Eukaryotic Ribosomal Proteins Research Articles

Structural basis for translational control by the human 48S initiation complex.

The selection of an open reading frame (ORF) for translation of eukaryotic mRNA relies on remodeling of the scanning 48S initiation complex into an elongation-ready 80S ribosome. Using cryo-electron microscopy, we visualize the key commitment steps orchestrating 48S remodeling in humans. The mRNA Kozak sequence facilitates mRNA scanning in the 48S open state and stabilizes the 48S closed state by organizing the contacts of eukaryotic initiation factors (eIFs) and ribosomal proteins and by reconfiguring mRNA structure. GTPase-triggered large-scale fluctuations of 48S-bound eIF2 facilitate eIF5B recruitment, transfer of initiator tRNA from eIF2 to eIF5B and the release of eIF5 and eIF2. The 48S-bound multisubunit eIF3 complex controls ribosomal subunit joining by coupling eIF exchange to gradual displacement of the eIF3c N-terminal domain from the intersubunit interface. These findings reveal the structural mechanism of ORF selection in human cells and explain how eIF3 could function in the context of the 80S ribosome.

Dual protection by Bcp1 and Rkm1 ensures incorporation of uL14 into pre-60S ribosomal subunits.

Eukaryotic ribosomal proteins contain extended regions essential for translation coordination. Dedicated chaperones stabilize the associated ribosomal proteins. We identified Bcp1 as the chaperone of uL14 in Saccharomyces cerevisiae. Rkm1, the lysine methyltransferase of uL14, forms a ternary complex with Bcp1 and uL14 to protect uL14. Rkm1 is transported with uL14 by importins to the nucleus, and Bcp1 disassembles Rkm1 and importin from uL14 simultaneously in a RanGTP-independent manner. Molecular docking, guided by crosslinking mass spectrometry and validated by a low-resolution cryo-EM map, reveals the correlation between Bcp1, Rkm1, and uL14, demonstrating the protection model. In addition, the ternary complex also serves as a surveillance point, whereas incorrect uL14 is retained on Rkm1 and prevented from loading to the pre-60S ribosomal subunits. This study reveals the molecular mechanism of how uL14 is protected and quality checked by serial steps to ensure its safe delivery from the cytoplasm until its incorporation into the 60S ribosomal subunit.

Dietary glycine supplementation activates mechanistic target of rapamycin signaling pathway in tissues of pigs with intrauterine growth restriction.

The mechanistic target of rapamycin (mTOR) cell signaling pathway serves as the central mechanism for the regulation of tissue protein synthesis and growth. We recently reported that supplementing 1% glycine to corn- and soybean meal-based diets enhanced growth performance between weaning and market weights in pigs with intrauterine growth restriction (IUGR). Results of recent studies have revealed an important role for glycine in activating mTOR and protein synthesis in C2C12 muscle cells. Therefore, the present study tested the hypothesis that dietary glycine supplementation enhanced the mTOR cell signaling pathway in skeletal muscle and other tissues of IUGR pigs. At weaning (21 d of age), IUGR pigs and litter mates with normal birth weights (NBW) were assigned randomly to one of the two groups: supplementation with either 1% glycine or 1.19% l-alanine (isonitrogenous control) to a corn- and soybean meal-based diet. Tissues were obtained from the pigs within 1wk after the feeding trial ended at 188 d of age to determine the abundances of total and phosphorylated forms of mTOR and its two major downstream proteins: eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and ribosomal protein S6 kinase-1 (p70S6K). Results showed that IUGR decreased (P < 0.05) the abundances of both total and phosphorylated mTOR, 4EBP1, and p70S6K in the gastrocnemius muscle and jejunum. In the longissimus lumborum muscle of IUGR pigs, the abundances of total mTOR did not differ (P > 0.05) but those for phosphorylated mTOR and both total and phosphorylated 4EBP1 and p70S6K were downregulated (P < 0.05), when compared to NBW pigs. These adverse effects of IUGR in the gastrocnemius muscle, longissimus lumborum muscle, and jejunum were prevented (P < 0.05) by dietary glycine supplementation. Interestingly, the abundances of total or phosphorylated mTOR, 4EBP1, and p70S6K in the liver were not affected (P > 0.05) by IUGR or glycine supplementation. Collectively, our findings indicate that IUGR impaired the mTOR cell signaling pathway in the tissues of pigs and that adequate glycine intake was crucial for maintaining active mTOR-dependent protein synthesis for the growth and development of skeletal muscle.

