Analysis of tcRNA102 associated with myosin heavy chain-mRNPs in control and dystrophic chick pectoralis muscle.

D J Zezza,S M Heywood

doi:10.1016/s0021-9258(17)38414-4

Abstract

Translational control RNA (tcRNA102) is closely associated with nonpolysomal myosin heavy chain-mRNA in mRNP particles. The nucleotide sequence of tcRNA102 has revealed a heterogeneity at the 3' end. This heterogeneity is mostly with regard to an ambiguity between adenine and guanine residues. tcRNA102 (obtained from pectoralis muscle) runs as a single band on denaturing acrylamide gels. When this band is extracted and rerun on a native gel at low voltage, two individual bands appear (A the slower moving and B the faster moving). From the partial RNase U2 sequence analysis and our previous sequence determinations (McCarthy, T. L., Siegel, E., Mrockowski, B., and Heywood, S. M. (1983) Biochemistry 22, 935-941), we may now assign tcRNA102 (A) the 3'-terminal sequence ... GGUUGGACGG-3' and tcRNA102(B) and 3' terminal sequence ... GAUUAAGCAA-3'. Analysis of the tcRNA102s indicates that dystrophic pectoralis muscle contains much less tcRNA102 than a similar preparation from control muscle. The tcRNA102 found in dystrophic pectoralis muscle is of the "A" type while normal pectoralis muscle contains predominantly the "B" type. In addition, control leg muscle from dystrophic chick contains predominantly "B" type. These results suggest that the differences observed at the DNA level (see accompanying paper, Zezza, D. J., and Heywood, S. M. (1986) J. Biol. Chem. 261, 7455-7460) may be reflected in the RNA transcripts.

Highlights

This heterogeneity is mostly with regard to an ambi- mutation may affect a molecule involvedin theexpression of guity between adenineandguanineresiduest.cmuscle-specificproteins at thetranscriptional and/or trans
Been reported that avian dystrophic muscle continues to ex- had suggested that tcRNAlO2 was likely composed of more press a neonatal isoform of myosin heavy chain af%r control than one species with different 3’-OH-terminal sequences. muscle had ceased expressing this isoform of myosin (Band- We have been able to distinguish two species (A and B)
The small is synthesized at a fasterrate indystrophic chicken pectoralis amount of tcRNA102 found in dystrophic muscle is largely of muscle; it fails to accumulate as a result of a faster the A form while that found in control muscle or leg muscle degradationrate (Young and Schneible, 1984)

Summary

EXPERIMENTAL PROCEDURES

Animals-The dystrophic chicken embryos were obtained from a dystrophic strain maintained at the University of Connecticut, Storrs. The MHC mRNP fraction was isolated as previously described (Havaranis and Heywood, 1981).All operations were performed at 4 “C. RNA was extracted from the mRNPs or total cytoplasmic fraction as previously described (McCarthy et al, 1983) and 3’ end-labeled using RNA ligase (Bethesda Research Laboratories and P-LBiochemicals) and [32P]pCp. Northern Blot Analyses-MHC mRNPs were collected from gram equivalent weights of control and dystrophic pectoralis muscle as previously described (Havaranis and Heywood, 1981).The RNA was phenol extractedand precipitated in 2.5 volumes of 95% ethanol. For analysis under denaturing conditions, the RNAs were electrophoresed on 10% acrylamide gels containing 8 M urea as previously described (McCarthy et al, 1983). RNA sequencing gels were as previously described using either 10 or 20% denaturing gels (McCarthy et al, 1983). The reactions were performed at 55 “cfor 15 min in 7 M urea

RESULTS

Recovery of RNA and DNA from normal and dystrophic chicks

CD tRNA