Informosomes

In 1964 Spirin et al. discovered in embryonic cells cytoplasmic ribonucleoprotein particles containing rapidly labeled RNA with many properties of mRNA. The particles contain RNA and protein in a ratio of about 1 3 or 1 4. These particles, which are free cytoplasmic mRNA-protein complexes not combined with ribosomes, were named informosomes. Under ultracentrifugation they form a number of discrete peaks with sedimentation coefficients equal to 20, 30, 40, 50, 55, 65, and 75S (Spirin et al., 1964). 

Recently, informosomes with sedimentation coefficients up to 200S have been observed (Spirin, 1969 Ovchinnikov et al., 1969a). 

Thus three types of nucleoproteins containing D-RNA have been described up to now nuclear particles, informosomes (free cytoplasmic particles), and polysome-bound mRNA-proteins. Only the nuclear particles have been obtained in a purified state, and this has made it possible to study their protein composition and structural organization more extensively. Unfortunately, free informosomes and polysome-bound particles have not been characterized to a similar extent due to the difficulties in their isolation. This does not allow one to compare directly these three types of particles and to establish whether they represent the same structural elements or not. To answer this question it will be necessary to characterize the proteins constituting the cytoplasmic particles. 

The term nuclear informosomes should be used with caution. 8ince the informosomes described by 8pirin in embryonic cytoplasm seem to be well-characterized cell structures, one should compare nuclear D-RNP and informosomes before extending this term to the nuclear structures. 8ince even a comparison of the proteins of these two structures has not yet been made, for the time being we prefer to call the nuclear particles nuclear D-RNP. ... 

The isolation of informosomes. The best material for the studies of free, cytoplasmic D-RNA-containing particles are embryonic cells in early stages of development. In such cells the synthesis of ribosomal RNA is absent (Aitkhozhin et al. 1964), and all of the high molecular weight RNA that is synthesized is D-RNA. This facilitates studies of this RNA species. 

Unfortunately, it is not yet possible to purify informosomes free of contaminating ribosomes, subribosomal particles, and soluble proteins of the cytoplasm. The only useful purification method is centrifugation in a CsCl density gradient, but this requires a prefixation with formaldehyde or with other cross-linking agents (Spirin et al., 1965). The fixation makes it impossible to study adequately the proteins of particles or their activities. Another possible approach is to use free flow electrophoresis, which should differentiate ribosomes and D-RNP, but such work has only just started (Schweiger and Hannig, 1970). 

Although no direct determination of base composition has been done, some data indicate that the RNA of informosomes is D-RNA. Deproteinized RNA of the extract is heterogeneous, and its sedimentation properties are different from those of ribosomal RNA. This RNA has a very high hybridizability with homologous DNA, which is typical of D-RNA (Spirin and Nemer, 1965). It is labeled at those periods of early development when only D-RNA is synthesized. 

The sedimentation coefficients of RNA isolated from loach informosomes are 4S, 6S, lOS, 15S, 21S, 26S, 30S, and 35S. It is not possible to correlate precisely the definite classes of informosomes with definite RNA classes, but in general the higher the sedimentation coefficient of the informosome, the higher the molecular weight of the RNA isolated from the particles (Ovchinnikov et al., 1969a). More careful measurement... 

Fig. 12. Informosomes from loach embryo extracts labeled for 3 hours with uridine. Dotted lines, radioactivity solid lines, optical density. A. Sucrose density gradient centrifugation of cytoplasmic extract (22,000 rpm for 18 hours). B. Recentrifugation of the material of 73 to 62S peak in CsCI density gradient. C. The same but of 45 to 36S zone. (From Ovchinnikov et al. 1960a. Molec. Biot. (U.S.S.RJ, 3 448 3.)...

The buoyant density of informosomes from sea urchins was found to be higher, 1.50 to 1.75 g/cm, which corresponds to a much lower protein content (Infante and Nemer, 1968). It may be the result of the higher salt content in sea urchin cells (0.5 A/KCl, for example, may produce a partial dissociation of proteins from the 308 particles). Another explanation is the possible removal of some proteins from informosomes during CsCl density gradient centrifugation in the absence of formaldehyde (8pirin, 1969). 

The RNA of informosomes is sensitive to RNase, although somewhat more resistant than free D-RNA (8pirin, 1969). It is not clear whether mild RNase treatment transforms large informosomes to some definite minimal subunits as in the case of nuclear D-RNP. 

Many properties of informosomes can be studied only after isolation of the particles in a pure state. As we can see, free informosomes of the embryonic cytoplasm resemble in some respects nuclear D-RNPs. They are relatively homogeneous with respect to buoyant density, at least in the case of large informosomes there is a good correlation between the sizes of the informosomes and the RNAs isolated from them. They form a number of discrete components in the sucrose density gradient. All the above-mentioned properties may be explained in terms of a polysomelike structure. However, for such a conclusion it is necessary to have data about the presence of minimal units in informosomes, which data are unavailable at the moment. On the other hand, there are some differences between the nuclear D-RNPs and informosomes with respect to their sedimentation coefficients. The question of the relation between the two types of particles is discussed in detail in the section on The Problem of Messenger RNA Transport (p. 96). 

Normal cells. It is much more difficult to demonstrate free informosomes in the cells of adult organisms than in embryos since in adult cells there is intensive synthesis of rRNA and ribosomal precursors. Hoagland and Askonas (1963) first observed nucleoprotein particles containing RNA in rat liver cytoplasm and were able to use them to stimulate protein synthesis in a cell-free system. 

