conflrctmg reports have indicated that the sodmm dodecyl sulfate (SDS)-gel electrophoretrc patterns of the protein components of PmRNP and CmRNP are erther identrcal [4-61 or different [7,8] . This varrabrhty may be partly due to differences m the methods of rsolatron and also to possible contammatron by non-mRNP components [2,3]. Whether or not some common proteins, e.g P78, remam associated with mRNA sequences m

In vitro synthesis of light and heavy polypeptide chains of myosin.

Abstract Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that preparations of highly purified chicken skeletal myosin contain four polypeptide chains: a “heavy” chain (MW about 200,000) and three “light” chains of molecular weights 25,500; 17,600; and 15,200. Three classes of polysomes that differ in size were obtained from homogenates of embryonic chicken leg muscle. The heavy and light chains of myosin are synthesized on two different classes of polysomes from messenger RNAs of different lengths. This suggests that the messages for myosin subunits are monocistronic.

Highly purified mRNA for myosin heavy chain: size and polyadenylic acid content.

Summary The polyadenylic acid segment of highly purified preparations of myosin heavy chain mRNA consists of about 170 nucleotides-about 2.92% of the intact mRNA. The molecular weight of the mRNA is calculated from these values as 2.05 × 106. This is in good agreement with the value of 2.23 × 106 obtained by formamide-gel electrophoresis. These values are 10–20% higher than the estimated minimum size of the mRNA suggesting that some “non-translatable” segment(s) may be present. No apparent correlation was observed between the sizes of mRNAs present in muscle polysomes of different sizes and their poly(A) contents.

Changes in tropomyosin subunit pattern in chronic electrically stimulated rabbit fast muscles

Abstract The tropomyosin subunit ratio of rabbit fast muscle (α:β = 80:20) changes to that characteristic of skeletal slow muscles (α:β = 55:45) on continuous (10 Hz) stimulation for 3 weeks. The altered myosin light chain pattern and histochemical ATPase stain also show clear changes of fast → slow transformation. However, the rate of changes in the light chain patterns of myosin are slower than those of tropomyosin subunits. These results do not support the previous finding (Amphlett et al., Nature 257 , 602, 1975) that the tropomyosin subunit pattern remains unaltered during transformation of skeletal muscles and the conclusion that the genetic expression of tropomyosin is regulated under separate control from other myofibrillar proteins. Rather, our results suggest that the polymorphic patterns of all myofibrillar proteins in skeletal muscles undergo changes in a temporal manner during skeletal muscle transformation.

Characterization of the Ca2+-regulatory complex of chick embryonic muscles: Polymorphism of tropomyosin in adult and embryonic fibers

Abstract The Ca 2+ -regulatory tropomyosin-troponin complex was purified from chick embryonic muscles by a combination of DEAE-cellulose chromatography and (NH 4 ) 2 SO 4 fractionation. The embryonic complex was very similar to that obtained from adult chicken muscles with respect to stoichiometry of components and biological activity. Tropomyosin of embryonic skeletal muscles contains both α and β subunits, the β form being the major species. In the adult stage the β form is decreased with a concomitant increase in the α form. These results indicate that i) the Ca 2+ -regulatory proteins are not deficient in early embryonic muscles as previously thought (Hitchcock, S.E., Develop. Biol. 23 , 399, 1970), and ii) different structural genes coding for tropomyosin subunits are expressed differentially in embryonic and adult muscle fibers.

Translational control involving a novel cytoplasmic RNA and ribonucleoprotein.

