Comparison of proteins bound to the different functional classes of messenger RNA.

Proteins from nuclear ribonucleoproteins, informosomes, polysomal messenger ribonucleoproteins and cytoplasmic "binding factor" are characterized. 1. Nuclear ribonucleoproteins are purified from nuclei disrupted by ultrasonication. Possible contamination by nucleoplasm, histones or remaining cytoplasmic structures is controlled. 2. Informosomal proteins are obtained by mild RNAase degradation. This method gives informosomal proteins without appreciable contamination. 3. Polysomal messenger ribonucleoproteins are obtained from cells where the initiation of protein synthesis is arrested in order to release the messenger ribonucleoproteins from the polysomes. Their proteins are obtained like the informosomal proteins by mild RNAase digestion. No contamination by informosomes could be detected by sodium dodecyl sulfate gel electrophoresis. 4. Cytoplasmic "binding factor" proteins are purified by affinity chromatography. 5. The four sets of proteins are analysed by sodium dodecylsulfate acrylamide gel electrophoresis. In spite of the fact that some proteins from one or another kind of messenger ribonucleoprotein, have apparently the same molecular weight, the majority of proteins differ.     

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.     

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.     

Storage of messenger RNA in eukaryotes: Envelopment with protein,translational barrier at 5′ side,or conformational masking by 3′ side?

Messenger RNA can be stored in the cytoplasm of higher Eukaryotes in the form of masked messenger ribonucleoprotein particles (masked mRNPs, or informosomes). The typical example is the storage of mRNPs in germ cells (oocytes and spermatocytes). The masked mRNPs are inactive in translation, stable, i.e., protected against degradation, and unavailable for poly(A) tail processing, such as cytoplasmic polyadenylation and deadenylation. The major nonspecific mRNA-binding protein forming mRNPs and belonging to a special p50 family of basic, glycine-rich, phosphorylatable proteins seems to be necessary, but not sufficient for the masking. In some cases, mRNA-specific repressor proteins bound to the 5′-untranslated regions (5′-UTR) of mRNAs may be involved. Interactions of the 3′-untranslated regions (3′-UTR) with sequence-specific proteins seem to be of decisive importance for the masking of mRNPs. The hypothesis is proposed that the masking is achieved through a 3′-UTR–induced conformational rearrangement of mRNP; closing into a circle and condensation of mRNP are considered plausible. © 1994 Wiley-Liss, Inc.     

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.     

Identification and isolation of casein kinase type II from RNA-binding proteins of amphibian oocytes

Protein kinase previously detected in RNA-binding proteins of amphibian oocytes phosphorylates casein far more efficiently than histones to form phosphoserine and phosphothreonine and utilizes both ATP and GTP. Heparin in concentrations below 1 microgram/ml inhibits protein kinase. This allows to relate the enzyme to casein kinases II. Protein kinase was extensively purified (more than 15000-fold) with respect to proteins of ribosome-free extract. The homogeneous enzyme consists of three polypeptide chains (Mr 43,000, 41,000, and 29,000). The 125I-labelled enzyme possessing casein kinase and RNA-binding activities when injected into amphibian oocytes was detected in the particles identical to free cytoplasmic informosomes in terms of their sedimentation properties.     

RNA-cytoskeletal associations.

It has become evident over the past years that a large fraction of messenger RNAs is tightly associated with the cytoskeleton. Whereas microtubules are involved in RNA-cytoskeletal association in large cells like oocytes, neurons, or oligodendrocytes, microfilaments play the major role in smaller somatic cell types. Association of RNA with cytoskeletal filaments clearly is required for mRNA transport, but also appears to be crucial for efficient protein synthesis. Recent data now shed light on how mRNAs attach to the cytoskeleton. Messenger RNA seems to interact with microtubules or microfilaments in the form of large ribonucleoprotein particles, which in some cases also contain components of the protein synthesis apparatus. Recently, a number of RNA binding proteins have been identified in flies, amphibians, and mammals that are essential for the interaction of mRNA with cytoskeletal filaments or with microtubule- or actin-associated proteins. Such proteins include heterologous ribonucleoproteins, which are also involved in nuclear export of 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...     

mRNP proteins,initiation factors and phosphorylation

Information has been collected to stimulate interest regarding the nature and the possible role of mRNP protein phosphorylation in a cytoplasmic control mechanism for protein synthesis. It does not imply a direct relationship between mRNP protein and initiation factors. These proteins have some properties in common (e.g. molecular weight, phosphorylation, protein kinase, mRNA binding activity). We emphasize that some free mRNP may be translatable after modification of their protein by interference factors belonging to other cellular compartments. Thus, some mRNP proteins might possess initiation factor or protein synthetic activity if we accept Spirin's theory, i.e., Eukaryotic messenger RNA and informosomes omnia mea mecum porto     

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.     

