Informosomes and polyribosome-associated proteins in eukaryotes

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Informosomes and polyribosome-associated proteins in eukaryotes Alexander S. Spirin and Murat A. Ajtkhozhin Proteins of unknown function are complexed with all mRNA in the cytoplasm of animal and plant cells, thus forming messenger ribonucleoproteins or informosomes. There are also a large number of other proteins which are loosely associated around eukaryotic polyribosomes as a result of their ability to bind RNA. These include elongation factors, aminoacyl-tRNA synthetases and protein kinases. It is suggested that modifications of the polyribosome-associated proteins can modulate protein synthesis by altering the affinity of the proteins for RNA. higher plants4, suggesting that the nucleoprotein form of mRNA is a common feature of higher eukaryotes. The mRNPs which are not bound with ribosomes (the so-called free cytoplasmic informosomes) contain nontranslatable mRNA. This may be stored (masked) mRNA (which is to be translated only at a certain later stage of cell differentiation), or temporarily untranslated mRNA on its way from the nucleus to polyribosomes, or 'run-off" mRNA released from a translational complex, or simply the excess mRNA of the cytoplasm. A striking example of the transition of the translatable mRNA from polyribosomes into the nontranslatable state as stored informosomes occurs in wheat grains: as protein synthesis declines during ripening, an increasing fraction of the wheat embryo mRNA is found separate from ribosomes in free mRNPss. Free mRNP particles The nontranslatable state of mRNA That mRNA could exist as a nucleoprotein was discovered 20 years in free mRNPs suggested that the proago when cytoplasmic extracts of tein moiety of the particles is involved embryonic fish and sea urchin cells were in inhibiting (repressing) translation°. investigatedL2. The nonribosomal par- Studies of the protein moiety of free ticles were shown to be mRNA-protein mRNPs have met, however, several complexes of a defined stoichiometry technical difficulties, so that its composi(protein to RNA mass ratio of about tion and function are still unknown. 3:1, buoyant density in CsC1 of about Scherrer's group has demonstrated the 1.4 g cm-3)13. They were called inforrno- presence of a repressor activity among somes but are now more generally proteins of the free mRNP fraction of referred to as messenger ribonucleopro- duck reticulocytes 7 but the repressor teins (mRNPs). The size of the particles protein itself has not been isolated. On the other hand, a repressor activity has depended on the size of their mRNA. Similar particles were later detected been reported for some small cytoby different workers in all the animal plasmic RNAs (see for example Ref. 8) cells studied and in the cytoplasm of or their complexes with proteins9 bound with free mRNPs. An interesting posA. S. Spirin ia at the Institute of Protein Research, sibility has been advanced by BrawerAcademy of Sciences of the USSR, Pushchino, Moscow Region, USSR and M. A. A]tkhozhin is at man et al. ~° and by Bag ~ that: the the Institute of Molecular Biology and Biochem- products of translation of some mRNAs can specifically bind to their mRNA istry, Academy of Sciences o f the Kazakh SSR, Alma-Ata, USSR. such that they repress further translation The cytoplasm of eukaryotic cells is much more organized structurally than that of baeteria and other prokaryotes. The internal membrane network subdivides the eukaryotic cytoplasm into specific compartments, such as nucleus, endoplasmic reticulum, golgi apparatus mitochondria and chloroplasts. More dynamic polymer protein structures, such as microtubuli, microfilaments and intermediate filaments, form the so-called 'cytoskeleton'. The tendency to form multienzyme complexes and aggregates is especially displayed in eukaryotes. It has also been found that messenger RNA (mRNA) and dynamic mRNA-ribosome complexes (polyribosomes) of the eukaryotic cytoplasm are accompanied by many bound proteins, some of whose functions are still to be found.

~ ) 1985, Elsevier Science Publishers B.V., Amsterdam 0~76 - 5067t85ttO2.00

of the mRNA. Correspondingly, free mRNPs should contain the protein products of translation of the constituent mRNA. Indeed, it has been shown that actin and other muscle-specific polypeptides can be detected in free mRNPs of cultured muscle cellsn. Upon heat shock, the fraction of free mRNPs of the same cells contains one of the heatshock proteinsix. Such a feed-back regulation may selectively switch off translation and promote transition from polyribosomes into free mRNPs of mRNAs whose translational products are excessively accumulated in the cytoplasm. At the same time, just one or a few molecules of a specifically-bound protein per mRNA molecule seem to be sufficient to prevent the initiation of translation of mRNA. Hence, repressor proteins can hardly comprise the bulk of the protein of free mRNPs; rather, they must be minor protein components. The proteins of free mRNPs vary significantly, and include up to 10 major proteins (20-100 kDa). Among the major components of free mRNPs, the 78 kDa polypeptide should be mentioned; there is evidence that it is complexed with 3'-terminal poly(A)sequences of mRNAs. In general, the functions of the major protein components of free mRNPs are unclear. Polyribosome-bound mRNPs The translatable portion of cytoplasmic mRNA is localized in polyribosomes. The dissociation of polyribosomes was reported to result in the release of the translatable mRNA in the form of mRNA-protein complexes 12. The polyribosome-bound mRNPs are somewhat less loaded with proteins than free mRNPs (protein to RNA mass ratio is about 2:1, buoyant density in CsC1 is about 1.45 g cm-3). The protein composition of polyribosomal mRNPs does not coincide with that of free mRNPs, though some components may be common'3. The 78 kDa polypeptide which appears to be bound to poly(A)-sequences is a major protein component of both types of mRNP. The characteristic major component of polyribosome-bound mRNPs of both animals and plants is a protein with a molecular weight of 50 000. Both the 50 and the 78 kDa proteins are firmly bound with translatable mRNA and often comprise a significant fraction of the total protein of polyribosomal mRNPs. Functions of the proteins of polyribosome-bound mRNPs are not known. The elongation and initiation