Publisher Summary To investigate the role of cellular regulatory mechanisms involving inhibitory RNA (iRNA) and 10-S ribonucleoproteins (iRNP), this chapter presents a detailed study on the characterization and mechanism of action of these macromolecules. It also outlines recent progress made in this area. The results presented in the chapter strongly imply that iRNA and iRNP are genuine cytoplasmic macromolecular entities involved in the modulation of mRNA translation in a subtle manner at a post-transcriptional level. The iRNA and iRNP are quite distinct from the various eukaryotic RNA species that have modulating effects on in vitro translation systems. The potent inhibitory effect of iRNA and iRNP on mRNA translation is not exerted in an absolutely discriminatory manner. Rather, they act as specific inhibitors of mRNA binding to 43-S complex. This mode of inhibition distinguishes iRNA and iRNP from other cellular inhibitors that act through the phosphorylation of the eIF2 a-subunit. Because iRNA inhibits mRNA binding to ribosomes, it is quite likely that the relative affinities of mRNA and iRNA for this rate-limiting step in polypeptide synthesis may function as a subtle parameter in the cytoplasmic regulation of translation. Thus, a quantitative modulation of the translation of various mRNAs by iRNA rather than a specific discriminatory effect can lead to striking effects on cellular translation patterns.

Correlation between the protein and mRNA levels for myosin light chains and tropomyosin subunits during chick fast muscle development in vivo

Using myosin light chains and tropomyosin subunits as representative myofibrillar proteins, we have characterized their isoprotein forms and also correlated them with the accumulation of the corresponding mRNAs during development of a fast muscle in chicken, viz, pectoralis. Both slow and fast myosin light chain isoforms, except fast myosin light chain LC3, and the two subunits of tropomyosin are present in early embryonic muscle. During development, the slow myosin light chains and β‐tropomyosin appear in reduced amounts in pectoralis muscle and finally they disappear in adult muscle. Translation studies with total cellular RNA from developing muscle indicates that while the protein levels of the above isoforms, in general, correlate with the accumulation of corresponding mRNAs, for LC3, additional post‐transcriptional control appears to modulate the expression of this isoprotein skeletal muscle development in vivo.

The translational inhibitory 10 S cytoplasmic ribonucleoprotein of chicken embryonic muscle is distinct from messenger ribonucleoproteins

A class of cytoplasmic RNA species, about 70-90 nucleotides in size, and sedimenting as 4 S in sucrose gradients, which are potent inhibitors of in vitro mRNA translation, have been isolated from chick embryonic muscle [l-3]. The inhibitory RNA differs from the (U)-rich low molecular mass (jkft) RNA species of chick embryonic muscle, which inhibits specific mRNA translation (referred to as translational control RNA, tcRNA) [4-61 with respect to size, base composition, lack of oligo(U) tract, and its ability to inhibit the translation of a variety of mRNAs in a nondiscriminatory manner [l-3]. The association of tcRNA with specific mRNA (e.g., mRNA is complexed with specific tcRNA to form a translationally repressed mRNP) has been postulated as a negative control of translation [4-61. A cytoplasmic 1 O-l 5 S RNP particle containing a 4.5 S RNA from chick embryonic muscle which inhibits mRNA translation in vitro was isolated in [7]. It was concluded that except for the presence of a single polypeptide, M, 36 000 in the 10-l 5 S RNP, the protein patterns of the inhibitory RNP and mRNP are very similar [7]. It was suggested that the inability of some free mRNP to be translated in vitro may be related to the presence of lo-15 S RNP [7]. However, neither the inhibitory RNA was characterized, nor the RNP was purified in [7]. We describe here the characterization of a purified 10 S cytoplasmic RNP (iRNP) containing an inhibitory 4 S RNA (iRNA) species from chick embryonic muscle. The properties of iRNA indicate that it is unrelated to cytoplasmic mRNP. Furthermore, the inhibitory action of iRNP

Association of cap binding protein-related polypeptides with cytoplasmic RNP particles of chick embryonic muscle

Cap binding protein (CBP)‐related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide (M r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low‐M r immunoreactive peptides (M r 43000 and M r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M r 43000 CBP‐related protein was present in polysomes. The relevance of the association of different CBP‐related polypeptides with cytoplasmic RNP particles and polysomes are discussed.