Comparisons of Proteins Associated with Duck‐Globin mRNA and Its Polyadenylated Segment in Polyribosomal and Repressed Free Messenger Ribonucleoprotein Complexes

Abstract

EDTA dissociation of polyribosomes from duck erythroblasts allowed us to isolate the 15‐S globin messenger ribonucleoproteins (mRNP) by sucrose gradient centrifugation or affinity chromatography on poly(U)‐Sepharose or oligo(dT)‐ce1lu1ose columns. Their protein composition was compared by one and two‐dimensional electrophoresis in sodium dodecyl sulfate to the free 20‐S mRNP containing the repressed fraction of globin mRNA [Vincent, A., Civelli, O., Maundrell, K., and Scherrer, K. (1980) Eur. J. Biochem. 112, 617–633]. The protein composition of the 15‐S mRNP isolated by these methods in different ionic strength conditions, was characterized by a major 73000‐Mr polypeptide and seven minor polypeptides with Mr ranging from 45000 to 68000, all of which are slightly basic, and about five acidic ones in the 80000–130000‐Mr range. All these are retained in the 15‐S mRNP core particle isolated at 0.5 M KCl. At low ionic strength, in addition, a specific group of acidic polypeptides in the Mr range 35000–105000 was also found associated with globin mRNA. Oligo(dT)‐cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73000‐Mr major protein is bound to the poly(A) segment; some other proteins resolved as minor components interact with both the poly(A) and non‐poly(A) regions of globin mRNA. Characterization of proteins interacting with the poly(A) segment of non‐polyribosomal globin mRNA in 20‐S free mRNP demonstrated the absence of the polyribosomal 73000‐Mr poly(A)‐binding protein. Furthermore, it confirmed that the protein compositions of translatable polyribosomal and repressed free globin mRNP are very different. Indeed, the respective core (0.5 M KCl) particles contain only two possibly common polypeptides. The specificity of proteins associated with globin mRNA in two different functional states shown here supports the hypothesis of a role of mRNP proteins in translational control of mRNA.

The translation of the messenger for the poly(A)‐binding protein‐associated with translated mRNA is suppressed

“…The absence of de novo synthesis of this protein in duck erythroblast confirms the data of in vitro translation of mRNA of polyribosomal and free mRNP; i.e., the mRNA coding for this protein is predominantly found in the translationally not expressed mRNP (cf. The drawback of this quick mRNP isolation procedure is the large contamination of mRNA by ribosomal subunits (discussion in [13]) which, however, did not interfere in the present experiments; on the contrary, the ribosomal proteins LETTERS October 1983 provided an interesting control. As observed with total cytosolic proteins ( fig.2), no labeiling of the 73 kDa protein bound to translated mRNA could be detected ( fig.3).…”

“…Pellets of polyribosomes isolated from duck erythroblasts (section 2.1) were resuspended in 10 mM TEA-HCl (pH 7.4), 50 mM KCI, 5 mM pmercaptoethanol and dissociated with EDTA prior to affinity chromatography on oligo(dT)-cellulose as detailed in [13]. Elution of bound material was by 50% formamide containing 10 mM TEA-HCl (pH 7.4), 10 mM EDTA (pH 7.4) and 50 mM NaCl.…”

“…As observed with total cytosolic proteins ( fig.2), no labeiling of the 73 kDa protein bound to translated mRNA could be detected ( fig.3). Some other polypeptides with M, 50~120~, previously identified as acidic mRNP proteins [13] are synthesized de novo contrary to the poly(A)-binding protein and the ribosomal proteins (A& I5 OOO-50000 [16]). The absence of synthesis of ribosomal proteins in duck erythroblasts contrasts with what occurs in dividing cells [19- Comparison by polyacrylamide gel electrophoresis of cytoplasmic proteins labelled in vivo with the in vitro translation products of mRNA isolated from polyribosomal and free mRNP.…”

The poly(A)‐binding protein facilitates in vitro translation of poly(A)‐rich mRNA

“…Since the translation of complete polyribosomal mRNP was not inhibited by poly(A), it is likely that the component of the lysate which interacts with poly(A) is already present in the polyribosomal mRNP. A good candidate for this component was poly(A)-binding protein which is present, bound in the mRNP [23,35] and free in the cytosol [24]. For this reason we purified the 73-kDa poly(A)-binding protein of duck erythroblasts in order to check its effect in the lysate supplemented with globin mRNA and poly(A).…”

“…Its mechanism of action in mRNA metabolism and function is, however, not known. Comparison of the translational efficiency in vitro between phenol-extracted globin mRNA and the corresponding mRNP has shown no difference [23]. In view of the evolutionary conservation of the poly(A)-binding protein and its abundance in a free form in the cytoplasm [20, 241, the possibility that exogenous mRNA immediately reconstitutes an mRNP when added to the cell-free lysate had to be taken into consideration [25].…”