Rbg1–Tma46 dimer structure reveals new functional domains and their role in polysome recruitment

Developmentally Regulated GTP-binding (DRG) proteins are highly conserved GTPases that associate with DRG Family Regulatory Proteins (DFRP). The resulting complexes have recently been shown to participate in eukaryotic translation. The structure of the Rbg1 GTPase, a yeast DRG protein, in complex with the C-terminal region of its DFRP partner, Tma46, was solved by X-ray diffraction. These data reveal that DRG proteins are multimodular factors with three additional domains, helix–turn–helix (HTH), S5D2L and TGS, packing against the GTPase platform. Surprisingly, the S5D2L domain is inserted in the middle of the GTPase sequence. In contrast, the region of Tma46 interacting with Rbg1 adopts an extended conformation typical of intrinsically unstructured proteins and contacts the GTPase and TGS domains. Functional analyses demonstrate that the various domains of Rbg1, as well as Tma46, modulate the GTPase activity of Rbg1 and contribute to the function of these proteins in vivo. Dissecting the role of the different domains revealed that the Rbg1 TGS domain is essential for the recruitment of this factor in polysomes, supporting further the implication of these conserved factors in translation.     

Heterologous complementation reveals that mutant alleles of QSR1 render 60S ribosomal subunits unstable and translationally inactive.

QSR1 is a highly conserved gene which encodes a 60S ribosomal subunit protein that is required for joining of large and small ribosomal subunits. In this report we demonstrate heterologous complementation of a yeast QSR1 deletion strain with both the human and corn homologs and show that the human and corn proteins are assembled into hybrid yeast/human and yeast/corn ribosomes. While the homologous genes complement lethality of the QSR1 deletion, they also result in a diminished growth rate. Analyses of the translation rates of ribosomes containing the human and corn proteins reveal a partial loss of function. Velocity gradient analyses of the hybrid ribosomes after exposure to high concentrations of salt indicate that the decreased activity is due to lability of the hybrid 60S subunits.     

A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ-like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK-like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ-like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J-domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co-chaperone, mediating transmembrane recruitment of DnaK-like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK-like chaperones of the endoplasmic reticulum.     

Association of protein biogenesis factors at the yeast ribosomal tunnel exit is affected by the translational status and nascent polypeptide sequence

Ribosome-associated protein biogenesis factors (RPBs) act during a short but critical period of protein biogenesis. The action of RPBs starts as soon as a nascent polypeptide becomes accessible from the outside of the ribosome and ends upon termination of translation. In yeast, RPBs include the chaperones Ssb1/2 and ribosome-associated complex, signal recognition particle, nascent polypeptide-associated complex (NAC), the aminopeptidases Map1 and Map2, and the Nalpha-terminal acetyltransferase NatA. Here, we provide the first comprehensive analysis of RPB binding at the yeast ribosomal tunnel exit as a function of translational status and polypeptide sequence. We measured the ratios of RPBs to ribosomes in yeast cells and determined RPB occupation of translating and non-translating ribosomes. The combined results imply a requirement for dynamic and coordinated interactions at the tunnel exit. Exclusively, NAC was associated with the majority of ribosomes regardless of their translational status. All other RPBs occupied only ribosomal subpopulations, binding with increased apparent affinity to randomly translating ribosomes as compared with non-translating ones. Analysis of RPB interaction with homogenous ribosome populations engaged in the translation of specific nascent polypeptides revealed that the affinities of Ssb1/2, NAC, and, as expected, signal recognition particle, were influenced by the amino acid sequence of the nascent polypeptide. Complementary cross-linking data suggest that not only affinity of RPBs to the ribosome but also positioning can be influenced in a nascent polypeptide-dependent manner.     

Saccharomyces cerevisiae translational activator Cbs1p is associated with translationally active mitochondrial ribosomes

In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.     

Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3.

Termination of translation in higher organisms is a GTP-dependent process. However, in the structure of the single polypeptide chain release factor known so far (eRF1) there are no GTP binding motifs. Moreover, in prokaryotes, a GTP binding protein, RF3, stimulates translation termination. From these observations we proposed that a second eRF should exist, conferring GTP dependence for translation termination. Here, we have shown that the newly sequenced GTP binding Sup35-like protein from Xenopus laevis, termed eRF3, exhibits in vitro three important functional properties: (i) although being inactive as an eRF on its own, it greatly stimulates eRF1 activity in the presence of GTP and low concentrations of stop codons, resembling the properties of prokaryotic RF3; (ii) it binds and probably hydrolyses GTP; and (iii) it binds to eRF1. The structure of the C-domain of the X.laevis eRF3 protein is highly conserved with other Sup35-like proteins, as was also shown earlier for the eRF1 protein family. From these and our previous data, we propose that yeast Sup45 and Sup35 proteins belonging to eRF1 and eRF3 protein families respectively are also yeast termination factors. The absence of structural resemblance of eRF1 and eRF3 to prokaryotic RF1/2 and RF3 respectively, may point to the different evolutionary origin of the translation termination machinery in eukaryotes and prokaryotes. It is proposed that a quaternary complex composed of eRF1, eRF3, GTP and a stop codon of the mRNA is involved in termination of polypeptide synthesis in ribosomes.     

