Small cytoplasmic poly(A)+RNA, homologous to the B2 repetitive sequence of the mouse genome, is present in ribonucleoproteins

Small cytoplasmic poly(A) + RNA homologous to a highly repeated sequence B2 of the mouse genome (scB2 RNA) was not found as free RNA within a cell. This RNA is bound to small (12-18S) ribonucleoprotein particles as well as to heavy RNP particles, apparently informosomes. After deproteinization of the heavy RNP the major part of scB2 RNA molecules cosedimented with heavy RNAs. It seems that scB2 RNA is associated with mRNA in informosomes via short complementary regions. About half of the scB2 RNA molecules was revealed in the cytoskeleton fraction. The possibility that scB2 RNAs are involved in mRNA transport or in the regulation of mRNA translation is discussed.     

Properties of informosomes fixed by ultraviolet irradiation

The conditions for UV irradiation (lambda = 254 nm) of ribonucleoprotein preparations containing informosomes were elaborated which provide for complete fixation of informosomal proteins on RNA. The degree of fixation was controlled by centrifugation in a CsCl density gradient. It was found that complete fixation of informosomes from loach embryos was achieved by irradiation with greater than or equal to 250 quanta per nucleotide. Fixation of informosomes isolated from Krebs ascite carcinoma II was observed during irradiation with higher (720 quanta per nucleotide) doses. It was shown that the irradiation doses used do not cause the destruction of ribonucleoprotein particles of RNA degradation. No fixation of ribosomal particles was observed during irradiation with the above doses. The method described can be used for the isolation of informosomes with the use of density gradients for the analysis of polypeptide composition of these particles.     

RNA-binding proteins of Rana temporaria oocytes: incorporation into informosomes of oocytes in vivo]

The participation of RNA-binding protein in the formation of informosomes in vivo was studied using an intracellular microinjection technique. The RNA-binding protein of the frog Rana temporaria oocytes was isolated by affinity chromatography and was labelled in vitro without any loss of its activity. It was shown that during cultivation of the oocytes the specific incorporation of the injected RNA -- binding [3H]-protein into the ribonucleoprotein particles occurred. These particles were further described as informosomes, characteristic ribonucleoprotein particles of animal cells.     

Informosome formation in maturing amphibian oocytes

[14C]Uridine and/or RNA-binding [3H]protein preparations were microinjected into the oocytes of Rana temporaria frog. It was shown that both labelled compounds are incorporated into RNP particles with the buoyant density in CsCl characteristic of informosomes and with a homogeneous sedimentation distribution in sucrose gradients. Injection of actinomycin D into recipient oocyte leads to inhibition of synthesis of the informosome RNA component as well as to the incorporation of RNA-binding [3H] proteins into free informosomes. The results obtained provide experimental proofs for the assumption that the RNA-binding proteins under study form complexes predominantly with newly synthesized RNA.     

Informosomes Travel in Time: An Early mRNA Concept in the Current mRNP Landscape

Biochemistry (Moscow) - Messenger RNA is complexed with proteins throughout its life cycle. The first mRNA-containing particles of non-ribosomal nature, named informosomes, were discovered in...     

Inhibition of casein kinase type 2 activity by formation of complexes with mRNA in vivo

It is known that casein kinase 2 possesses, besides the protein kinase, an RNA-binding activity. Using ligand blotting it has been demonstrated that the both activities are localized on the alpha- and alpha'-subunits of the enzyme. Casein kinase 2 is suppressed in vitro by polyuridylic acid. A part of the intracellular pool of casein kinase 2 is found in the informosomes. The informosomes and free proteins were separated by centrifugation in a sucrose density gradient, and each fraction was incubated with casein and [gamma-32P]ATP. The informosome-bound protein kinase is completely inhibited, while the free protein kinases heavily phosphorylate casein. It is concluded that the activity of casein kinase 2 can be regulated by the reversible formation of complexes with RNA.     

RNA isolation from the ribonucleoproteins fixed with formaldehyde

A method for isolating RNA from the polyribosomes and informosomes fixed with formaldehyde was developed. The ribonucleoproteins were obtained by centrifugation in CsCl density gradient. It has been demonstrated that this method makes it possible to obtain full-sized rRNA and mRNA appropriate for molecular hybridization. We succeeded in amplifying 150-nucleotide sequences of individual mRNA and demonstrating the applicability of these RNA preparations for synthesis of labeled probes for RNA arrays. The method proposed is recommended for the search for untranslated mRNA and the study of the changes in translation of individual proteins during early ontogenesis and various pathologies.     