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Dn..~ {~ T~,S ~ T~8. factors of translation are not amongst the major, firmly bound proteins of polyribosomal mRNPs 14. Despite their apparent stability, the mRNA-protein complexes seem to be dynamic structures: during their life history the proteins are not permanently anchored but exchange with free proteins of the cytoplasmic poop5.

RNA-binding proteins of the cytoplamn A fraction of free proteins with an affinity for high-polymer polynucleotides (the so-called RNA-binding proteins) has been discovered in the cytoplasm of animal~6 and plant 17 cells. For a long time these proteins were suspected to compose a pool of free informosomal proteins in the cytoplasm.

Indeed, the injection of the total fraction of labeled free RNA-binding proteins into frog oocytes resulted in the direct incorporation (without preliminary degradation) of some of them into mRNPs 18. The major components of free RNAbinding proteins, however, do not coincide with the main polypeptides of free or polyribosomal mRNPs 14. In other words, only a small fraction of free RNA-binding proteins can be considered as a pool of mRNPs - most of them seem to have a different destination. The cytoplasmic RNA-binding proteins vary with cell type. Rabbit reticulocytes have a relatively simple set of free RNA-binding proteins: there are

three major polypeptides (95, 49 and 36 kDa) and several minor components. The 95 kDa (or the homologous 70 kDa) and 49 kDa proteins occur in all types of animal and plant cells. These two proteins have been identified as elongation factors of translation, EF-2 and EF-let, respectively19. Correspondingly, the isolated eukaryotic elongation factors, EF-1 and EF-2, behave as typical RNA-binding proteins in all standard tests, in contrast to their prokaryotic analogs, EF-T, and EF-G, which display no appreciable affinity for highpolymer polynucleotides2°. It is interesting that aminoacyl-tRNA synthetases have also been found among RNAbinding proteins of the eukaryotic cytoplasm (bacterial aminoacyl-tRNA

CYTOPLASM POLYRIBOSOMES

FREE INFORMOSOMES (NON-TRANSLATABLE mRNA.PROTEIN COMPLEXES)

POLYRIBOSOME-BOUND INFORMOSOMES (TRANSLATABLE MESSENGER RIBONUCLEOPROTEINS)

ADDITIONAL PROTEIN LOOSELYBOUNDTO POLYRIBOSOMES

FREE RNA-BINDING PROTEINS

(EF-I, EF-2, ARSases, SPECIFIC KINASES ANn TRANSFERASES,ETC

(EF-I, EF-2, ARSases, ETC.)

Fig. 1. Compartmentation of messenger ribonucleoproteins and RNA-binding proteins in the cytoplasm of the eukaryotic cell.

V DYNAMIC PROTEIN CONSTELLATIONS AROUND POLYRIBOSOMES

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synthetases lack this nonspecific RNAbinding capability)2L What, then, is the biological significance of the nonspecific RNA-binding capability of elongation factors, aminoacyl-tRNA synthetases and several other proteins in the cytoplasm of eukaryotic cells? 'Clouds' of proteins around

polyribosomes In addition to ribosomal proteins and to the proteins more or less firmly bound with mRNA (i.e. the proteins of polyribosomal mRNPs), the polyribosomal fraction of eukaryotic cells contains many loosely associated proteins which seem to be in a dynamic equilibrium with a cytoplasmic pool of free proteins22. This accompanying protein material comprises up to 30% of the total polyribosome mass (whereas the protein of polyfibosomal mRNPs contributes no more than 2%). The additional proteins which are loosely and transiently associated with polyribosomes can be fixed on them by formaldehyde crosslinking. Their presence on polyribosomes can then be revealed by a shift in buoyant density in CsC1, from 1.59 g cm -3 in the case of pure eukaryotic ribosomes or their complexes with mRNPs to 1.50 g cm -3 for polyribosomes with the additionally associated proteins3m (the buoyant density shift by 0.09 g cm -3 is equivalent to about 1.5 x 106 Da of additional protein per ribosome). Zonal centrifugation used for detecting and isolating polyribosomes results in the loss of this additional protein material from polyribosomes. This is a result of the removal of free proteins and, hence, of the shift of the equilibrium towards dissociation. The loss of the additional protein from polyribosomes can also be induced by adding excess exogenous RNA to the cytoplasmic extract. In contrast, lowering the ionic strength in the extract below physiological level favours the retention of the proteins on polyribosomes because of their increased affinity for RNA under such conditions. It has been shown that almost all proteins loosely associated with polyribosomes are identical to the RNA-binding proteins present in the cytoplasm in the free state22. In connection with this, it should be mentioned that a significant portion of aminoacyl-tRNA synthetases has been repeatedly reported to be associated with polyribosomes of eukaryotic cell extracts. Association of translation factors, primarily the elongation factors

