MRNPs associated proteins

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. 

Because the export machinery selectively binds transport-competent mRNPs, i.e., mRNAs associated with the appropriate nuclear proteins, the diffusion-based model for intranuclear RNA movement provides an additional checkpoint for gene expression at the level of mRNA export. This type of control has been demonstrated for tRNA export only mature tRNAs are transported to the cytoplasm because incompletely processed tRNAs do not efficiently bind to exportin-t, the tRNA-specific export receptor. 

Le Hir, H., Moore, M.J. and Maquat, L.E. (2000b) Pre-mRNA splicing alters mRNP composition evidence for stable association of proteins at exon-exon junctions. Genes Dev., 14, 1098-1108. 

Once the processing of an mRNA is completed in the nucleus, it remains associated with specific hnRNP proteins in a messenger ribonuclear protein complex, or mRNP. Before it can be translated into the encoded protein, it must be exported out of the nucleus into the cytoplasm. The nucleus is separated from the cytoplasm by two membranes, which form the nuclear envelope (see Figure 5-19). Like the plasma membrane surrounding cells, each nuclear membrane consists of a water-impermeable phospholipid bllayer and vari-... 

Other Proteins That Assist in mRNP Export In addition to the mRNA-exporter and FG-nucleoporins, several other types of proteins are involved in the transport of mRNPs by this mechanism. As mentioned earlier, the mRNA-exporter is thought to bind to mRNAs cooperatively with specific mRNP proteins. For example, SR proteins associated with exons appear to stimulate the binding of the mRNA-exporter to processed mRNAs in mRNPs. Thus SR proteins not only... 

Another participant in mRNP transport to the cytoplasm Is the nuclear cap-bIndIng complex, mentioned earlier as protection against exonuclease attack on the 5 end of nascent transcripts and pre-mRNAs. Electron microscopy experiments discussed below have demonstrated that the 5 end of mRNAs lead the way through the nuclear pore complex. Recent experiments in yeast indicate that the 3 poly(A) tail plays an Important role In mRNP transport, suggesting that a poly(A)-binding protein participates. Nucleoporins associated with the NPC cytoplasmic filaments In addition to FG-nucleoporins are required for mRNA export and may function to dissociate the mRNA-exporter and other mRNP proteins that accompany the mRNP through the pore. 

Once the mRNP reaches the cytoplasm, most of the mRNP proteins that associated with the mRNA in the nucleus, the nuclear cap-binding complex, and the nuclear poly (A)-binding protein (PABPII) dissociate and are shuttled back to the nucleus. In the cytoplasm, the 5 cap of an exported mRNA is bound by the eIF4E translation initiation factor, the poly(A) tail is bound by multiple copies of the cytoplasmic poly(A)-binding protein (PABPI), and other RNA-binding proteins associate with the body of the mRNA, forming a cytoplasmic mRNP that has a lower ratio of protein to RNA than nuclear mRNPs. 

It was shown that the polyribosomal form of mRNP complexes is actively translated, whereas the free form is not. One mi t expect that a covalent chemical modification of some of the mRNA proteins, such as ADP-ribosylation, will render the mRNA available for translation. We characterized the mRNA-associated ADP-iibosyl transferase in plasmac) oma, in Krebs II, ascite tumor cells, and in liver. Several auto(ADP-ribosylated) proteins could be obtained when mRNP particles were incubated with NAD. It is unlikely that we are dealing with a contamination of chromatin since in plasmocytoma the enzymatic activity in mRNP represent 34% of the total cellular activity, while the maximum DNA contamination is only 4%. Moreover, after DNAse hydrolysis the enzymatic activity remains unchanged and addition of DNA is without effect [31]. More information on these mRNP particles will be given by Thomassin et al. (this volume). 

Recently, we have demonstrated the existence of a poly(ADPR) polymerase activity associated with cytoplasmic free messenger ribonucleoprotein particles (mRNP) isolated from mouse plasmacytoma cells [4]. The enzyme does not require DNA for activity and is able to produce an ADP-ribosylation of some of the mRNP proteins. We have extended our observations to Krebs II ascites-tumor cells and to rat liver. In the present report, we will discuss some properties of this enzyme, particularly the activation by RNase A. 