Specific template activity of ribonucleoprotein particles isolated from germinating Triticum aestivum embryo

A ribonucleoprotein (RNP) complex formed in wheat embryo at early germination stage was shown to be composed of 40-S RNP particle monomers and oligomers. RNA purified from this complex stimulated incorporation in vitro of various amino acids with efficiences dependent upon the frequencies of the corresponding codons calculated for this RNA from its base composition. Methionine was incorporated over the expected rate when the crude RNP complex, instead of the purified RNA, was used as the template. It is believed that the RNP complex represents a crude informosome fraction. The informosomes seem to contain a protein component that promotes the initiation of translation but does not involve the subsequent production of protein.     

Distribution of elongation factors EF-1 and EF-2 among components of rabbit reticulocyte lysate

The distribution of activity of the elongation factors EF-1 and EF-2 among the components of rabbit reticulocyte lysate separated by sucrose density gradient centrifugation was studied. At low ionic strength (0.01 M KCl) about 30% of the EF-1 activity was found in polyribosomes. At moderate ionic strength (0.1 M KCl) the EF-1 activity was absent in the polyribosomes. An addition of RNA excess to the lysate prior to centrifugation at low ionic strength resulted in elimination of the EF-1 activity from the polyribosomes. This indicates that EF-1 is reversibly bound to the polyribosomes and that EF-1 may be retained on them due to interaction with RNA of polysomes mediated by its RNA-binding site. After dissociation of polyribosomes containing EF-1 in the presence of EDTA and subsequent fractionation of the dissociation products at low ionic strength (0.01 M KCl) the EF-1 activity was revealed in the ribosomal subparticles (predominantly in 60S). At 0.1 M KCl EF-1 mainly sedimented in the zone of distribution of polyribosomal informosomes. The elongation factor EF-2 was not revealed in polyribosomes during lysate centrifugation even at low ionic strength which corresponds to its lower affinity for RNA.     

hnRNP particles

This article describes the discovery of nuclear DNA-like RNA (dRNA or hnRNA) and ribonucleoprotein particles in eukaryotes. Native hnRNA particles were isolated by sucrose gradient sedimentation and their structural organisation – nucleic acid (i.e. RNA) wrapped in a regular way on the surface of a series of globular protein particles – was determined. This led to the formulation of the informofer cycle hypothesis for the synthesis of hnRNA as a giant precursor molecule, its transport in informosomes within the nucleus, and subsequent splicing before export from the nucleus as free mRNA.     

Monoclonal antibodies against polyribosome informosomes from rabbit reticulocytes

Monoclonal antibodies to proteins of polyribosome-bound informosomes from rabbit reticulocytes were obtained. Using the immunoblotting technique, it was shown that antibodies produced by one of the clones react with several polypeptides of polyribosome-bound informosomes.     

Phosphorylation of cytoplasmic ribonucleoproteins in guinea pig adrenal cells. Effect of ACTH

Phosphorylation of proteins bound with cytoplasmic ribonucleoproteins (RNP) of guinea pig adrenal cells activated by ACTH was studied. Incorporation of 32P into ribosomes and mRNP (informosomes) in check samples is not high. Phosphorylation of ribosomes, informosomes and free proteins which have separated from ribonucleoproteins sharply grow after corticotropin addition. Specificity of translation in the activated adrenal cells is supposed to be related to mRNP phosphorylation.     

Messenger ribonucleoproteins (informosomes) and RNA-binding proteins

Messenger ribonucleoproteins, first discovered in 1964 in our laboratory as free mRNA-containing particles of fish embryo cytoplasm and designated as informosomes, proved to have a universal occurrence in eukaryotic cells. Messenger ribonucleoproteins of different intracellular localization such as free cytoplasmic non-translatable informosomes, translatable messenger ribonucleoproteins in polyribosomes and nuclear pre-mRNA-containing particles are characterized by a number of features common for all of them. However, the transport from the nucleus into the cytoplasm as well as the transition from the free non-translatable state into the polyribosome-bound translatable state are accompanied by essential changes in the protein moiety of the particles. The existance of free RNA-binding proteins in eukaryotic cells has also been shown. These proteins seem to represent a pool for the formation of messenger ribonucleoproteins (informosomes).It has recently been demonstrated that the eukaryotic translation factors and, in particular, both the elongation factors and some initiation factors are among the cytoplasmic RNA-binding proteis. It is suggested that the mRNA in eukaryotic cells at different stages of its life time carries on itself the proteins which are required for its own biogenesis, processing and transport (nuclear informosomes), for its existence in a temporarily inactive state (free cytoplasmic informosomes) and for its functioning as a template (polyribosomal informosomes):omnia mea mecum porto.     