Insights into translocation mechanism and ribosome evolution from cryo-EM structures of translocation intermediates of Giardia intestinalis.

Giardia intestinalis is a protozoan parasite that causes diarrhea in humans. Using single-particle cryo-electron microscopy, we have determined high-resolution structures of six naturally populated translocation intermediates, from ribosomes isolated directly from actively growing Giardia cells. The highly compact and uniquely GC-rich Giardia ribosomes possess eukaryotic rRNAs and ribosomal proteins, but retain some bacterial features. The translocation intermediates, with naturally bound tRNAs and eukaryotic elongation factor 2 (eEF2), display characteristic ribosomal intersubunit rotation and small subunit's head swiveling-universal for translocation. In addition, we observe the eukaryote-specific 'subunit rolling' dynamics, albeit with limited features. Finally, the eEF2·GDP state features a uniquely positioned 'leaving phosphate (Pi)' that proposes hitherto unknown molecular events of Pi and eEF2 release from the ribosome at the final stage of translocation. In summary, our study elucidates the mechanism of translocation in the protists and illustrates evolution of the translation machinery from bacteria to eukaryotes from both the structural and mechanistic perspectives.

Abstract 16: Cardiac Mitochondrial Transcription Factor A Overexpression Protects Against Stunning and Improves Mortality in a Mouse Model of Cardiac Arrest

Background: Cardiac arrest (CA) affects over 600,000 Americans every year and is associated with overwhelming mortality. After CA, myocardial stunning is a common occurrence leading to reduced cardiac output, and nearly 25% of CA die from cardiogenic shock. This cardiac injury is driven by ischemia-reperfusion injury. Mitochondria make up 30% of the heart by volume and are highly prone to ischemia-reperfusion damage. This damage may impair mitochondrial DNA (mtDNA), which is necessary for maintaining mitochondrial function. Mitochondrial transcription factor A (TFAM) can bind and protect mtDNA from damage. In this study, we investigated the role of cardiac-specific TFAM overexpression on cardiac function after CA in a mouse model. Methods: Wild-type (WT) and transgenic mice featuring cardiac-specific TFAM overexpression (TFAM OE) were subjected to 8-min of asystole by direct injection of potassium chloride and were revived by cardiopulmonary resuscitation. Surviving mice were assessed by echocardiography and monitored for survival over 4 weeks frame. Hearts were collected and sent for bulk RNA sequencing as well as assessment of mtDNA copy number, mitochondrial morphology, and protein expression. Results: TFAM OE mice express 40% more TFAM than WT littermates. TFAM OE mice had protected cardiac EF (51.11±2.95%) one day after CA when compared to WT mice (38.49±3.76%) and improved survival over 4 weeks. Bulk-RNA sequencing implicated dramatic changes to ribosomal expression and protein synthesis in the arrest TFAM OE mice. Western blot analysis of 1-day post-arrest hearts demonstrated increased markers of mRNA translation, including increased phosphorylation of eukaryotic translation initiation factor 4E-binding protein (4E-BP1) and ribosomal protein S6, suggesting that TFAM OE mice support protein translation to aid in cardiac recovery after arrest. Conclusions: Cardiac TFAM overexpression protects cardiac function and survival after cardiac arrest. Early evidence suggests that this protection may be mediated by supporting protein translation, which may offer a novel pathway for improving outcomes after arrest.

De Novo Transcriptome Assembly and Analysis of Longevity Genes Using Subterranean Termite (Reticulitermes chinensis) Castes.

