[Major mRNP proteins in the structural organization and function of mRNA in eukaryotic cells]. / Mazhornye belki mRNP v strukturnoi organizatsii i funktsionirovanii mRNK v kletkakh éukariot.

The properties of two universal major proteins of cytoplasmic mRNP, p50 and the poly(A)-binding protein (PABP), are summarized. Their roles in formation of polyribosomal and free inactive mRNP are considered, with the focus on the authors' studies of p50. The parts these mRNP proteins play in translation regulation, stability, and localization of mRNA are described, and the the possible mechanisms of their function are discussed.

Interaction of the universal mRNA-binding protein, p50, with actin: a possible link between mRNA and microfilaments.

We have shown previously that p50 is the most abundant protein associated with a variety of eukaryotic mRNAs and exhibits about 98% amino acid sequence identity to mammalian Y-box binding transcription factors. The dual function of p50 in the cell as a regulator of both transcription and translation has been suggested. To gain insight into the role of p50 in these processes, we performed the yeast two-hybrid screen to identify p50 molecular partners. Here we report the identification of actin as a p50-interacting protein. Coimmunoprecipitation of p50 and actin from HeLa extracts as well as in vitro binding studies indicate specificity and a high affinity for the interaction between p50 and actin. Interestingly, p50 binding to actin is affected by mRNA; binding was observed at a low p50/mRNA ratio and was greatly reduced at higher ratios. Since the p50/mRNA ratio appears to be important for mRNA translatability, we speculate that p50 can regulate the attachment of mRNA to the actin network depending on its translational activity. Using immunofluorescence, we show that p50 binds to actin filaments in permeabilized cells and causes actin fibers to bundle in vitro. Together, these findings support the view that p50 may play an important role in mRNA transport, anchoring, and localization on actin filaments in the cell.

Translational regulation by Y-box transcription factor: involvement of the major mRNA-associated protein, p50.

p50, the major core protein of messenger ribonucleoprotein particles (mRNPs), is a universal protein found exclusively in association with different mRNA species in the cytoplasm of somatic mammalian cells. Furthermore, p50 is the most abundant and tightly bound protein within both inactive mRNPs and active mRNPs derived from polysomes, although the latter contain a lower level of p50. Recent experiments have revealed that, depending on the p50 to mRNA ratio, p50 may either act as a repressor or an activator of protein synthesis. On the other hand, p50 exhibits about 98% amino acid sequence identity to mammalian transcription factors that bind specifically to Y-box containing DNA. Thus, it is a counterpart of the Y-box binding proteins which are found in bacteria, plants and animals, exhibiting multiple biological activities ranging from transcriptional regulation of a wide variety of genes to 'masking' mRNA activity in germinal cells. This review summarizes our current knowledge of p50 structure and function. It also discusses the biological roles of p50 and related proteins in gene expression and describes the likely mechanisms of their action.

The major core protein of messenger ribonucleoprotein particles (p50) promotes initiation of protein biosynthesis in vitro.

The major core protein of cytoplasmic messenger ribonucleoprotein particles (p50) has been shown previously to inhibit protein synthesis in vitro and in vivo. Furthermore, p50 is highly homologous to the Y-box-binding transcription factor family of proteins, binds DNA containing the Y-box motif, and thus may have a dual function in cells as a regulator of both transcription and translation. Here we show that binding or removal of p50 from rabbit reticulocyte lysate by monospecific antibodies to p50 strongly inhibits translation of endogenous and exogenous globin mRNAs as well as prokaryotic beta-galactosidase mRNA in a rabbit reticulocyte cell-free system. Thus, depending on the conditions, p50 not only may act as a translational repressor, but may also be required for protein synthesis. Translation inhibition with anti-p50 antibodies is not a result of mRNA degradation or its functional inactivation. The inhibition does not change the ribosome transit time, and therefore, it does not affect elongation/termination of polypeptide chains. The inhibition with anti-p50 antibodies is followed by a decay of polysomes and accumulation of the 48 S preinitiation complex. These results suggest that p50 participates in initiation of protein biosynthesis. Although uninvolved in the formation of the 48 S preinitiation complex, p50 is necessary either for attachment of the 60 S ribosomal subunit or for previous 5'-untranslated region scanning by the 43 S preinitiation complex.

