Eur. J. Biochem. 77, 287-295 (1977)
Characterization of the Protein Moiety of Messenger Ribonucleoprotein Complexes from Duck Reticulocytes by Two-Dimensional Polyacrylamide Gel Electrophoresis Rita U. MUELLER, Virginia CHOW, and Eugen S. GANDER Departamento de Biologia Celular, Universidade de Brasilia (Received November 15, 1976)
The protein moiety of duck globin messenger ribonucleoprotein complexes isolated by oligo(dT)cellulose chromatography or by sucrose gradient centrifugation was analysed by two-dimensional polyacrylamide gel electrophoresis under conditions where the separation in the first dimension occurs according to charge and in the second according to molecular weight. By comparing the pattern of protein from the mRNA . protein complex with that of ribosomal subunits we found that two acidic proteins with an identical molecular weight of about 49000 and three basic proteins of about M , 56000, 64000 and 73000 were associated with the duck globin mRNA but were absent from either puromycin/high-salt-derived or ‘run-off ribosomal subunits. The comparison of the proteins from the complex with mRNA with those found in the 0.5 M KCI wash, commonly used as the source of initiation factors, showed also that only the 49000-M, protein from the complex could possibly be present in the 0.5 M KC1 wash of polyribosomes; proteins with mobilities similar to the other three proteins complexed with mRNA were not detected in the salt wash of polyribosomes.
There is now good evidence that eukaryotic messenger RNA, whether occurring ribosome-free in the cytoplasm or whether incorporated into polyribosomes, is associated with specific proteins forming the so-called messenger ribonucleoprotein complexes or mRNA . protein [l]. The analysis of the protein moieties of polyribosoma1 mRNA . protein from a variety of species and tissues reveals the presence of two main proteins with very similar molecular weights of around 49000 and 73 000 and several minor components [2 - 71. It has been shown recently that the main proteins of duck and rabbit mRNA . protein [S] and of mRNA . protein isolated from polyribosomes of mouse L-cells, rat hepatocytes and rabbit reticulocytes migrate identically when coelectrophoresed on sodium dodecylsulphate/polyacrylamide gels [ 5 ] . These results justify the conclusion that the slight differences reported for the molecular weights of the main proteins isolated from polyribosomal mRNA . protein from various sources are due to the limitation of the sodium dodecylsulphate/polyacrylamide gel method for an exact determination of molecular weights and that it is highly probable that at least the main proteins of
all ribosomal m R N A . proteins so far isolated have the same molecular weights. Hence these proteins might occupy similar or identical regions in the various mRNAs as proposed previously [ 5 ] . Contrary to the general agreement in respect of the two main proteins bound to polyribosomal mRNA, the specificity and number of the minor protein components has been the subject of considerable discussions and the reports vary widely concerning presence, number and molecular weight of these components. For example in the case of the duck globin mRNA . protein we reported the presence of six minor components in the molecular weight range of 50000- 120000 in addition to the two main components [2]. Protein populations comparable to this situation have been reported for mouse kidney mRNA . protein [9] and for mRNA . protein isolated from KB-cell polyribosomes [4]. On the other hand no minor components at all were found to be associated with rabbit globin mRNA by Blobel [ 5 ] , a result that is in contrast to our recent finding of at least two minor components of molecular weights around 60000 and 64000 associated with the rabbit globin mRNA [S].
