A testis cytoplasmic RNA-binding protein that has the properties of a translational repressor.

Translation of the mouse protamine 1 (Prm-1) mRNA is repressed for several days during male germ cell differentiation. With the hope of cloning genes that regulate the translational repression of Prm-1, we screened male germ cell cDNA expression libraries with the 3' untranslated region of the Prm-1 RNA. From this screen we obtained two independent clones that encode Prbp, a Prm-1 RNA-binding protein. Prbp contains two copies of a double-stranded-RNA-binding domain. In vitro, the protein binds to a portion of the Prm-1 3' untranslated region previously shown to be sufficient for translational repression in transgenic mice, as well as to poly(I). poly(C). Prbp protein is present in multiple forms in cytoplasmic extracts prepared from wild-type mouse testes and is absent from testes of germ cell-deficient mouse mutants, suggesting that Prbp is restricted to the germ cells of the testis. Immunocytochemical localization confirmed that Prbp is present in the cytoplasmic compartment of late-stage meiotic cells and haploid round spermatids. Recombinant Prbp protein inhibits the translation of multiple mRNAs in a wheat germ lysate, suggesting that Prbp acts to repress translation in round spermatids. While this protein lacks complete specificity for Prm-1-containing RNAs in vitro, the properties of Prbp are consistent with it acting as a general repressor of translation.     

The chloroplast protein disulfide isomerase RB60 reacts with a regulatory disulfide of the RNA-binding protein RB47

Biochemical studies have identified two proteins, RB47 and RB60, that are involved in the light-regulated translation of the psbA mRNA in the chloroplast of the unicellular alga Chlamydomonas reinhardtii. RB47, a member of the eukaryotic poly(A)-binding protein family, binds directly to the 5' untranslated region of the mRNA, whereas RB60, a protein disulfide isomerase (PDI), is thought to bind to RB47 and to modulate its activity via redox and phosphorylation events. Our present studies show that RB47 forms a single disulfide bridge that most probably involves Cys143 and Cys259. We found that RB60 reacts with high selectivity with the disulfide of RB47, suggesting that the redox states of these two redox partners are coupled. Kinetics analysis indicated that RB47 contains two fast reacting cysteines, of which at least one is sensitive to changes in pH conditions. The results support the notion that light controls the redox regulation of RB47 function via the coupling of RB47 and RB60 redox states, and suggest that light-induced changes in stromal pH might contribute to the regulation.     

RNA-binding properties of a translational activator, the adenovirus L4 100-kilodalton protein.

The adenovirus L4 100-kDa nonstructural protein (100K protein) is required for efficient initiation of translation of viral late mRNA species during the late mRNA species during the late phase of infection (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). The RNA-binding properties of this protein were analyzed in an immunoprecipitation assay with the 100K-specific monoclonal antibody 2100K-1 (C. L. Cepko and P. A. Sharp, Virology 129:137-154, 1983). Coprecipitation of the 100K protein and 3H-infected cell RNA was demonstrated. The RNA-binding activity of the 100K protein was inhibited by single-stranded DNA but not by double-stranded DNA, double-stranded RNA, or tRNA. Competition assays were used to investigate the specificity with which the 100K protein binds to RNA in vitro. Although the protein exhibited a strong preference for the ribohomopolymer poly(U) or poly(G), no specific binding to viral mRNA species could be detected; uninfected or adenovirus type 5-infected HeLa cell poly(A)-containing and poly(A)-lacking RNAs were all effective inhibitors of binding of the protein to viral late mRNA. Similar results were obtained when the binding of the 100K protein to a single, in vitro-synthesized L2 mRNA was assessed. The poly(U)-binding activity of the 100K protein was used to compare the RNA-binding properties of the 100K protein prepared from cells infected by adenovirus type 5 and the H5ts1 mutant (B. W. Hayes, G. C. Telling, M. M. Myat, J. F. Williams, and S. J. Flint, J. Virol. 64:2732-2742, 1990). A temperature-dependent decrease in H5ts1 100K protein binding was observed, correlating with the impaired translational function of this protein in vivo. By contrast, wild-type 100K protein RNA binding was unaffected by temperature. These data suggest that the 100K protein acts to increase the translational efficiency of viral late mRNA species by a mechanism that involves binding to RNA.     

