Free RNA-binding cytoplasmic proteins are detected in a labile association with polyribosomes

A special fraction of RNA-binding proteins with a non-specific affinity for RNA is present in the extracts of eukaryotic cells. Earlier these proteins were considered exclusively as a pool of free informosomal proteins. It has been shown that a significant part (about 1/3) of RNA-binding proteins is found in labile association with mono- and polyribosome mass, respectively. The labile-associated proteins dissociate from the complex with mono- and polyribosomes with an increase in the ionic RNA-binding proteins bind to particles due to the non-specific affinity for the exposed part of RNA of mono- and polyribosomes. The decrease of the ionic strength leads to the stabilization of the RNA-binding proteins-polyribosomes complexes and enables purification of these complexes. A direct comparison by the O'Farrell two-dimensional analysis has shown that practically all the proteins that are labile-associated with polyribosomes are present within the preparation of free RNA-binding proteins.     

The role of RNA-binding and ribosomal proteins as specific RNA translation regulators in cellular differentiation and carcinogenesis

Tight control of mRNA expression is required for cell differentiation; imbalanced regulation may lead to developmental disorders and cancer. The activity of the translational machinery (including ribosomes and translation factors) regulates the rate (slow or fast) of translation of encoded proteins, and the quality of these proteins highly depends on which mRNAs are available for translation. Specific RNA-binding and ribosomal proteins seem to play a key role in controlling gene expression to determine the differentiation fate of the cell. This demonstrates the important role of RNA-binding proteins, specific ribosome-binding proteins and microRNAs as key molecules in controlling the specific proteins required for the differentiation or dedifferentiation of cells. This delicate balance between specific proteins (in terms of quality and availability) and post-translational modifications occurring in the cytoplasm is crucial for cell differentiation, dedifferentiation and oncogenic potential. In this review, we report how defects in the regulation of mRNA translation can be dependent on specific proteins and can induce an imbalance between differentiation and dedifferentiation in cell fate determination.     

Expression of TAR RNA-binding protein in baculovirus and co-immunoprecipitation with insect cell protein kinase

TAR RNA-binding protein TRBP was originally isolated by its binding affinity for radiolabeled HIV-1 leader RNA. Subsequent studies have suggested that this protein is one member of a family of double-stranded RNA-binding proteins. Recent findings indicate that TRBP might function to antagonize the translational inhibitory effect that can be mediated through cellular protein kinase, PKR. Here, we report on the over-expression of a cDNA coding for TRBP in eukaryotic SF9 cells using baculovirus. We characterized the nuclear localization of TRBP in insect cells, and we demonstrate that TRBP co-immunoprecipitates with a protein in these cells antigenically related to human PKR.     

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.     

Identification of mRNA/protein (mRNP) complexes containing Puralpha,mStaufen, fragile X protein,and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.     

Translational control in cellular and developmental processes

Growing evidence indicates that translational control of specific mRNAs contributes importantly to genetic regulation across the breadth of cellular and developmental processes. Synthesis of protein from a specific mRNA can be controlled by RNA-binding proteins at the level of translational initiation and elongation, and translational control is also sometimes coupled to mRNA localization mechanisms. Recent discoveries from invertebrate and vertebrate systems have uncovered novel modes of translational regulation, have provided new insights into how specific regulators target the general translational machinery and have identified several new links between translational control and human disease.     

Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system

We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH ~7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression.     

FUS contributes to mTOR-dependent inhibition of translation

The amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)–linked RNA-binding protein called FUS (fused in sarcoma) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian target of rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation because deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants R521G and P525L associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA-binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling and that ALS-linked FUS mutants can cause a toxic gain of function in the cytoplasm by repressing the translation of mRNA at polyribosomes.     

Dynamic association of the fragile X mental retardation protein as a messenger ribonucleoprotein between microtubules and polyribosomes

The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that regulates translation and plays essential roles in synaptic function. FMRP is bound to specific mRNA ligands, actively transported into neuronal processes in a microtubule-dependent manner, and associated with polyribosomes engaged in translation elongation. However, the biochemical relationship between FMRP-microtubule association and FMRP-polyribosome association remains elusive. Here, we report that although the majority of FMRP is incorporated into elongating polyribosomes in the soluble cytoplasm, microtubule-associated FMRP is predominantly retained in translationally dormant, polyribosome-free messenger ribonucleoprotein (mRNP) complexes. Interestingly, FMRP-microtubule association is increased when mRNPs are dynamically released from polyribosomes as a result of inhibiting translation initiation. Furthermore, the I304N mutant FMRP that fails to be incorporated into polyribosomes is associated with microtubules in mRNP particles and transported into neuronal dendrites in a microtubule-dependent, 3,5-dihydroxyphenylglycine-stimulated manner with similar kinetics to that of wild-type FMRP. Hence, polyribosome-free FMRP-mRNP complexes travel on microtubules and wait for activity-dependent translational derepression at the site of function. The dual participation of FMRP in dormant mRNPs and polyribosomes suggests distinct roles of FMRP in dendritic transport and translational regulation, two distinct phases that control local protein production to accommodate synaptic plasticity.     

The archaeal origins of the eukaryotic translational system

Among the 78 eukaryotic ribosomal proteins, eleven are specific to Eukarya, 33 are common only to Archaea and Eukarya and 34 are homologous (at least in part) to those of both Bacteria and Archaea. Several other translational proteins are common only to Eukarya and Archaea (e.g., IF2a, SRP19, etc.), whereas others are shared by the three phyla (e.g., EFTu/EF1A and SRP54). Although this and other analyses strongly support an archaeal origin for a substantial fraction of the eukaryotic translational machinery, especially the ribosomal proteins, there have been numerous unique and ubiquitous additions to the eukaryotic translational system besides the 11 unique eukaryotic ribosomal proteins. These include peptide additions to most of the 67 archaeal homolog proteins, rRNA insertions, the 5.8S RNA and the Alu extension to the SRP RNA. Our comparative analysis of these and other eukaryotic features among the three different cellular phylodomains supports the idea that an archaeal translational system was most likely incorporated by means of endosymbiosis into a host cell that was neither bacterial nor archaeal in any modern sense. Phylogenetic analyses provide support for the timing of this acquisition coinciding with an ancient bottleneck in prokaryotic diversity.     

Proteins binding to 5'' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.     

Activation of a cellular tyrosine-specific protein kinase by phosphorylation

One of the 3 major RNA-binding proteins of rabbit reticulocytes, a polypeptide of 36 kDa, is identified as glyceraldehyde-3-phosphate dehydrogenase (GAPD). This fact was deduced from the identity of molecular masses, one-dimensional peptide maps and isoelectric points of the 36 kDa protein and GAPD from rabbit muscle. It is concluded that GAPD can bind rather unspecifically different RNAs and polynucleotides. This means that GAPD, like other RNA-binding proteins, can form loose dynamic complexes with polyribosomes. Association of such a kind may be used for compartmentation of glycolysis near polyribosomes.