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Two-Dimensional Gel Analysis of Messenger Ribonucleoprotein Complexes
In addition to this discrepancy concerning presence, number and molecular weight of the minor components, little is known concerning their origin. While it is well documented that the main proteins almost certainly are not of ribosomal origin nor contaminating soluble proteins, as they stick firmly to the mRNA even under high-salt conditions where unspecifically bound proteins would dissociate from the mRNA, the evidence about the minor proteins remains questionable. This is due to a large extent to the fact that the techniques currently used for the liberation and isolation of polyribosomal mRNA . proteins do not yield complexes that are free of contaminations with small ribosomal subunits and/or ribosomal-split soluble proteins, all of which could bind to the mRNA [8, 101 (and our unpublished results). Another unsolved question related to the mRNAassociated proteins is their physiological role. According to some authors mRNA-associated proteins might be involved with the binding of mRNA to the small ribosomal subunit and therefore might be initiation factors [ l l - 131. The molecular weights of the proteins associated with mRNA overlap those of several polypeptide components of eukaryotic initiation factors recently isolated and purified to homogeneity [14]. As a biological assay for the function of the mRNA-associated proteins is lacking, one is left with extrapolations based entirely on comparison of their molecular weights with those of proteins with known functions. The comparison of proteins from mRNA . protein with other cellular proteins has been done until now by one-dimensional sodium dodecylsulphate/polyacrylamide gel electrophoresis. As any one-dimensional electrophoresis system is inadequate for the analysis and interpretation of complex protein patterns, where inevitably the proteins in question could overlap with either ribosomal or soluble proteins, we decided to investigate the protein population of duck globin mRNA . protein using a two-dimensional system. An optimal separation in such a system is only obtained if each dimension separates proteins according to different parameters; we therefore chose for the first dimension a separation of the proteins according to their charge and in the second dimension according to their molecular weights. Using this technique for the comparison of the mRNA-associated proteins with proteins of polyribosomes, salt-washes of polyribosomes and active subunits, we hoped to gain better insight into the problem of the origin of the mRNA-associated proteins as well as into their possible relationship with initiation factors and/or ribosomal proteins. Here we show that besides the main proteins only two minor components are attached to the duck globin mRNA. Under our conditions one of the main proteins, that with a molecular weight of 49000, is acidic while the other three are basic. None of these
proteins is of ribosomal origin, as they cannot be found in preparations of purified active ribosomal subunits. Our results also suggest that only the 49000-M, main band could possible be contained in the initiation factor fraction as all the other globin proteins associated with mRNA were not detectable in 0.5 M KC1 washes of polyribosomes.
MATERIALS AND METHODS Substances RNase-free sucrose was purchased from Sigma Chemical Company, sodium dodecylsulphate from Fischer Scientific Company, acrylamide and bisacrylamide from Eastman-Kodak and oligo(dT)-cellulose from Collaborative Research Inc. All the other chemicals were reagent grade. S o h tions High-Salt B u f f r . 0.02 M Tris-HC1, pH 7.5; 0.5 M KCl; 0.003 M MgC12. Buffer A : 0.01 M Tris-HC1, pH 7.4; 0.2 M NaCl; 0.01 M EDTA; 0.2% Brij-35. Spacer Gel 1st Dimension. 4 acrylamide; 0.2 % bisacrylamide; 0.05 M boric acid; 0.02 M Na2EDTA; 8.0 M urea; 0.02 ammonium persulfate; 0.2 7; N,N’,N’-tetramethylethylenediamine, pH 6.1. Separation Gel 1st Dimension. 6 o/, acrylamide; 0.2% bisacrylamide; 0.5 M boric acid; 0.02 M NazEDTA; 0.4 M Tris; 6.0 M urea; 0.02% ammonium persulfate; 0.2 N , N , N ’,N’-tetramethylethylenediamine, pH 8.5. Sample Buffer 1st Dimension. The same as the spacer gel but without acrylamide/bisacrylamide and containing 10 % sucrose. Electrophoresis Buifer. 1st Dimension. 0.12 M triethanolamine; 0.06 M Na2EDTA; 0.155 M boric acid, pH 8.6. Spacer Gel 2nd Dimension. 0.125 M Tris-HC1, pH 6.8; 6 % acrylamide; 0.16% bisacrylamide; l.Oo/o sodium dodecylsulphate; 0.1 7;ammonium persulfate; 0.1 7; N , N , N ‘ , N ‘-tetramethylethylenediamine. Separation Gel 2nd Dimension. 0.375 M Tris-HC1, pH 8.8; 10% acrylamide; 0.26 bisacrylamide; 1.0% sodium dodecylsulphate; 0.1 ammonium persulphate; 0.1 % N , N , N ’,N’-tetramethylethylenediamine. Sample Buffer 2nd Dimension. 0.06 M Tris-HC1, pH 6.8; 5 % 2-mercaptoethanol; 2.3 % sodium dodecylsulphate; 10 % glycerol. Electrophoresis Buffer. 2nd Dimension. 0.3 ?< Tris ; 1.45 % glycine; 0.1 ‘%;sodium dodecylsulphate.