The PUF family of RNA-binding proteins: does evolutionarily conserved structure equal conserved function?

Drosophila Pumilio (Pum) protein is a founder member of a novel family of RNA-binding proteins, known as the PUF family. The PUF proteins constitute an evolutionarily highly conserved family of proteins present from yeast to humans and plants, and are characterized by a highly conserved C-terminal RNA-binding domain, composed of eight tandem repeats. The conserved biochemical features and genetic function of PUF family members have emerged from studies of model organisms. PUF proteins bind to related sequence motifs in the 3' untranslated region (3'UTR) of specific target mRNAs and repress their translation. Frequently, PUF proteins function asymmetrically to create protein gradients, thus causing asymmetric cell division and regulating cell fate specification. Thus, it was recently proposed that the primordial role of PUF proteins is to sustain mitotic proliferation of stem cells. Here we review the evolution, conserved genetic and biochemical properties of PUF family of proteins, and discuss protein interactions, upstream regulators and downstream targets of PUF proteins. We also suggest that a conserved mechanism of PUF function extends to the newly described mammalian members of the PUF family (human PUM1 and PUM2, and mouse Pum1 and Pum2), that show extensive homology to Drosophila Pum, and could have an important role in cell development, fate specification and differentiation.     

Structural analysis of translating ribosomes. Proton magnetic resonance method

Comparison has been made of the proton magnetic resonance (PMR) spectra of translating ribosomes in the pre-translocation and post-translocation states as well as of the complexes of translating ribosomes with elongation factors Tu (EF-Tu) or G (EF-G) in the presence of the uncleavable analogue of GTP--guanylyl-imidodiphosphate (GMP-PNP). It is shown that proteins L7/L12 within the translating ribosomes possess a high intramolecular mobility both in the pre-translocation and in the post-translocation states. The interaction of EF-G with translating ribosomes results in a decrease of the mobility of the L7/L12 proteins. The interaction of EF-Tu with translating ribosomes leads to slight changes in the PMR spectra different from the changes caused by EF-G.     

The translation machinery and 70 kd heat shock protein cooperate in protein synthesis.

The function of the yeast SSB 70 kd heatshock proteins (hsp70s) was investigated by a variety of approaches. The SSB hsp70s (Ssb1/2p) are associated with translating ribosomes. This association is disrupted by puromycin, suggesting that Ssb1/2p may bind directly to the nascent polypeptide. Mutant ssb1 ssb2 strains grow slowly, contain a low number of translating ribosomes, and are hypersensitive to several inhibitors of protein synthesis. The slow growth phenotype of ssb1 ssb2 mutants is suppressed by increased copy number of a gene encoding a novel translation elongation factor 1 alpha (EF-1 alpha)-like protein. We suggest that cytosolic hsp70 aids in the passage of the nascent polypeptide chain through the ribosome in a manner analogous to the role played by organelle-localized hsp70 in the transport of proteins across membranes.     

MURF-1 and MURF-2 target a specific subset of myofibrillar proteins redundantly: towards understanding MURF-dependent muscle ubiquitination

MURF-1, MURF-2 and MURF-3 are a specific class of RING finger proteins that are expressed in striated muscle tissues. MURF-1 has been suggested to act as an ubiquitin ligase, thereby controlling proteasome-dependent degradation of muscle proteins. Here, we performed yeast two-hybrid (YTH) screens of skeletal muscle cDNA libraries with MURF-1 baits to identify potential myocellular targets of MURF-1-dependent ubiquitination. This identified eight myofibrillar proteins as binding partners of MURF-1: titin, nebulin, the nebulin-related protein NRAP, troponin-I (TnI), troponin-T (TnT), myosin light chain 2 (MLC-2), myotilin and T-cap. YTH mating studies with MURF-1,2,3 baits indicated that these eight myofibrillar proteins are all targeted redundantly by both MURF-1 and MURF-2. Western blot studies on cardiac tissues from wild-type and MURF-1-deficient mice suggested that titin and nebulin were ubiquitinated at similar levels, and MLC-2 and TnI at reduced levels in MURF-1 KO mice. Mapping of the TnI and titin binding sites on MURF-1 peptide scans demonstrated their binding to motifs highly conserved between MURF-1 and MURF-2. Our data are consistent with a model in which MURF-1 and MURF-2 together target a specific set of myofibrillar proteins redundantly, most likely to control their ubiquitination-dependent degradation. Finally, our YTH screens identified the interaction of MURF-1 with 11 enzymes required for ATP/energy production in muscle including the mitochondrial ATP synthase and cytoplasmic creatine kinase. These data raise the possibility that MURF-1 may coordinately regulate the energy metabolism of mitochondrial and cytoplasmic compartments.     

