Polysomes mRNPs

Thus, in the case of reticulocytes it is possible to isolate polysomal mRNPs in a pure state, indicating a good prospect for characterizing them and understanding their structure. From the ratio of the sedimentation coefficients of RNP and free RNA, the RNA content in polysomal mRNP appears to be higher than in nuclear complexes mRNA with a molecular weight of about 150,000 to 200,000 is present in polysomes in particles with a sedimentation coefficient of 14S and in the 308 particles in nuclei. 

The same conclusion may be drawn from the results of density gradient ultracentrifugation in CsCl. The buoyant density of polysomal mRNP isolated from reticulocyte polysomes is equal to 1.455 g/cm (Bumy et al., 1969). This corresponds to a content of about 30 percent RNA and means that the amount of protein per RNA unit in polysomal mRNP is 1.5 to 2 times lower than in nuclear D-RNP complexes (Fig. 15). In polysomes from tissues involved in the synthesis of many different proteins, the polysomal mRNPs... 

Fig. 14. Polysomal mRNP from rat liver. A. Sucrose gradient analysis of the polyribosomes preparation treated with EDTA. Labeling period, 40 minutes. Centrifugation was at 21.000 rpm for 16 hours. —A A Cts/min -------, absorbance. B. CsCI equilibrium gradient analysis of the poly-...

Fig. 15. Polysomal mRNP from reticulocytes. A. Sucrose gradient sedimentation of the polyribosomal suspension after EDTA treatment (40 hours centrifugation). B.Sucrose gradient sedimentation of unlabeled, sodium dodecyl sulfate-extracted polyribosomal RNA supplemented with the high specific radioactivity RNA detached from labeled polyribosomes by EDTA treatment. C. CsCi equilibrium sedimentation of the messenger ribonucleoprotein complex. Ribosomal subparticles have been added as Internal markers. The centrifugation was carried out at 4°C for 18 hours at 36,000 rpm in an SW-39 rotor. (From Huez et al. 1967. Biochim. Biophys. Acta, 145 629-636 Burny et al. 1969. Biochim, Biophys. Acta, 190 228-231.)...

The RNA of the mRNPs is sensitive to RNase and easily degraded. An increase in ionic strength leads to the dissociation of proteins from the particles. For example, in 0.55 M LiCl with 0.01 M MgCl2 the buoyant density of the particles increases and reaches 1.61 g/cm, which implies the loss of about three quarters of their total protein. A further increase in the salt concentration produces complete dissociation of the particles. After the removal of salt by dialysis the original particles can be reconstituted. The original and reconstituted particles have the same sedimentation properties and the same buoyant density (Perry and Kelley, 1968). Thus polysomal mRNP may be dissociated and reconstituted in a manner similar to that for nuclear complexes. 

A very important question concerns the nature of the proteins of polysomal mRNP. This question has not yet been answered because of the difficulties in the isolation of polysomal mRNPs. Since reticulocyte mRNPs can now be obtained in a pure state, the question may be answered in the near future. In another approach, Schweiger and Hannig (1970) tried to find proteins typical of nuclear D-RNP by direct analysis of polysomes. They separated the total polysomal protein into several fractions using free flow electrophoresis and then compared the fractions with nuclear D-RNPs obtained by means of polyacrylamide gel electrophoresis. In free polysomes and in membrane-bound polysomes, proteins similar to proteins of the nuclear D-RNP were found. If the membrane-bound polysomes are separated from the membranes by treatment with pure deoxycholate, these proteins are dissociated from polysomes and thus resemble the proteins of nuclear D-RNP in their sensitivity to deoxycholate. 

Thus, in general, the question about the similarity of the proteins composing the nuclear and polysomal particles remains open pending the isolation of pure polysomal mRNP. Free flow electrophoresis may be a good approach since, according to preliminary data by Schweiger and Hannig (1970), it permits the separation of ribosomes and mRNP. 

A number of experiments have been done to prove that polysomal mRNPs are not artifacts (Leytin et al., 1970). It is easy to imagine that some ribosomal proteins dissociate from ribosomes during EDTA treatment and then reassociate with free mRNA. To check this possibility, free D-RNA was added to polysomes at different stages of mRNP isolation. It was found that the added RNA does not interact with the proteins of undissociated polysomes if the polysomes are freed extensively from contaminating proteins. However, after dissociation of polysomes mixed with D-RNA before the addition of EDTA, one can observe an interaction of D-RNA with some proteins. In the CsCl density gradient these latter complexes occupy a wide zone, the position of whidi depends on the amount of polysomes and D-RNA. On the other hand, if D-RNA is added to the material already dissociated by EDTA most of it remains free and has a tendency to sediment to the bottom in CsCl density gradients. 