The mitochondrial protein targeting suppressor (mts1) mutation maps to the mRNA-binding domain of Npl3p and affects translation on cytoplasmic polysomes

In all eukaryotic organisms, messenger RNA (mRNA) is synthesized in the nucleus and then exported to the cytoplasm for translation. The export reaction requires the concerted action of a large number of protein components, including a set of shuttle proteins that can exit and re-enter the nucleus through the nuclear pore complex. Here, we show that, in Saccharomyces cerevisiae, the shuttle protein Npl3p leaves the nuclear pore complex entirely and continues to function in the cytoplasm. A mutation at position 219 in its RNA-binding domain leaves Npl3p lingering in the cytoplasm associated with polysomes. Yeast cells expressing the mutant Npl3(L-219S) protein show alterations in mRNA stability that can affect protein synthesis. As a result, defects in nascent polypeptide targeting to subcellular compartments such as the mitochondria are also suppressed.     

The nuclear nurture and cytoplasmic nature of localized mRNPs

From yeast to mammals, evidence has emerged in recent years highlighting the essential role played by the nuclear "history" of a messenger RNA in determining its cytoplasmic fate. mRNA localization, translation and stability in the cytoplasm are often pre-destined in the nucleus, and directed by the composition and architecture of nuclear assembled mRNA-protein complexes. In this review we focus on nuclear-acquired RNA-binding proteins and complexes that participate in determining the journey of localized mRNAs.     

Characterization of a messenger RNA transport protein

A cytoplasmic protein which facilitates the energy-dependent transport of mRNA from isolated nuclei to a specified medium has been further characterized, since it could have relevance to the mechanism of mRNA nucleo-cytoplasmic transport in vivo. This protein is now shown, by cDNA hybridization analysis using appropriate recombinant probes, to be obligatory for the transport of alpha 2u-globulin and albumin mRNA from male rat liver nuclei. It is concentrated in the cytoplasm. When isolated under conditions where they retain nuclear proteins, the nuclei contain less than 2% of the total mRNA transport activity. Approx. 20% is recovered in the cytosol, while the rest (80%) copurifies with the messenger ribonucleoproteins in the polyribosome fraction. The protein is eluted from the poly A-messenger ribonucleoproteins between 0.25 and 0.50 M NaCl. The activities of the cytosolic- and messenger ribonucleoprotein-derived transport proteins were mutually additive below saturation of the transport system. Further, the activities of both fractions were increased when they were fortified with the catalytic subunit of the cAMP-dependent protein kinase in the presence of ATP. On the other hand, protein kinase-induced thiophosphorylation of the protein with ATP[S] decreased transport activity. The molecular weight of the transport protein from either cell compartment as judged by molecular sieving is approx. 35,000. It has now been purified 2000-fold and requires manganese ions and serum albumin for stabilization of activity. The highly purified transport factor from the cytosol is tentatively assigned a molecular weight of 32,000 by SDS-polyacrylamide gel electrophoresis.     

Polysome-associated proteins in herpes simplex virus-infected cells

In the cytoplasm of eucaryotic cells, mRNA is associated with proteins. These mRNA-protein complexes, termed messenger ribonucleoprotein (mRNP) particles, are divided into two functional classes. The first class contains free (non-ribosome-associated) mRNPs which have been termed informosomes by others. The second class of mRNPs, those associated with polysomes, are actively engaged in protein synthesis and are termed polysomal mRNPs. The experiments described in this paper examined the proteins associated with polyribosomes in uninfected and herpes simplex virus type 1-infected cells. The data indicate that after infection with herpes simplex virus type 1, specific changes occur in the proteins which normally are found associated with these polysomal mRNPs. These changes include both the appearance of new and possibly virus-specific proteins and the loss of normal host-specific proteins. The relationship of these changes to the patterns of protein synthesis in these cells is also discussed.     

Assembly and nuclear export of pre-ribosomal particles in budding yeast

The ribosome is responsible for the final step of decoding genetic information into proteins. Therefore, correct assembly of ribosomes is a fundamental task for all living cells. In eukaryotes, the construction of the ribosome which begins in the nucleolus requires coordinated efforts of >350 specialized factors that associate with pre-ribosomal particles at distinct stages to perform specific assembly steps. On their way through the nucleus, diverse energy-consuming enzymes are thought to release assembly factors from maturing pre-ribosomal particles after accomplishing their task(s). Subsequently, recruitment of export factors prepares pre-ribosomal particles for transport through nuclear pore complexes. Pre-ribosomes are exported into the cytoplasm in a functionally inactive state, where they undergo final maturation before initiating translation. Accumulating evidence indicates a tight coupling between nuclear export, cytoplasmic maturation, and final proofreading of the ribosome. In this review, we summarize our current understanding of nuclear export of pre-ribosomal subunits and cytoplasmic maturation steps that render pre-ribosomal subunits translation-competent.