RegA proteins from phage T4 and RB69 have conserved helix-loop groove RNA binding motifs but different RNA binding specificities

The RegA proteins from the bacteriophage T4 and RB69 are translational repressors that control the expression of multiple phage mRNAs. RegA proteins from the two phages share 78% sequence identity; however, in vivo expression studies have suggested that the RB69 RegA protein binds target RNAs with a higher affinity than T4 RegA protein. To study the RNA binding properties of T4 and RB69 RegA proteins more directly, the binding sites of RB69 RegA protein on synthetic RNAs corresponding to the translation initiation region of two RB69 target genes were mapped by RNase protection assays. These assays revealed that RB69 RegA protein protects nucleotides –9 to –3 (relative to the start codon) on RB69 gene 44, which contains the sequence GAAAAUU. On RB69 gene 45, the protected site (nucleotides –8 to –3) contains a similar purine-rich sequence: GAAAUA. Interestingly, T4 RegA protein protected the same nucleotides on these RNAs. To examine the specificity of RNA binding, quantitative RNA gel shift assays were performed with synthetic RNAs corresponding to recognition elements (REs) in three T4 and three RB69 mRNAs. Comparative gel shift assays demonstrated that RB69 RegA protein has an ~7-fold higher affinity for T4 gene 44 RE RNA than T4 RegA protein. RB69 RegA protein also binds RB69 gene 44 RE RNA with a 4-fold higher affinity than T4 RegA protein. On the other hand, T4 RegA exhibited a higher affinity than RB69 RegA protein for RB69 gene 45 RE RNA. With respect to their affinities for cognate RNAs, both RegA proteins exhibited the following hierarchy of affinities: gene 44 > gene 45 > regA. Interestingly, T4 RegA exhibited the highest affinity towards RB69 gene 45 RE RNA, whereas RB69 RegA protein had the highest affinity for T4 gene 44 RE RNA. The helix–loop groove RNA binding motif of T4 RegA protein is fully conserved in RB69 RegA protein. However, homology modeling of the structure of RB69 RegA protein reveals that the divergent residues are clustered in two areas of the surface, and that there are two large areas of high conservation near the helix–loop groove, which may also play a role in RNA binding.     

RNA determinants of translational operator recognition by the DNA polymerases of bacteriophages T4 and RB69

The DNA polymerases (gp43s) of the two related phages T4 and RB69 are DNA-binding proteins that also function as mRNA-binding autogenous translational repressors. As repressors, T4 gp43 is narrowly specific to its own mRNA whereas RB69 gp43 is equally effective against mRNA for either protein. We used in vitro RNase-sensitivity and RNA footprinting assays to identify features of the non-identical T4 and RB69 mRNA targets (translational operators) that allow for their identical binding affinities and biological responses to RB69 gp43. We observed that T4 gp43 and RB69 gp43 produce identical footprints on RNA substrates bearing the T4-derived operator, suggesting that the two gp43s make identical contacts with this operator. In contrast, the footprint produced by RB69 gp43 on its autogenous RNA target was shorter than its footprint on operator RNA from T4. As expected, we also observed only weak protection of RB69-derived operator RNA from RNase by T4 gp43; however, photocross-linking studies suggested that T4 gp43 recognizes structural features of the RB69-derived operator that are not detected by RNase- sensitivity assays. The results suggest that RB69 gp43 and T4 gp43 differ in their abilities to use RNA-sequence-independent interactions to configure potential RNA targets for translational repression.     