The longevity phenomenon is entirely controlled by the insulin signaling pathway (IIS-pathway). Both vertebrates and invertebrates have IIS-pathways that are comparable to one another, though no one has previously described de novo transcriptome assembly of IIS-pathway-associated genes in termites. In this research, we analyzed the transcriptomes of both reproductive (primary kings “PK” and queens “PQ”, secondary worker reproductive kings “SWRK” and queens “SWRQ”) and non-reproductive (male “WM” and female “WF” workers) castes of the subterranean termite Reticulitermes chinensis. The goal was to identify the genes responsible for longevity in the reproductive and non-reproductive castes. Through transcriptome analysis, we annotated 103,589,264 sequence reads and 184,436 (7G) unigenes were assembled, GC performance was measured at 43.02%, and 64,046 sequences were reported as CDs sequences. Of which 35 IIS-pathway-associated genes were identified, among 35 genes, we focused on the phosphoinositide-dependent kinase-1 (Pdk1), protein kinase B2 (akt2-a), tuberous sclerosis-2 (Tsc2), mammalian target of rapamycin (mTOR), eukaryotic translation initiation factor 4E (EIF4E) and ribosomal protein S6 (RPS6) genes. Previously these genes (Pdk1, akt2-a, mTOR, EIF4E, and RPS6) were investigated in various organisms, that regulate physiological effects, growth factors, protein translation, cell survival, proliferation, protein synthesis, cell metabolism and survival, autophagy, fecundity rate, egg size, and follicle number, although the critical reason for longevity is still unclear in the termite castes. However, based on transcriptome profiling, the IIS-pathway-associated genes could prolong the reproductive caste lifespan and health span. Therefore, the transcriptomic shreds of evidence related to IIS-pathway genes provide new insights into the maintenance and relationships between biomolecular homeostasis and remarkable longevity. Finally, we propose a strategy for future research to decrypt the hidden costs associated with termite aging in reproductive and non-reproductive castes.

Optimal methionine supplementation improved the growth, hepatic protein synthesis and lipolysis of grass carp fry (Ctenopharyngodon idella)

Six experimental diets with graded methionine levels (0.33%, 0.55%, 0.71%, 0.87%, 1.03%, 1.20%, dry matter) were formulated to quantify the dietary methionine requirement, and assess the influence of dietary methionine levels on the metabolism of protein and lipid in Ctenopharyngodon Idella fry (initial weight 0.36 ± 0.01 g) during an 8-week feeding trial. The results showed that 1.03% dietary methionine significantly improved the growth performance, protein efficiency ratio and feed utilization, compared with 0.33% methionine. Dietary methionine supplementation slightly increased survival rate. 0.55%–0.87% methionine level increased the phosphorylated protein and mRNA levels of phosphatidylinositol 3-kinase (PI3K), target of rapamycin (TOR), eukaryotic initiation factor 4E binding protein-1 and ribosomal protein S6 kinase 1. Compared with 1.20% methionine, 0.55%–0.87% methionine levels significantly increased mRNA levels of hepatic insulin-like growth factor-1 (IGF-1), carnitine palmitoyl transferase-1, peroxisome proliferator-activated receptor α, adipose triglyceride lipase and hormone sensitive lipase. The highest mRNA levels of hepatic acetyl-CoA carboxylase, sterol regulatory element-binding protein 1, stearyl-CoA desaturase and fatty acid synthetase were observed in 0.33% methionine diet. Based on specific growth rate and feed conversion ratio, the dietary methionine requirement of grass carp fry was 1.03% of diet (2.67% of dietary protein), using the broken-line regression analysis. Suitable dietary methionine could improve the growth, feed utilization and lipolysis of fry and promote protein synthesis via the IGF-1/PI3K/TOR pathway. While methionine deficiency tended to enhance hepatic lipid synthesis.

Towards the Idea of Molecular Brains.