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EF-1 and EF-2, with eukaryotic poly- other RNA-binding proteins; the phosribosomes has also been claimed. Thus, phorylation results in the loss or the RNA-binding proteins of the decrease of their ability to interact with eukaryotic cytoplasm may have a tend- RNA2L The polyribosomal fraction of ency to be localized on polyribosomes; rabbit reticulocytes contains a latent evidently, the proteins loosely asso- protein kinase activity which is capable, ciated with polyribosomes are in after activation, of specifically phosdynamic equilibrium with free RNA- phorylating the elongation factor 1 (EFbinding proteins (see Fig. 1). The result 1); the phosphorylation induces its can be an increased local concentration release from polyfibosomes~. The polyribosome fraction also conof these proteins around polyribosomes tains a specific ADP-ribosyl transferase ('clouds'). It has been proposed that the non- which catalyses the ADP-ribosylation of specific affinity for RNA of proteins the elongation factor 2 (EF-2)26. It is serving translation is an evolutionary known that an analogous ADP-ribosylaacquisition. It may provide for their tion of EF-2 by diphtheria toxin results increased local concentration or partial in the loss of its affinity for RNA and its compartmentalization near the sites of release from the polyribosome fractheir function, i.e. around polyribo- tion27. Several eukaryotic aminoacyl-tRNA somes, in a big and complex volume of the eukaryotic cell23. This affinity should synthetases have been found to exist in not be too strong, to avoid the formation two forms: one possesses a nonspecific of stable complexes. In other words, it is affinity for high-polymer RNAs and the advantageous to have diffuse 'clouds' of other lacks it2]~. Whether the loss (or appropriate proteins around RNA-con- the acquisition) of the affinity for RNA taining structures, primarily polyribo- involves modification of the protein somes. The relatively low affinity of the remains to be seen. Noncovalent modifications of RNAproteins for both mRNA and ribosomal RNA may result in the formation of binding proteins are also possible. such loose dynamic constellations. The Recently, the plant hormone kinetin experimental data available seem to sup- (6-furfurylaminopurine) has been shown port the idea of local 'clouds' around to form specific complexes with some polyfibosomes of the eukaryotic cell, at RNA-binding proteins of wheat embryo least for the translation factors and extracts29. At the same time, this horaminoacyl-tRNA synthetases. mone has been reported to activate elongation factor 1 (EF-1) in wheat embryos during germination3°. Perhaps Regulation of protein synthesis: the specific effect of cytokinins on transnew posa'bllities lation in plants is mediated by the interSince the proteins controlling trans- action of the hormone with translation lation are complexed with mRNA factors. Generally, modifications of the (mRNPs) or loosely clustered around RNA-binding proteins may significantly polyribosomes (dynamic constellations), change the polyribosomal 'suite' and protein synthesis can be regulated thereby modulate the protein synthesis through alterations of the affinities of in the eukaryotic cell. these proteins for RNA. For example, the removal of a repressor protein from Acknowledgement The authors express their gratitude to nontranslatable mRNA may be a result of some modification of the protein, Lev Ovchinnikov and Waldemar Minich reducing or abolishing its affinity for for discussions and comments, as well as RNA. On the other hand, the decrease for the use of their manuscripts before of the nonspecific affinity of a transla- publication. tion factor, or an aminoacyl-tRNA synthetase for RNA as a result of a References modification could induce the decelera- 1 Spirin, A. S., Belitsina, N. V. and Ajtkhozhin, M. A. (1964) J. Gen. Biol. 25, 321-337 (Rustion of total protein synthesis. Though sian); (1965) Fed. Proc. (Translation Suppl.) direct evidence in favour of such regula24, "I907-T922 (English translation) tory mechanisms is not yet available, 2 Spirin, A. S. and Nemer, M. (1965) Science several recent observations support this 150, 214-217 3 Spifin, A. S. (1969) Eur. Z Biochern. 10, hypothesis. 20-35 Thus, in addition to the translation factors and aminoacyl-tRNA syn- 4 Ajtkhozhin, M. A., Akhanov, A. U. and Doschanov, Kh. I. (1973) FEBS Lett. 31,104thetases, the fraction of RNA-binding 106; Ajtkhozhin, M. A. and Iskhakov, B. K. proteins of the eukaryotic cell has been (1982) Informosomes of Plants (Russian), shown to contain protein phosNauka, Alma-Ata phokinases capable of phosphorylating 5 Ajtkhozhin, M. A., Doschanov, Kh. I. and

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