RNA-binding properties and translation repression in vitro by germ cell-specific MSY2 protein

The large amount of MSY2 protein, a mouse germ cell-specific Y-box protein, in oocytes and its degradation by the late two-cell stage suggest that MSY2 may stabilize and/or regulate the translation of maternal mRNAs. We report here the ability of bacterially expressed recombinant MSY2 protein to bind to mRNA and repress translation in vitro. Although MSY2 displays some sequence specificity in binding to short RNA sequences derived from the 3' untranslated region (UTR) of the protamine 1 (Prm1) mRNA, as determined by both gel shift and filter binding assays, essentially no sequence specificity is observed when full-length Prm1 mRNA is used. The binding of MSY2 is approximately 10-fold greater to the full-length Prm1 mRNA than to a 37-nucleotide sequence derived from the 3' UTR, and gel shift assays indicate that multiple MSY2 molecules bind to a single Prm1 mRNA. MSY2 binding to luciferase mRNA at ratios of protein to mRNA that are likely to exist in the oocyte also leads to a moderate inhibition of protein synthesis in vitro. Given the abundance of MSY2 in mouse oocytes (2% of total oocyte protein), these data suggest that MSY2 packages mRNAs in vivo with relatively little sequence specificity, which may lead to both stabilization and translation repression of maternal mRNAs.     

The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor

The cold-inducible RNA-binding protein (CIRP) is a nuclear 18-kDa protein consisting of an amino-terminal RNA Recognition Motif (RRM) and a carboxyl-terminal domain containing several RGG motifs. First characterized for its overexpression upon cold shock, CIRP is also induced by stresses such as UV irradiation and hypoxia. Here, we investigated the expression as well as the subcellular localization of CIRP in response to other stress conditions. We demonstrate that oxidative stress leads to the migration of CIRP to stress granules (SGs) without alteration of expression. Stress granules are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. Relocalization of CIRP into SGs also occurs upon other cytoplasmic stresses (osmotic pressure or heat shock) as well as in response to stresses of the endoplasmic reticulum. CIRP migration into SGs is independent from TIA-1 which has been previously reported to be a general mediator of SG formation, thereby suggesting the existence of multiple pathways leading to SG formation. Moreover, deletion mutants revealed that both RGG and RRM domains can independently promote CIRP migration into SGs. However, the methylation of arginine residues in the RGG domain is necessary for CIRP to exit the nucleus to be further recruited into SGs. By RNA-tethering experiments, we also show that CIRP down-regulates mRNA translation and that this activity is carried by the carboxyl-terminal RG-enriched domain. Altogether, our findings further reveal the diversity of mechanisms by which CIRP is regulated by environmental stresses and provide new insights into CIRP cytoplasmic function.     

Unusual mRNA pseudoknot structure is recognized by a protein translational repressor

Translation of ribosomal proteins in the alpha operon of E. coli is repressed by one of the encoded proteins, S4; it specifically recognizes an RNA fragment containing the translational initiation site for the first gene in the operon. RNA structure mapping experiments have suggested a pseudoknot structure for the S4 binding site: the loop of a hairpin is base paired to sequences downstream of the hairpin. Here, we systematically test this proposed structure by measuring S4 binding to an extensive set of site-directed mutations that create compensatory base pair changes in potential helices. The pseudoknot folding is confirmed, and two additional, unexpected interactions within the pseudoknot are also detected. The overall structure is an unusual "double pseudoknot" linking a hairpin upstream of the ribosome binding site with sequences 2-10 codons downstream of the initiation codon. Stabilization of this structure by S4 could account for translational repression.     

Biochemical characterization of interactions between DNA polymerase and single-stranded DNA-binding protein in bacteriophage RB69

The organization and proper assembly of proteins to the primer-template junction during DNA replication is essential for accurate and processive DNA synthesis. DNA replication in RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and archaea and has been a prototype for studies on DNA replication and assembly of the functional replisome. To examine protein-protein interactions at the DNA replication fork, we have established solution conditions for the formation of a discrete and homogeneous complex of RB69 DNA polymerase (gp43), primer-template DNA, and RB69 single-stranded DNA-binding protein (gp32) using equilibrium fluorescence and light scattering. We have characterized the interaction between DNA polymerase and single-stranded DNA-binding protein and measured a 60-fold increase in the overall affinity of RB69 single-stranded DNA-binding protein (SSB) for template strand DNA in the presence of DNA polymerase that is the result of specific protein-protein interactions. Our data further suggest that the cooperative binding of the RB69 DNA polymerase and SSB to the primer-template junction is a simple but functionally important means of regulatory assembly of replication proteins at the site of action. We have also shown that a functional domain of RB69 single-stranded DNA-binding protein suggested previously to be the site of RB69 DNA polymerase-SSB interactions is dispensable. The data from these studies have been used to model the RB69 DNA polymerase-SSB interaction at the primer-template junction.     