Incubation of mRNP particles with NAD, electrophoresis on lithium dodecyl sulfate polyacrylamide gels, and autoradiography reveal several ADP-ribosylated proteins with a predominant acceptor of mol. wt. 115,000 [4]. Using the protein blotting technique, we have observed that the antiserum directed against the nuclear calf thymus poly(ADPR) polymerase reacts with the 115,000 mol. wt. protein, associated with free mRNP particles. 

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. 

ADP-Ribosylation in Cytoplasmic Ribonucleoprotein Particles Which Contain Silent mRNA. We demonstrated that ADP-ribosyl transferase activity is associated with cytoplasmic free mRNP isolated from a variety of organs and tumor cells mouse plasmacytoma, rat liver, rat brain, Krebs n cell rat brain cultured neurons and astrocytes (25-28). Table 1 summarizes the mRNP poly(ADP-ribose) polymerase activity. On a protein basis, in contrast to the tumor cells, the activity associated with rat liver and whole rat brain free mRNP is very low. Nevertheless, it is 12 fold higher than the activity associated with the microsomal ribosomal fraction reported by Burizo et al. (19). In brain mRNP and mainly in neuronal mRNP die activity is much higher than in the rat liver mRNP on a DNA basis (not shown). 

Poly(ADP-ribosyl)ation is generally described as a nuclear event. However, extranuclear poly(ADP-ribose) polymerase activities have been detected in the cytosol of baby hamster kidney ceUs (1), the ribosomal fraction of HeLa cells (2) and rat testis (3). The activities looked like the nuclear enzyme in diat they totally or partially depended on DNA. We have previously reported the association of a poly(ADP-ribose) polymerase with a specific ribonucleoprotein complex, namely free messenger ribonucleoprotein particles (free mRNP) (4-6). The enzyme has the particularity to be DNA-independent. In diis paper, we have b n interested in the proteins which are poly(ADP-ribosyl)ated in free mRNP. We also present evidence of the existence of a poly(ADP-ribose) glycohydrolase in free mRNP. 

It appears from our results that free mRNP possesses both the enzymatic activities of synthesis and degradation of poly(ADP-ribose). A cytoplasmic poly(ADP-ribose) glycohydrolase has also recently been described by Tanuma et al. (12). ITiis suggests a balance between the ADP-ribosylation/de-ADP-ribosylation states of free mRNP proteins. In addition, free mRNP poly(ADP-ribosyl)ated proteins seem associated with different ribonucleoprotein particles which are removed from free mRNP by a 0.5 M KCl treatment. It is noteworthy that low rnoLwt. RNA able to inhibit mRNA translation in vitro have been isolated from such ribonucleoprotein particles (13). Free mRNP proteins have also been suggested to play a role in the mechanisms and regulation of the translation of mRNA (7, 8). One could speculate a role of poly(ADP-ribosyl)ation in the stmctural changes that may occur in the free mRNP to permit the translation of the mRNA repressed in these particles. 

Throughout the ribosome cycle, dynamic protein-mRNA interactions are functionally important in the initiation, elongation, and termination of polypeptide synthesis. In addition, more stable associations between proteins and mRNAs have been observed, particularly in eukaryotic cells. These messenger ribonucleoprotein complexes (mRNPs) occur both in polyribosomes and free in the cytosol, some of the latter being either temporarily or permanently unavailable for translation. Thus, protein-mRNA interactions contribute to the efficiency with which mRNAs are translated. 

Some proteins, such as the poly(A)-binding protein (p78), are present in most if not all mRNPs, whereas others appear to be cell specific and mRNA selective. In unfertilized sea urchin eggs and Xenopus oocytes, for example, untranslated messenger is sequestered by association with proteins that prevent translation until later stages of development. Duck reticulocytes contain globin mRNP, which cannot be translated in vitro, whereas the mRNA obtained by deproteinizing the complex can be translated, showing that in this case translation is prevented by the mRNP proteins. 

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