Genetic depletion of the RNA helicase DDX3 leads to impaired elongation of translating ribosomes triggering co-translational quality control of newly synthesized polypeptides

DDX3 is a multifaceted RNA helicase of the DEAD-box family that plays central roles in all aspects of RNA metabolism including translation initiation. Here, we provide evidence that the Leishmania DDX3 ortholog functions in post-initiation steps of translation. We show that genetic depletion of DDX3 slows down ribosome movement resulting in elongation-stalled ribosomes, impaired translation elongation and decreased de novo protein synthesis. We also demonstrate that the essential ribosome recycling factor Rli1/ABCE1 and termination factors eRF3 and GTPBP1 are less recruited to ribosomes upon DDX3 loss, suggesting that arrested ribosomes may be inefficiently dissociated and recycled. Furthermore, we show that prolonged ribosome stalling triggers co-translational ubiquitination of nascent polypeptide chains and a higher recruitment of E3 ubiquitin ligases and proteasome components to ribosomes of DDX3 knockout cells, which further supports that ribosomes are not elongating optimally. Impaired elongation of translating ribosomes also results in the accumulation of cytoplasmic protein aggregates, which implies that defects in translation overwhelm the normal quality controls. The partial recovery of translation by overexpressing Hsp70 supports this possibility. Collectively, these results suggest an important novel contribution of DDX3 to optimal elongation of translating ribosomes by preventing prolonged translation stalls and stimulating recycling of arrested ribosomes.     

Multiple portions of poly(A)-binding protein stimulate translation in vivo

Translational stimulation of mRNAs during early development is often accompanied by increases in poly(A) tail length. Poly(A)-binding protein (PAB) is an evolutionarily conserved protein that binds to the poly(A) tails of eukaryotic mRNAs. We examined PAB's role in living cells, using both Xenopus laevis oocytes and Saccharomyces cerevisiae, by tethering it to the 3'-untranslated region of reporter mRNAs. Tethered PAB stimulates translation in vivo. Neither a poly(A) tail nor PAB's poly(A)-binding activity is required. Multiple domains of PAB act redundantly in oocytes to stimulate translation: the interaction of RNA recognition motifs (RRMs) 1 and 2 with eukaryotic initiation factor-4G correlates with translational stimulation. Interaction with Paip-1 is insufficient for stimulation. RRMs 3 and 4 also stimulate, but bind neither factor. The regions of tethered PAB required in yeast to stimulate translation and stabilize mRNAs differ, implying that the two functions are distinct. Our results establish that oocytes contain the machinery necessary to support PAB-mediated translation and suggest that PAB may be an important participant in translational regulation during early development.     

Stoichiometry and Change of the mRNA Closed-Loop Factors as Translating Ribosomes Transit from Initiation to Elongation

Protein synthesis is a highly efficient process and is under exacting control. Yet, the actual abundance of translation factors present in translating complexes and how these abundances change during the transit of a ribosome across an mRNA remains unknown. Using analytical ultracentrifugation with fluorescent detection we have determined the stoichiometry of the closed-loop translation factors for translating ribosomes. A variety of pools of translating polysomes and monosomes were identified, each containing different abundances of the closed-loop factors eIF4E, eIF4G, and PAB1 and that of the translational repressor, SBP1. We establish that closed-loop factors eIF4E/eIF4G dissociated both as ribosomes transited polyadenylated mRNA from initiation to elongation and as translation changed from the polysomal to monosomal state prior to cessation of translation. eIF4G was found to particularly dissociate from polyadenylated mRNA as polysomes moved to the monosomal state, suggesting an active role for translational repressors in this process. Consistent with this suggestion, translating complexes generally did not simultaneously contain eIF4E/eIF4G and SBP1, implying mutual exclusivity in such complexes. For substantially deadenylated mRNA, however, a second type of closed-loop structure was identified that contained just eIF4E and eIF4G. More than one eIF4G molecule per polysome appeared to be present in these complexes, supporting the importance of eIF4G interactions with the mRNA independent of PAB1. These latter closed-loop structures, which were particularly stable in polysomes, may be playing specific roles in both normal and disease states for specific mRNA that are deadenylated and/or lacking PAB1. These analyses establish a dynamic snapshot of molecular abundance changes during ribosomal transit across an mRNA in what are likely to be critical targets of regulation.     