Smaug, a novel RNA-binding protein that operates a translational switch in Drosophila.

During Drosophila embryogenesis, a gradient of Nanos protein emanating from the posterior pole organizes abdominal segmentation. This gradient arises from translational regulation of nanos mRNA, which is activated in the specialized cytoplasm at the posterior pole of the embryo and repressed elsewhere. Previously, we have defined cis-acting elements in the mRNA that mediate this translational switch. In this report, we identify a factor named Smaug that binds to these elements and represses translation in the bulk cytoplasm. Smaug interacts gentically and biochemically with Oskar, a key component of the pole plasm for activation of nanos mRNA and specification of the germline precursors. These observations suggest that Smaug operates a translational switch that governs the distribution of Nanos protein.     

RNA-binding protein-mediated translational repression of transgene expression in plants

We have demonstrated that RNA-binding proteins from coliphages and yeast can function as translational repressors in plants. RNA sequences called translational operators were inserted at a cap-proximal position in the 5-UTR of mRNAs of two reporter genes, gusor aroA:CP4. Translation of the reporter mRNAs was efficiently repressed when the RNA binding protein that specifically binds to its cognate operator was co-expressed. The efficiency of translational repression by RNA-binding protein positively correlated with the amount of binding protein in transformed plant cells. Detailed studies on coliphage MS2 coat protein-mediated translational repression also suggested that the efficiency of translational repression was position-dependent. A translational operator situated at the cap-proximal position was more efficient in conferring repression than one that was placed cap-distal. Translational repression can be an efficient means for regulation of transgene expression, thereby broadening current approaches for transgene regulation in plants.these authors contributed equally to this workthese authors contributed equally to this work     

RNA-binding properties of the mitochondrial Y-box protein RBP16

We have previously identified a mitochondrial Y-box protein in Trypanosoma brucei that we designated RBP16. The predicted RBP16 amino acid sequence revealed the presence of a cold-shock domain at its N-terminus and a glycine- and arginine-rich C-terminus reminiscent of an RGG RNA-binding motif. Since RBP16 is capable of interacting with different guide RNAs (gRNAs) in vitro and in vivo primarily via the oligo(U) tail, as well as with ribosomal RNAs, possible functions of RBP16 may be in kinetoplastid RNA editing and/or translation. Herein, we report experiments that further define the RNA-binding properties of RBP16. RBP16 forms a single stable complex with the gRNA gA6[14] at low protein concentration, while at higher protein concentration two stable complexes that possibly represent two different conformations are observed. Both complexes are stable at relatively high salt and moderate heparin concentrations indicating that the binding of RBP16 to gA6[14] does not rely primarily on ionic interactions. Phenylglyoxal treatment of the protein indicates that arginine residues are important in RNA binding. The minimal length of RNA sequence necessary for the binding of RBP16 was assessed by gel retardation and UV cross-linking competition assays using oligo(U) ribonucleotides of varying lengths (4–40 nt). Although RBP16 can bind to oligonucleotides as small as U₄, its affinity increases with the length of the oligo(U) ribonucleotide, with a dramatic increase in binding efficiency observed when the length is increased to 10 nt. Gel retardation assays employing T.brucei mRNAs demonstrated that, although it acts as a major binding determinant, a 3′ U tail is not an absolute requirement for efficient RBP16–RNA binding. Experiments with oligonucleotides containing U stretches embedded at different positions in oligo(dC) indicated that high-affinity binding requires both a uridine stretch, as well as 5′ and 3′ non-specific sequences. These results suggest a model for the molecular interactions involved in RBP16–RNA binding.     