How can single cells without nervous systems perform complex behaviours such as habituation, associative learning and decision making, which are considered the hallmark of animals with a brain? Are there molecular systems that underlie cognitive properties equivalent to those of the brain? This review follows the development of the idea of molecular brains from Darwin’s “root brain hypothesis”, through bacterial chemotaxis, to the recent discovery of neuron-like r-protein networks in the ribosome. By combining a structural biology view with a Bayesian brain approach, this review explores the evolutionary labyrinth of information processing systems across scales. Ribosomal protein networks open a window into what were probably the earliest signalling systems to emerge before the radiation of the three kingdoms. While ribosomal networks are characterised by long-lasting interactions between their protein nodes, cell signalling networks are essentially based on transient interactions. As a corollary, while signals propagated in persistent networks may be ephemeral, networks whose interactions are transient constrain signals diffusing into the cytoplasm to be durable in time, such as post-translational modifications of proteins or second messenger synthesis. The duration and nature of the signals, in turn, implies different mechanisms for the integration of multiple signals and decision making. Evolution then reinvented networks with persistent interactions with the development of nervous systems in metazoans. Ribosomal protein networks and simple nervous systems display architectural and functional analogies whose comparison could suggest scale invariance in information processing. At the molecular level, the significant complexification of eukaryotic ribosomal protein networks is associated with a burst in the acquisition of new conserved aromatic amino acids. Knowing that aromatic residues play a critical role in allosteric receptors and channels, this observation suggests a general role of π systems and their interactions with charged amino acids in multiple signal integration and information processing. We think that these findings may provide the molecular basis for designing future computers with organic processors.

Arginine supplementation in plant‐rich diets affects growth, feed utilization, body composition, blood biochemical indices and gene expressions of the target of rapamycin signaling pathway in juvenile Asian red‐tailed catfish (Hemibagrus wyckoiides)

AbstractArginine (Arg) is an important essential amino acid required for optimum fish growth. However, its role in plant‐rich diets on growth and the required level in juvenile Hemibagrus wyckioides are currently unknown. This study evaluated the effects of Arg supplementation in plant‐rich diets on growth, feed utilization, whole body composition, blood biochemical indicators and gene expressions of the target of rapamycin (TOR) signaling pathway in juvenile H. wyckioides. A total of 450 fish (6.40 ± 0.06 g) were randomized into six equal groups and fed on diets with graded Arg levels: 0 (control), 3, 6, 9, 12 and 15 g/kg of dry feed for eight weeks. The results revealed that specific growth rate (SGR) and feed conversion ratio of juvenile H. wyckioides were significantly improved and reduced, respectively in fish fed on 9 g/kg of Arg supplementation (p &lt; .05). The optimum amount of dietary Arg content in plant‐rich diets for H. wyckioides was 63.77 g/kg diet based on SGR. Feeding fish with dietary Arg supplementation ranging from 9 to 12 g/kg improved significantly (p &lt; .05) whole body protein contents and decreased lipid deposition. The mRNA levels of TOR, eukaryotic translation initiation factor 4E (4EBP) and ribosomal protein S6 kinase 1 (S6K1) in muscle and liver were significantly affected by increasing dietary Arg levels. Meanwhile, the mRNA relative expression levels of TOR and S6K1 in the liver and muscle increased significantly as Arg supplementation increased. On the other hand, the mRNA relative expression levels of 4EBP was significantly higher in fish fed on the 9 g/kg Arg diet compared to those fed on the control and 15 g/kg Arg diets. In conclusion, our current results indicate that dietary Arg supplementation in plant‐rich diets, might activate the TOR signaling pathway, which promotes protein synthesis and reduces lipid accumulation of H. wyckioides, resulting in improved growth.

Identification of responsive genes and analysis of genes with bacterial-inducible cis-regulatory elements in the promoter regions in Oryza sativa L.

Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most critical diseases in rice. In order to study rice responsive genes to bacterial stress, microarray data were retrieved from GEO dataset. To identify the responsive genes to biotic stress (bacteria) bioinformatic tools were employed and the data presented in the forms of heatmap, gene ontology, gene network, and cis-element prediction were used. Almost all responsive genes were down-regulated at around 3 h time point and up-regulated 24 h time point in response to bacterial stress in rice varieties (Oryza sativa subs. japonica ‘IR64’, ‘IRBB5’, ‘IRBB7’ and ‘Y73’). Gene ontology showed that genes are involved in different biological processes including translation and cellular protein metabolic processes. Network analysis showed that genes expressed in response to pathogen infection (Xoo) included protein translation, eukaryotic initiation factors (eIFs), ribosomal proteins, protein ubiquitin, and MAPK genes. The genes expressed in response to bacterial stress can enable plant balance between synthesis and degradation of proteins which in turn allows plants for further growth and development. TATA-box and CAAT box had the highest number of cis elements involved in bacterial stress. These genes can provide novel insights into regulatory mechanisms in biotic stress responses in rice. Identification of bacterial stress response/tolerance genes of rice can assist the molecular breeding of new rice varieties tolerant to bacterial stress.

The functional role of the C-terminal tail of the human ribosomal protein uS19

The eukaryotic ribosomal protein uS19 has a C-terminal tail that is absent in its bacterial homologue. This tail has been shown to be involved in the formation of the decoding site of human ribosomes. We studied here the previously unexplored functional significance of the 15 C-terminal amino acid residues of human uS19 for the assembly of ribosomes and translation using HEK293-based cell cultures capable of producing FLAG-labeled uS19 (uS19FLAG) or its mutant form deprived of the mentioned amino acid ones. The examination of polysome profiles of cytoplasmic extracts from the respective cells revealed that the deletion of the above uS19 amino acid residues barely affected the assembly and maturation of 40S subunits and the initiation of translation, but completely prevented the formation of polysomes. This implied the crucial importance of the uS19 tail in the elongation process. Analysis of tRNAs associated with 40S subunits and 80S ribosomes containing wild type uS19FLAG or its truncated form showed that the deletion of the C-terminal pentadecapeptide fragment of uS19 did not interfere with the binding of aminoacyl-tRNA (aa-tRNA) at the ribosomal A site. The results led to the conclusion that the transpeptidation, which occurs on the large ribosomal subunit after decoding the A site codon by the incoming aa-tRNA, is the most likely elongation stage, where this uS19 fragment can play a critical role. Our findings suggest that the uS19 tail is a keystone player in the accommodation of aa-tRNA at the A site, which is a pre-requisite for the peptide transfer.

Mutually exclusive amino acid residues of L13a are responsible for its ribosomal incorporation and translational silencing leading to resolution of inflammation.

Eukaryotic ribosomal protein L13a is a member of the conserved universal ribosomal uL13 protein family. Structurally, L13a is distinguished from its prokaryotic counterparts by the presence of an ∼55 amino acid-long carboxy-terminal α-helical extension. The importance of these evolved residues in the carboxy-terminal extension for mammalian ribosome biogenesis as well as L13a's extraribosomal function in GAIT (γ interferon-activated inhibitor of translation) complex-mediated translation silencing during inflammation is not understood. Here, we present biochemical analyses of L13a mutant variants identifying several mutually exclusive amino acid residues in the eukaryote-specific carboxy-terminal extension of human L13a (Tyr149-Val203) important for ribosomal incorporation and translational silencing. Specifically, we show that mutation of Arg169, Lys170, and Lys171 to Ala abrogate GAIT-mediated translational silencing, but not L13a incorporation into ribosomes. Moreover, we show that the carboxy-terminal helix alone can silence translation of GAIT element-containing mRNAs in vitro. We also show through cellular immunofluorescence experiments that nuclear but not nucleolar localization of L13a is resistant to extensive amino acid alterations, suggesting that multiple complex nuclear import signals are present within this protein. These studies provide new insights into L13a structure and its ribosomal and extraribosomal functions in model human cells.