This means that the artificial interaction of RNA with polysomal proteins proceeds at the moment of dissociation of the polysomes. After dissociation these proteins combine irreversibly with mRNA or with ribosomal subunits. These results complicate the question of whether mRNPs are native structures. However, if the mRNA in polysomes is free, then it shoidd be possible to obtain it in an almost free state after the dissociation of polysomes in the presence of a large excess of D-RNA. Such experiments have been done, but it was found that even in the presence of 2 mg of free D-RNA per 1 mg of RNA of polysomes the endogenous polysomal-mRNA is recovered in heterogeneous complexes with a buoyant density of 1.40 to 1.60 g/cm. This figure may correspond to the protein content that seems to be the lower limit of the protein content in polysomal mRNA-containing complexes. In any case the experiments by Leytin et al. (1970) indicate that it is necessary to be very cautious in the analysis of the composition of polysomal mRNP since many artifacts may be created during their isolation. 

Unfortunately the nature of the proteins of polysomal mRNPs is not yet quite clear. Information on this question should allow us to understand the relations between all the D RNA-containing particles of cells. 

It is noteworthy that free mRNP particles are a temporary untranslatable form of mRNA in the cytoplasm and that proteins associated with the active polysomal mRNP or the repressed mRNP are different [6, 7]. There is now some evidence that ribo-nucleoprotein particles are dynamic structures and that protein exchanges occur between the cytoplasmic mRNA-associated proteins and free proteins. Involvement of mRNA-associated proteins in the regulation of protein synthesis has been considered [7-10] and post-translational modification of these proteins as a regulatory mechanism might be considered. 

There are two predominant proteins (M.Wt. approximately 50>000 and 7Qf000) in polysomal mRNPs isolated from a wide variety of mammalian sources (reviewed in reference 59) Other proteins are present in minor amounts, and are far from consistent between different preparations. Those who wish to see complications at every level of mammalian protein synthesis like to hint that these minor components could be protein specific to particular mRNA species, perhaps involved in regulating the translation of the mRNA, whilst the 50,000 and J8,000 dalton proteins would be seen as invariant components common to all mRNAs. This idea seems unlikely to be true, at least as a generality, since there is no sign of the putative mRNA-specific proteins in mRNP preparations from cells... 

We have very little idea about the stoichiometry of the two major polysomal mRNP proteins, or their location on the mRNA, apart from the fact that the 8,000 dalton protein seems to be associated with the poly A tract at the 5 end (59) The function of the proteins is even more obscure. In cell-free translation assays the activity of polysomal mRNP is no greater than that of deproteinised mRNA (20). This result is open to the qualification that in crude cell-free systems the added deproteinised mRNA mi t pick up proteins from the cell-free extract and thereby be effectively converted into polysomal mRNP particles, but since the same result is obtained in highly fractionated systems (11) this reservation can probably be discounted. The major polysomal mRNP proteins are distinct from all seven recognised initiation factors (ll), and initiation complex formation is found to require the same set of seven factors regardless of whether polysomal mRNP or deproteinised mRNA is used (11). In short, there is no evidence that these proteins play a role in mRNA translation. 

If the free mRNPs represent mENAs in a state of specific repression, one mi t expect that the proteins would not only differ from the polysomal mRNP proteins, but would also differ according to the mENA species present in the mRNP. Virtually all free mRNPs, including the a-chain mRNP of rabbit reticulocytes which is almost certainly not a repressed form of a-globin mENA, lack the JdjOOO... 

As opposed to the above-reviewed studies, which all point to a negative role for proteins in free mRNP particles, polysome-derived mRNP particles contain proteins that are essential for translation. In a Krebs ascites tumor cell-free system from which RNA-binding proteins have been removed, at least in part, by passage over RNA-Sepharose, polysome-derived mRNPs are translated, while depro-teinized mRNA is not (Schmid et al., 1982). The polysomal mRNPs contain fewer proteins than free mRNPs, and are as active as depro-teinized mRNA in whole cell-free systems for translation (reviewed by Hershey, 1982 ). 

Association of carrier Nascent RNP Transfer RNP (informopher) (informosome) Polysome (mRNP) ... 

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