The ratio of coat protein to bacteriophage f2 RNA in the translational repressor complex]

One of the mechanisms underlying the regulation of the bacteriophage f2 RNA translation is the repression of the phage RNA-replicase formation by coat protein. This repression is due to the formation of a complex between f2 RNA and coat protein (complex I). In this work the mechanism of complex I formation as well as the effect of this complex on the f2 RNA-replicase formation was followed by inhibition of alanine incorporation into RNA-replicase polypeptide which was separated by polyacrylamide gel electrophoresis. The molar ratios of protein to f2 RNA in complex I were analyzed by sucrose gradient sedimentation. It was been found that complex I consists of six molecules of coat protein bound per one molecule of RNA. Ribonuclease digestion of the glutaraldehyde-fixed complex resulted in a mixture of products in which the hexamers of coat protein molecules were predominant. This indicates that the six molecules of coat protein bound to f2 RNA are neighbouring. It has been also shown that under conditions required for phage protein synthesis, coat protein occurs in solution is dimer. The results show that the translational repression of the RNA-replicase cistron is due to the cooperative attachment of three dimers of coat protein to phage template, forming a hexameric cluster on the RNA strand. The proposed mechanism of the complex I formation seems to be in good agreement with the sequence of events in the phage F2 life cycle. It is known that shortly after infection of the host cell the coat protein and phage RNA-replicase begin to be synthesised. According to our findings, the first portions of coat protein do not affect the translation of the RNA-replicase gene since at low concentration the coat protein occure in the form of monomers. At a later period of phage development, when the concentration of coat protein is sufficiently high to promote the formation of protein dimers, the translational repressor complex is formed and the RNA-replicase gene becomes inoperative.     

Additional evidence that the hemin-controlled translational repressor from rabbit reticulocytes is a protein kinase

Recent reports have suggested that the hemin-controlled translational repressor (HCR) which mediates the hemin control of protein synthesis in reticulocyte lysates, acts as a specific protein kinase, phosphorylating a subunit of the Met-tRNA_f binding factor (IF-1). We have found that crude and highly purified HCR can phosphorylate a 38,000 molecular weight component of IF-1, but that crude prorepressor (the precursor of HCR), which is not inhibitory, does not phosphorylate this component. Prolonged warming of the prorepressor induces the formation of the inhibitor and the protein kinase that phosphorylates the 38,000 molecular weight protein, and the formation of both is blocked by hemin. In addition, a brief incubation of the prorepressor with N-ethylmaleimide, which produces maximal inhibitory activity within 5 minutes, also induces formation of the protein kinase. These findings suggest that HCR and the protein kinase are the same protein and provide additional support for the concept that HCR controls protein synthesis by phosphorylating the Met-tRNA_f binding factor.     

RNA-binding properties of the plant protein Nt-4/1

The tobacco α-helical protein Nt-4/1 with unknown function forms ribonucleoprotein (RNP) complexes in vitro. Results obtained by retardation of RNP complexes in agarose gel were confirmed by Western-Northern hybridization. Several deletion and point mutants of Nt-4/1 were constructed, and the RNA-binding site was mapped in a positively charged region of the C-terminal domain of the protein. The results of this study and those described earlier support our hypothesis of the participation of Nt-4/1 protein in spreading RNA-containing pathogens in the plant.     

Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo(-) enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr(567) (Pol(Y567(A/S/T)) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr(567) is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol(Y567A) Exo(-) enzyme generates mispairs more frequently but extends them less efficiently than does a Pol(+) Exo(-) enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43.     

An RNA-binding protein, AtRBP1, is expressed in actively proliferative regions in Arabidopsis thaliana

A cDNA clone, named XF41, that encodes an RNA-binding protein was isolated from Arabidopsis thaliana. The deduced protein, named AtRBP1, contains two conserved consensus sequence-type RNA-binding domains (CS-RBDs) in the N-terminal half, a putative PY motif (a target of a WW domain) in the center, and uncharacterized C-terminal domain. A binding assay demonstrated that the AtRBP1 can bind to single-stranded nucleic acids in vitro. Analysis of localization of the GUS activity of transgenic Arabidopsis thaliana plants that have the chimeric gene containing the upstream sequence of the AtRBP1 gene and GUS gene demonstrated that the AtRBP1 gene is expressed in meristematic tissues such as the vegetative shoot apex and root tips, developing organs such as floral buds and pistils of young flowers, abscission layers of immature siliques and junctions of pedicels. Considering the specificity of the expression, AtRBP1 may be required in the progress of cell proliferation.