The yeast hsp70 homologue Ssa is required for translation and interacts with Sis1 and Pab1 on translating ribosomes

The 70-kDa heat shock proteins are molecular chaperones that participate in a variety of cellular functions. This chaperone function is stimulated by interaction with hsp40 proteins. The Saccharomyces cerevisiae gene encoding the essential hsp40 homologue, SIS1, appears to function in translation initiation. Mutations in ribosomal protein L39 (rpl39) complement loss-of-function mutations in SIS1 as well as PAB1 (poly(A)-binding protein), suggesting a functional interaction between these proteins. However, while a direct interaction between Sis1 and Pab1 is not detectable, both of these proteins physically interact with the essential Ssa (and not Ssb) family of hsp70 proteins. This interaction is mediated by the variable C-terminal domain of Ssa. Subcellular fractionations demonstrate that the binding of Ssa to ribosomes is dependent upon its C terminus and that its interaction with Sis1 and Pab1 occurs preferentially on translating ribosomes. Consistent with a function in translation, depletion of Ssa protein produces a general translational defect that appears similar to loss of Sis1 and Pab1 function. This translational effect of Ssa appears mediated, at least in part, by its affect on the interaction of Pab1 with the translation initiation factor, eIF4G, which is dramatically reduced in the absence of functional Ssa protein.     

Dom34‐Hbs1 mediated dissociation of inactive 80S ribosomes promotes restart of translation after stress

Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation‐inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non‐translating 80S ribosomes stabilized by the ‘clamping’ Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34‐Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34‐Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34‐Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.     

Biochemical characterization of arabidopsis developmentally regulated G-proteins (DRGs)

Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as several medical conditions. Individual subfamilies within the G-protein superfamily possess unique nucleotide binding and hydrolysis rates that are intrinsic to their cellular function, and so characterization of these rates for a particular G-protein may provide insight into its cellular activity. We have produced recombinant active DRG protein using a bacterial expression system and refolding, and performed biochemical characterization of their GTP binding and hydrolysis. We show that recombinant Arabidopsis thaliana atDRG1 and atDRG2a are able to bind GDP and GTP. We also show that DRGs can hydrolyze GTP in vitro without the assistance of GTPase-activating proteins and guanine exchange factors. The atDRG proteins hydrolyze GTP at a relatively slow rate (0.94 × 10⁻³ min⁻¹ for DRG1 and 1.36 × 10⁻³ min⁻¹ for DRG2) that is consistent with their nearest characterized relatives, the Obg subfamily. The ability of DRGs to bind nucleotide substrates without assistance, their slow rate of GTP hydrolysis, heat stress activation and domain conservation suggest a possible role as a chaperone in ribosome assembly in response to stress as it has been suggested for the Obg proteins, a different but related G-protein subfamily.     

Characterization of ATDRG1, a member of a new class of GTP-binding proteins in plants

We report the initial characterization of an Arabidopsis thaliana cDNA (atdrg1), a member of a new class of GTP-binding proteins (G-proteins) in plants. The predicted ATDRG1 protein contains all five structural motifs characteristic of the G-protein superfamily. Apart from these motifs, the amino acid sequence differs substantially from all known G-proteins except for a recently discovered new family named developmentally regulated G-proteins (DRGs). Sequences closely related to atdrg1 are found in species as distant as human (80% amino acid conservation), Drosophila (74%), yeast (77%) and Caenorhabditis elegans (77%). The remarkable evolutionary conservation of these proteins suggests an important, but as yet unclear role. Phylogenetic analysis of the available homologous sequences strongly suggests a diphyletic origin of the eukaryotic DRG proteins. Northern analysis shows high levels of atdrg1 mRNA in all Arabidopsis tissues studied, and homologues of atdrg1 are present throughout the plant kingdom. In situ hybridization reveals that atdrg1 is highly expressed in actively growing tissues and reproductive organs. Southern analysis indicates the presence of either one or two copies of atdrg1 in the Arabidopsis genome. Immunolocalization studies show that the protein is present in cytoplasmic vesicles found mainly in actively growing tissues suggesting a putative role for ATDRG1 in either the regulation of vesicle transport or the regulation of enzymes involved in storage protein processing.     

Linking the 3′ Poly(A) Tail to the Subunit Joining Step of Translation Initiation: Relations of Pab1p, Eukaryotic Translation Initiation Factor 5B (Fun12p), and Ski2p-Slh1p

The 3' poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A)(+) mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A)(+) mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.