Transition of Xwnt-11 mRNA from inactive form to polyribosomes in frogs during early embryogenesis

The distribution of Xwnt-11 mRNA between polysomes and informosomes was studied in Xenopus laevis and Rana temporaria during early embryogenesis. The ratio between polysomes and informosomes suggests their involvement in translation of these mRNAs. In eggs and immediately after fertilization the Xwnt-11 mRNAs are mostly positioned in informosomes. During the cleavage stage, these mRNAs have also been recognized in polysomes. Just before the onset of zygote genome functioning (at the stage of mid blastula), Xwnt-11 mRNA rapidly appears in polysomes of Rana embryos. However, in Xenopus, Xwnt-11 mRNA appears in polysomes only at the end of gastrula. Before this stage, the Xwnt-11 mRNA in Xenopus can be found mostly in informosomes.     

Comparison of cytoplasmic informosome proteins with RNA-binding proteins and translation initiation factors from rabbit reticulocytes.

Using one-and two-dimensional electrophoresis, the free and polyribosomal informosome proteins and a preparation of total RNA-binding proteins from rabbit reticulocytes were compared. It was shown that the major proteins of free and polyribosomal informosomes are similar only to the minor components of RNA-binding proteins. On the other hand, the major RNA-binding proteins, two of which are elongation translation factors EF-1L and EF2, can be present in informosome preparations only as minor components. The major proteins of polyribosomal informosomes do not coincide in terms of electrophoretic mobility with initiation factors eIF-2, eIF-2A, eIF-3, eIF-4A and eIF-4B. The major proteins of free informosomes differ in their electrophoretic mobility from initiation factors eIF-2A, eIF-4A and eIF-4B as well as from the alpha- and beta-subunits of initiation factor eIF-2.     

Poly(A)-containing RNA from germinating wheat embryos

Rapidly labelled mRNAs were isolated from informosomes and polyribosomes of imbibed wheat embryos. The distribution of poly(A) sequences in these fractions were studied by poly(U) Sepharose chromatography. It was shown that informosomes contain 11% polyadenylated mRNA while polyribosomes--38%. This fact suggests the important role of poly(A) sequences for translation of mRNA.     

The effect of cytisine on mRNA transport in the cytoplasm of eukaryotic cells (plants and animals)

The action of alkaloid cytisine on protein biosynthesis in eukaryotic cells was studied. It was shown that the alkaloid had no effect on mRNA translation. Cytisine inhibited the release of mRNP particles from rat liver and wheat embryos nuclei. Sedimentation properties and distribution in CsCl gradient of the material extracted from alkaloid-treated nuclei did not differ from control one and were similar to informosomes from animal and plant cells described earlier. The main part of mRNA with sedimentation coefficients 14-18S, capable for translation in the cytoplasm is retained in alkaloid-treated nuclei.     

Glutaraldehyde and glyoxal fixation of ribonucleoproteins for their analysis by cesium chloride equilibrium gradient centrifugation]

Conditions for fixation of different RNP (ribosomes, poliribosomes, informosomes) by glutaraldehyde and glyoxal for their subsequent analysis in CsCl density-gradient has been developed. Higher dialdehyde concentration and longer incubation time should be used for fixation of ribosomes and polyribosomes than for that of informosomes. For the fixation of all RNP studied their incubation with 0.01 M (0.1%) glutaraldehyde for several minutes is sufficient. Much higher concentration of the fixating agent (about 0.2-0.5 M i. e. 1-3%) and more prolonged time of incubation (in order of several 10 hours) are needed for the fixation of the RNP in the case of glyoxal. Conditions for selective aldehyde fixation of informosomes in the presence of ribosomes and polyribosomes has been developed.