Overexpression in COS cells of p50, the major core protein associated with mRNA, results in translation inhibition.

p50, the major core protein of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm of somatic mammalian cells, has been characterized previously as a member of the Y-box binding transcription factor family of proteins (YB-protein) by both high structural homology and ability to bind specifically the Y-box sequence in double-stranded DNA. YB proteins are present in a whole range of cell types and some have been identified as germ-specific cytoplasmic proteins masking stored mRNA from translation. Western blot analysis of the distribution of p50 in subcellular fractions of COS-1 cells shows that p50 is a cytoplasmic protein quantitatively associated with mRNA, both in polyribosomes and in free mRNPs. The level of p50 in COS-1 cells determined by Western immunoblotting is 0.10% of total protein, which is nearly equimolar to that of ribosomes and is approximately 5-10-fold higher than the mRNA level. Transient transfection of COS-1 cells with a p50-expressing vector results in a dramatic inhibition of protein synthesis. A control transfection with a vector expressing a frameshift mutant of p50 does not cause translation inhibition. Therefore the increase in p50 protein level is responsible for the inhibitory effect in these cells.

[The major cytoplasmic mRNP protein, p50, is required for efficient mRNA translation in vitro]. / Mazhornyi belok tsitoplazmaticheskikh mRNP, p50, neobkhodim dlia éffektivnoi transliatsii mRNK in vitro.

The major cytoplasmic mRNP protein of somatic cells, p50, is the member of the Y-box-binding transcription factor family and can control gene expression at two levels including mRNA transcription and translation. It has been demonstrated that p50 is responsible for the inactive state of mRNA within free mRNPs. In this work, we show that the Y-box-containing DNA (Y-DNA) predominantly binds to p50 in rabbit reticulocyte lysates and causes translation inhibition of the endogenous and exogenous globin mRNA and prokaryotic beta-galactosidase mRNA. Preincubation of Y-DNA with purified p50 prevents the inhibition. Inhibition of protein biosynthesis by the Y-DNA is not due to the degradation or functional inactivation of mRNA. The inhibition is associated with the decay of polyribosomes and dissociation of a newly synthesized protein from the ribosomes. The data indicate that Y-DNA inhibits protein biosynthesis predominantly at the initiation stage and that p50 is an essential component of the translation initiation apparatus.

[Major p50 protein of the somatic cell cytoplasmic mRNP: expression in Escherichia coli, isolation, and some properties of the recombinant protein]. / Mazhornyi belok p50 tsitoplazmaticheskikh mRNP somaticheskikh kletok: ékspressiia v Escherichia coli, vydelenie i nekotorye svoistva rekombinantnogo belka.

The major mRNP protein of rabbit reticulocytes, p50, belonging to the family of Y-box transcription factors has been expressed in E. coli. The isolation procedure of the recombinant protein has been described. The recombinant protein is similar to the protein isolated from rabbit mRNPs in SDS-PAGE mobility and interaction with specific antibodies. Similar to the natural protein, the recombinant protein forms homooligomeric complexes with a molecular mass of about 800 kDa, binds to the alpha-globin RNA and double-stranded DNA containing the Y-box. Both proteins can be phosphorylated in vitro.

The major protein of messenger ribonucleoprotein particles in somatic cells is a member of the Y-box binding transcription factor family.

A cDNA encoding the major core protein, p50, of cytoplasmic messenger ribonucleoprotein particles (mRNPs) of somatic cells was cloned from a rabbit reticulocyte cDNA library. From the derived 324-amino acid sequence, p50 is identified as a member of the Y-box binding transcription factor family. The protein was earlier described as a repressor of globin mRNA translation. These findings suggest that p50 may affect protein biosynthesis at two levels: mRNA transcription in the nucleus and mRNA translation in the cytoplasm. Together with recently published results showing that masked mRNA in germ cells also is associated with proteins of the Y-box binding protein family, the present finding indicates that these proteins are universal core proteins responsible for the formation of cytoplasmic mRNPs in eukaryotes. Highly purified p50 forms large 18 S homomultimeric complexes with a molecular mass of about 800 kilodaltons and melts RNA secondary structure. This suggests that p50 may affect translation by changing the overall structure of the mRNA.

Evidence for the appearance of a reticulocyte population low in lipoxygenase mRNA during the recovery from a phenylhydrazine-induced anemia in rabbits.

It is shown that during recovery from a phenylhydrazine-induced anemia in rabbits a selective decrease in lipoxygenase mRNA takes place with a corresponding shut-off of the synthesis of the enzyme. It is suggested that a new population, 'recovery'-reticulocytes, makes its appearance in the peripheral blood. Their cells are more mature than the stress macroreticulocytes. A cell-free system prepared from the recovery-reticulocytes exhibits low endogenous synthesis of non-globin polypeptides, even without nuclease treatment, but retains full capacity to be stimulated by exogenous mRNA.