Biochemistry 1983, 22, 935-941
935
Characterization of Translational-Control Ribonucleic Acid Isolated from Embryonic Chick Muscle? T. L. McCarthy, E. Siegel, B. Mroczkowski, and S. M. Heywood*
preincubation in stoichiometric amounts. Under these conditions, endogenous reticulocyte mRNA is not inhibited. Under appropriate salt and temperature conditions, tcRNAIo2 is capable of reassociating with myosin heavy chain (MHC) mRNA, thus altering the sedimentation characteristics of the mRNA. This suggests that the mRNA-tcRNA,,, interactions alter the secondary structure of the mRNA. In addition, tcRNAlO2does not associate with ribosomal RNA or globin mRNA, suggesting that some degree of specificity is involved with the RNA-RNA interactions.
ABSTRACT: Myosin heavy chain (MHC) mRNP particles have
been purified from 13-day chick embryonic skeletal muscle by a combination of sucrose density gradient centrifugation and metrizamide buoyant density centrifugation. Associated with the mRNPs are at least three distinct low molecular weight RNA molecules including translational-control RNA (tcRNA). This particular RNA contains 102 nucleotides and is uridine and guanine rich, and its nucleotide sequence has been determined. tcRNAlO2is capable of inhibiting the translation of the mRNAs with which it is associated upon n
b v i d e n c e for the control of gene expression at the translational level has continued to accumulate in recent years. Among the controls utilized by eukaryotic cells is the sequestering (in the cytoplasm) of mRNA into inactive particles (mRNPs) unassociated with ribosomes. We have previously reported that myosin heavy chain (MHC) mRNA can be isolated from such particles from both embryonic muscle and muscle cell cultures (Heywood & Kennedy, 1976; Bester et al., 1975; Havaranis & Heywood, 1981). In addition, we have suggested that a small RNA, designated translational-control RNA (tcRNA), is involved in maintaining the mRNA in an inactive state (Bester et al., 1975). Translational-controlRNA has been demonstrated to stoichiometrically inhibit the translation of MHC in vitro and upon hybridization dramatically increase its RNase resistance (Heywood & Kennedy, 1976). Recently, a number of reports have shown small RNAs to be associated with stored mRNPs in a variety of cell systems (Northeman, et al., 1980; Vincent et al., 1980; O’Loughlin & Gross, 1981). The MHC mRNP has been purified and shown to contain, in addition to MHC mRNA, a number of low molecular weight RNAs (Havaranis & Heywood, 1981). In this report, we confirm the existance of tcRNA among these small RNAs and characterize tcRNA with regard to its nucleotide sequence and function in the repression of mRNA transla tion. Experimental Procedures Metrizamide Gradient Purijkation of MHC mRNPs. The isolation of chick muscle MHC mRNP by sucrose density gradient centrifugation and subsequent metrizamide buoyant density centrifugation has been previously described (Bester et al., 1980; Havaranis & Heywood, 1981). Terminal 3’End Labeling of tcRNA. MHC mRNP RNA isolated from the leg muscle of 10 dozen 13-day-old chick embryos was used for end labeling. ’Ethanol-precipitated RNA was resuspended in 10 pL of ligation buffer (0.05 M Hepesl-KOH, pH 7.5, 0.015 M MgC12, 0.0033 M dithiothreitol, 10% Me2SO) heated to 65 OC for 5 min and then From the Genetics and Cell Biology Section U-125, University of Connecticut, Storrs, Connecticut 06268. Received April 22, 1982; revised manuscript received November 17, 1982. T.L.M. is supported by the Muscular Dystrophy Association of America., This research is supported by National Institutes of Health Grant 5 R01 HD03316 and Cancer Grant 20882.
0006-2960/83/0422-0935$0 1.5010
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rapidly cooled. An additional 5 pL of ligation buffer containing 100 pg/mL ATP was added, and the total reaction volume of 15 pL was transferred to a siliconized Eppendorf microtube in which 40 pmol of [5’-32P]-pCp(New England Nuclear) had been dried under vacuum according to the procedure of Donis-Keller et al. (1977). The equivalent of 10 units of T4 RNA ligase (P-L Biochemicals) was added, and the reaction was incubated at 4.0 OC for a minimum of 24 h at which time 5 pL of dye mix containing TBE (0.05 M Tris, 0.06 M boric acid, and 1 mM EDTA, pH 8.3), 0.05% bromophenol blue, 0.05% xylene cyanol, and 7 M urea was added. Gel Electrophoresis of 3’-End-Lubeled tcRNA. End-labeled tcRNA was electrophoresed on a 48-cm 10% polyacrylamide gel (made up in TBE buffer, pH 8.3, and 7 M urea). The running buffer was TBE, pH 8.3, and electrophoresis was carried out at room temperature at 800 V. tcRNA was excised from the gel and eluted by the addition of 3.0 mL of elution buffer (0.5 M sodium acetate, 0.010 M magnesium acetate, 0.0001 M EDTA, and 0.2% NaDodS04) and subsequently incubated at 37 OC for 24 h. At the end of the incubation period, the solution was passed through a sterile 5-cm3 disposable hypodermic syringe containing a glass wool plug to remove gel fragments. Finally, the [32P]-tcRNAwas ethanol precipitated in the presence of unlabeled tRNA used as a carrier. Sequencing tcRNA. Polyacrylamide gel purified, 3’-endlabeled tcRNA was pelleted at 10000 rpm for 1 h. The pellet was carefully resuspended in 50 pL of H 2 0 ; 10-pL aliquots of [32P]-tcRNAwere used for each sequencing reaction. One microliter of 13X digestion buffer (0.43 M sodium citrate, pH 5.0,0.022 M EDTA) was added to each 10-pL aliquot so that the final concentration was 0.033 M sodium citrate4017 M EDTA. The sequencing procedure used is a modification of the Donis-Keller et al. (1977) procedure. This buffering system was used for all the enzyme digestion reactions except the ribonuclease T1 reaction, which also contained 5 M urea. The units of enzyme used in each individual reactions were as follows: 0.026 unit of ribonuclease T1, 2.0 units of ribonuclease Bacillus cereus, 2.5 units of ribonuclease Phy M., I Abbreviations: Hepes, 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid; Tris, tris(hydroxymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid; NaDodSO,,, sodium dodecyl sulfate; PBS, phosphate-buffered saline; IgG, immunoglobulin G.
0 1983 American Chemical Society
936
BIOCHEMISTRY
M C C A R T H Y ET A L .
Table I: Oligo[d(A)]-cellulose Binding Properties of mRNP-Associated RNAsa RNA (nucleotides) ~~
~
150
102
89
exuerimental conditions
cum bound
%bound
cum bound
%bound
cDm bound
%bound
binding at 25 "C heat denaturation at 65 "C followed by slow cooling to 25 "C
158 195