On the other hand, component B seems to correspond to informosomes. The RNA isolated from this component is rather heterogeneous and hybridizes effectively with DNA (Perry and Kelley, 1966). Thus it probably represents true D-RNA, and the B component represents a complex of D-RNA and proteins, with a very low content of RNA. The latter may, of course, reflect some degradation of RNA during isolation. It is significant that the component B is observed not only in the 45S peak of sucrose gradients, but also in other sedimentation zones. The B component s buoyant density varies between 1.34 and 1.40 g/cm. ... 

Free informosomes also have been observed in the cytoplasm of cells of insect epidermis. In contrast to embryonic informosomes, they are very heterogeneous with respect to buoyant density (Katatos, 1968). Finally, free mRNP particles similar in properties to informosomes have been described in the membrane fraction of microsomes from rat brain. These particles have a heterogeneous distribution in a sucrose density gradient and a buoyant density of about 1.40 g/cm. In contrast to nuclear complexes they are not destroyed by deoxycholate treatment (Samec et al., 1968). The nature of these particles remain unclear. 

The presence of informosomelike RNPs has been demonstrated also in the case of Ehrlich ascites carcinoma cells infected with Sendai virus (Volkova et al., 1969). The cytoplasmic extracts of the cells labeled for 30 minutes with uridine contain virus-specific RNA in the form of particles with a sedimentation coefficient 45S (the sedimentation coefficient of the complete virus equals 57S). The buoyant density of the RNP peak in CsCI equals 1.43 to 1.44 g/cm. Although these properties are compatible with the idea that they are informosome, the particles, and in particular their protein component, should be characterized in more detail before reaching a definite conclusion. Recently SOS virus RNA-containing particles with p = 1.40 g/cm have been found in HeLa cells infected with poliovirus (Huang and Baltimore, 1970), although the authors have some doubts about the reality of these complexes. 

Free Informosome-Forming Protein in the Cytoplasm and the Problem of the Preexistence of I nformosomes. 

In attempts to check the in-vivo existence of informosomes, Girard and Baltimore (1966) mixed different RNAs with the cytoplasmic supernatant and observed the interaction of RNAs with the cytoplasmic proteins. The sedimentation coefficient of the RNA increased 1.5 to 2.5 times. These data were confirmed by (Ovchinnikov et al. (1968) and Spirin (1969) with cytoplasmic fractions from rat liver and loach embryo. Thus in the cytoplasm of animal cells a special protein able to form complexes with... 

Two questions arise. First, is the informosome-forming protein (IFF) also found in cytoplasmic informosomes and nuclear D-RNPs Second, are cytoplasmic informosomes preexisting structures, or are they produced artificially by the interaction of IFF with mRNA during the preparation of the cellular extract The answer to the first question has not yet been obtained, since the isolation of pure IFF meets with a number of difficulties. The first results obtained by Baltimore and Huang (1970) indicate the possibility of separating active material into a number of fractions by chromatography on DEAE-cellulose. 

In conclusion it should be pointed out that free D-RNPs or informosomes have been found in the cytoplasm of embryonic and adult cells, but the possibility of nuclear leakage at least in the case of the cells from adult organisms has not yet been excluded. 

Many other schemes may also be drawn. To determine which scheme is correct, it will be necessary to answer the following questions l.What is the exact localization of free cytoplasmic informosomes in the cell 2. Do nuclear D-RNP, cytoplasmic informo-somes, and polysome-bound mRNP have the same nature In particular do they contain the same protein moiety or not 3. What is the nature of intermediate complexes in the process of polysome formation and where are the polysomes formed ... 

The comparison of nuclear and cytoplasmic D-RNPs. In the above sections we described the properties of different types of cellular D-RNP. If nuclear D-RNA-containing particles and free informosomes are compared one can find a number of similarities. Both kinds of particles give several discrete peaks in a sucrose density gradient, which, at least in the case of nuclear D-RNPs reflects the polysomelike structure of the complexes. 

Both of them have the same buoyant density of CsCl density gradient ultracentrifugation, i.e., 1.40 g/cm. Although the informosomes from embryonic tissues are more heterogeneous and contain more than one component, the main component has this buoyant density. Informosomes containing virus-specific RNA synthesized on DNA of vaccinia virus are very homogeneous and have a buoyant density of 1.40 g/cm. Thus, the nuclear D-RNPs and free informosomes contain the same amount of protein per unit or RNA. 

Free informosomes of the cytoplasm are complexes of D-RNA with informatin, but in contrast to nuclear D-RNP they are in more extended form. 

Free informosomes are complexes of D-RNA with another protein, and this determines the differences in conformation. 

Regulation at the translational level and the role of informosomes. Many data indicate that regulation of gene expression takes place at the translational level in eukaryotic cells and that regulation of transcription is not the only mode of control of the kinds of proteins that are produced. Classic examples of translational control are the synthesis of such proteins as fibroin in the silk glands of the silkworm or hemoglobin production in reticulocytes. In both cases the synthesis of mRNA much precedes protein formation, and for a long period of time mRNA is accumulated without involvement in protein production. In fact, the peak of protein synthesis coincides with the period of depression of RNA production (Smirnov et al., 1964 see also review of Spirin, 1966). 

One can suggest that the cell contains a pool of free informatin, synthesized in the cytoplasm, which moves to the nucleus and combines there with the chromosomal sites involved in the synthesis of D-RNA. It corresponds to informosome-forming protein of the cytoplasm. 

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