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Comparisons of proteins associated with duck-globin mRNA and its polyadenylated segment in polyribosomal and
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)-cellulose 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 73 000-Mr polypeptide and seven minor polypeptides with Mr ranging from 45 000 to 68 000, all of which are slightly basic, and about five acidic ones in the 80 000--130 000-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 35 000--105 000 was also found associated with globin mRNA. Oligo(dT)-cellulose chromatography of mRNP digested with ribonucleases A and T1 indicated that the 73 000-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 73 000-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.
Insights
Messenger ribonucleoproteins (mRNP) associated with translatable globin mRNA differ significantly in protein composition from repressed mRNA. This suggests mRNP proteins play a key role in regulating gene translation.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
Background:
- Polyribosomes dissociate into messenger ribonucleoproteins (mRNP) upon EDTA treatment.
- Globin mRNA exists in both translatable (polyribosomal) and repressed (free) forms.
Purpose of the Study:
- To compare the protein composition of 15-S globin mRNP (translatable) with 20-S free mRNP (repressed).
- To investigate the role of mRNP proteins in translational control of globin mRNA.
Main Methods:
- Isolation of 15-S globin mRNP using sucrose gradient centrifugation and affinity chromatography.
- Protein analysis via one- and two-dimensional electrophoresis in sodium dodecyl sulfate.
- RNase digestion and oligo(dT)-cellulose chromatography to identify protein-RNA interactions.
Main Results:
- 15-S mRNP contains a major 73,000-Mr polypeptide bound to the poly(A) tail and several minor polypeptides.
- 20-S mRNP lacks the 73,000-Mr poly(A)-binding protein found in 15-S mRNP.
- Core particles of translatable and repressed mRNP share only two common polypeptides, indicating distinct protein compositions.
Conclusions:
- The protein composition of globin mRNP is functionally distinct for translatable and repressed mRNA.
- Specific mRNP proteins likely mediate translational control of globin mRNA, supporting a regulatory role.