The Identification of Collagen Messenger RNA

Abstract

RNA isolated from calvaria of 16-to 18-day-old chick embryos, assayed in rabbit reticulocyte lysates, programs the synthesis of a collagenase-sensitive protein with the molecular weight of collagen pro-achains. When RNA labeled with [3Hjuridine for 2 hr and chased for 1 or 2 hr was electrophoresed on aqueous polyacrylamide gels, most of the radioactivity not in 28S or 1S rRNA migrated with an apparent-molecular weight of about 1,800,000. After oligo(dT)-cellulose chromotography and analysis in 99% forinamide gels, this nonrihosomal, rapidly labeled calvaria RNA species migrates at 28S-30S and thus has a molecular weight of at least 1,600,000.Both the ability to-program the synthesis of collagenasesensitive protein in reticulocyte lysates and the presence of a single prominent rapidly labeled 30S peak in acrylamide gels strongly support the deduction that there is only one major mRNA species in calvaria and that this species is collagen messenger RNA.Understanding the control of collagen biosynthesis is essential to understanding normal growth and development in vertebrates. Not only is collagen their major structural protein, but the onset of collagen synthesis is coincident with the onset of major differentiational' events. An important step in the study of collagen biosynthesis must be the purification and characterization of the messenger RNAs which code for the five or more different collagen pro-a-chains.Collagen messenger RNA (mRNA) has already been detected in polysomes prepared from the wings and legs of 8-'or 9-day-old chick embryos by assaying for collagen synthesis in homologous cell-free extracts (1, 2) and in heterologous cellfree extracts prepared from Krebs II ascites cells (3). Collagen synthesizing polysomes have also been isolated from L-929 fibroblasts (4). However, collagen mRNA has not been succ'essfully isolated from any of these polysomes.More recently, Benveniste, Wilczek, and Stern (5) reported that total RNA'isolated from the calvaria of 15-day-old chick embryos contained active collagen mRNA. They found that more than half of the [3H]proline-labeled protein made in ascites tumor extracts when calvaria RNA was added could be solubilized by collagenase. Also, some of this protein cochromatographed with carrier collagen on carboxymethyl cellulose. We have thus isolated RNA from calvaria. From a combination of pulse-chase studies of this RNA, under both aqueous and denaturing conditions, and assays of its ability to code for the synthesis of intact procollagen in rabbit reticulocyte lysates, we have been able to show that calvaria contain predominantly one class of mRNA, having a molecular weight of about 1,600,000-1,800,000. Several arguments indicate that this is collagen mRNA. MATERIALS AND METHODSPreparation and Fractionation of RNA from Calvaria. Calvaria RNA was prepared from 16-to 18-day-old chick embryos according to the procedure described by Benveniste et al. (5). The RNA was further purified by centrifugation through 5.7 M CsCl as described by Glisin et al. (6).To prepar...

Poly(riboadenylate)-containing messenger ribonucleoprotein particles of chick embryonic muscles

“…For the preparation of poly(A)-containing in vivo pulselabeled RNA, the procedure of Boedtker et al (1974) was used with some modification. Minced leg and breast muscles obtained from three to five embryos were incubated in 20-25 m L of Dulbecco's modified Eagle's medium [ 10 g of Gibco solid medium and 30 mL of 1 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), pH 7.5, made to l L ] supplemented with 2-5 mCi of carrier-free H332P04 (New England Nuclear) for 60 min.…”

Translation of chick calvarial procollagen messenger RNAs by a messenger RNA dependent reticulocyte lysate

“…Several experiments have been reported in which procollagen messenger RNA activity has been detected in RNA preparations (Benveniste et al, 1973(Benveniste et al, , 1976Boedtker et al, 1974Boedtker et al, , 1976Harwood et al, 1975;Wang et al, 1975). The translation of procollagen mRNAs into complete proa chains has proven to be more difficult.…”

Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems

“…For example, cell-free systems from Krebs II ascites cells have the disadvantage of endogenous collagen-synthesizing abilities (Benveniste et al, 1976), and unprocessed reticulocyte lysates have a high endogenous messenger activity which hampers the efficient translation of procollagen mRNA (Boedtker et al, 1974). This latter problem is not encountered with the wheatgerm cell-free system (Roberts & Paterson, 1973), which has low endogenous mRNA content.…”