Isolation and Characterization of Ribosome‐Free Cytoplasmic Messenger‐Ribonucleoprotein Complexes from Avian Erythroblasts

Abstract

Two RNA . protein complexes were isolated from duck erythroblast postribosomal supernatants. Their nominal sedimentation values on sucrose gradients are 12 S and 20 s, respectively. The 12-S particle contains a 4-6 S RNA, in the 20-S particle a 9-S RNA is found. This 9-S RNA is shown to direct the synthesis of all duck globin chains in a cell-free, messenger RNA-dependent protein-synthesizing system.The protein moiety of these ribosome-free particles is described and compared with the proteins found in the mRNA * protein complex liberated by EDTA from polyribosomes. We show that in the free-cytoplasmic particles no protein can be found which is identical to any of the polypeptides associated with polyribosomal mRNA. Some of these proteins are phosphorylated and contain phosphoserine. The electrophoretic patterns of phosphorylated proteins from the two globin mRNA-containing complexes differ, as do those of the unlabelled polypeptides. We conclude that the mRNA-associated protein population is exchanged when the mRNA enters the translation machinery.The possible role of the RNA-associated proteins in the post-transcriptional and translational control of eukaryotic protein synthesis is discussed.On the basis of experiments concerning the nucleic acid metabolism in duck immature red cells and in HeLa cells we postulated [I] that the regulation of gene expression in animal cells resides in a multistep process including the three principal phases of cellular information transfer : (a) the synthesis in the nucleus of giant precursor molecules (pre-mRNA) to messenger RNA (mRNA), (b) the processing of the pre-mRNA which gives rise ultimately to mRNA that is transported (and stored) in the cytoplasm, (c) the translation of the mRNA into protein. This regulational system governing the multiple steps involved in synthesis, processing and translation of mRNA was termed "Cascade Regulation" [l -31. Evidence that the giant nuclear RNA of molecular weight more than 5~1 0~is an informative precursor to mRNA was given recently by Imaizumi et al. [5] who showed conclusively by molecular hybridization that these RNA molecules, isolated from duck erythroblasts, contain sequences homologous to polyribosomal 9-S duck globin mRNA. However, the processing of pre-mRNA and the transfer of mRNA to the cytoplam are still obscure.Abbreviations. pre-mRNA, precursor molecule to mRNA; mRNA * protein, messenger ribonucleoprotein complex. strate that newly synthesized mRNA appears in the cytoplasm first free from ribosomes but associated with proteins in the form of ribonucleoprotein complexes. Some mRNA types then associate with ribosomes; others may be degraded without being translated.

Identification and Characterization of the Translationally Repressed Cytoplasmic Globin Messenger‐Ribonucleoprotein Particles from Duck Erythroblasts

“…The 19-S particles with a sedimentation coefficient unmodified by the ionic strength changes contains a group of characteristic polypeptides with M , ranging from 22000 to 30000 and a single polypeptide of M , 52000. This protein pattern contains the main components of the pattern of free globin mRNP previously published [12]. Comparison of the absorbance at 254 and 280 nm of 19-S particles indicates, however, a very low RNA content; indeed, as we will show below it contains no globin mRNA.…”

“…These particles, which previously copurified with free globin mRNP [12,171, display a specific protein composition. Their role and possible relation to the 20-S complex is at present unknown.…”

“…Moreover, characterization of the protein composition of free mRNP complexes performed in several laboratories [9,11,12,15 -171 has led to quite different results. Particularly, the existence in free mRNP of the protein bound to the poly (A) segment of polyribosomal mRNA [18-211 is still subject to controversy.…”

“…In the case of duck immature red cells, cytoplasmic globin mRNA has been previously isolated as polyribosomal 15-S mRNP and 20-S free cytoplasmic mRNP [12,17,221. These two particles displayed different protein compositions.…”

“…We previously reported the identification of free cytoplasmic globin mRNP isolated from the post-poly- ribosomal supernatant of duck erythroblasts [12]. It was prepared by sedimentation on a zonal rotor and subsequently purified through several cycles of sedimentation on isokinetic sucrose gradients [17].…”