Communication between RACK1/Asc1 and uS3 (Rps3) is essential for RACK1/Asc1 function in yeast Saccharomyces cerevisiae

The receptor for activated c-kinase (RACK1, Asc1 in yeast) is a eukaryotic ribosomal protein located in the head region of the 40S subunit near the mRNA exit channel. This WD-repeat β-propeller protein acts as a signaling molecule and is involved in metabolic regulation, cell cycle progression, and translational control. However, the exact details of the RACK1 recruitment and stable association with the 40S ribosomal subunit remain only partially known. X-ray analyses of the yeast, Saccharomyces cerevisiae, ribosome revealed that the RACK1 propeller blade (4–5) interacts with the eukaryote-specific C-terminal domain (CTD) of ribosomal protein S3 (uS3 family). To check the functional significance of this interaction, we generated mutant yeast strains harboring C-terminal deletions of uS3. We found that deletion of the 20 C-terminal residues (interacting with blade 4–5) from the uS3-CTD abrogates RACK1 binding to the ribosome. Strains with truncated uS3-CTD exhibited compromised cellular growth and protein synthesis similar to that of RACK1Δ strain, thus suggesting that the uS3-CTD is crucial not only for the recruitment and association of RACK1 with the ribosome, but also for its intracellular function. We suggest that eukaryote-specific RACK1-uS3 interaction has evolved to act as a link between the ribosome and the cellular signaling pathways.

O-GlcNAcylation alters the selection of mRNAs for translation and promotes 4E-BP1–dependent mitochondrial dysfunction in the retina

Diabetes promotes the posttranslational modification of proteins by O-linked addition of GlcNAc (O-GlcNAcylation) to Ser/Thr residues of proteins and thereby contributes to diabetic complications. In the retina of diabetic mice, the repressor of mRNA translation, eIF4E-binding protein 1 (4E-BP1), is O-GlcNAcylated, and sequestration of the cap-binding protein eukaryotic translation initiation factor (eIF4E) is enhanced. O-GlcNAcylation has also been detected on several eukaryotic translation initiation factors and ribosomal proteins. However, the functional consequence of this modification is unknown. Here, using ribosome profiling, we evaluated the effect of enhanced O-GlcNAcylation on retinal gene expression. Mice receiving thiamet G (TMG), an inhibitor of the O-GlcNAc hydrolase O-GlcNAcase, exhibited enhanced retinal protein O-GlcNAcylation. The principal effect of TMG on retinal gene expression was observed in ribosome-associated mRNAs (i.e. mRNAs undergoing translation), as less than 1% of mRNAs exhibited changes in abundance. Remarkably, ∼19% of the transcriptome exhibited TMG-induced changes in ribosome occupancy, with 1912 mRNAs having reduced and 1683 mRNAs having increased translational rates. In the retina, the effect of O-GlcNAcase inhibition on translation of specific mitochondrial proteins, including superoxide dismutase 2 (SOD2), depended on 4E-BP1/2. O-GlcNAcylation enhanced cellular respiration and promoted mitochondrial superoxide levels in WT cells, and 4E-BP1/2 deletion prevented O-GlcNAcylation-induced mitochondrial superoxide in cells in culture and in the retina. The retina of diabetic WT mice exhibited increased reactive oxygen species levels, an effect not observed in diabetic 4E-BP1/2-deficient mice. These findings provide evidence for a mechanism whereby diabetes-induced O-GlcNAcylation promotes oxidative stress in the retina by altering the selection of mRNAs for translation.

Exploitation of nuclear protein SFPQ by the encephalomyocarditis virus to facilitate its replication

The encephalomyocarditis virus (EMCV) is a single-stranded RNA virus that induces sudden death, diabetes, myocarditis and nervous disorders in non-human primates. The rapid development of xenografts such as heart transplantation from pig to human raises the issue of EMCV safety in human cells. SFPQ, a proline and glutamine rich splicing factor that participates in diverse molecular functions including paraspeckle formation, microRNA synthesis and transcription regulation, is known to regulate host innate immune response to viruses. However, the role of SFPQ in EMCV infection remains unclear. Here we reported that the SFPQ was essential for EMCV replication. Depletion of SFPQ impaired EMCV production, while forced expression of SFPQ promoted viral replication. Mechanistically, loss of SFPQ affected the transcription profile of host mitochondria pathway related genes. In addition, cellular SFPQ was exploited by EMCV and accumulated in cytoplasm and it interacted with eukaryotic initiation factors and ribosomal proteins to facilitate internal ribosome entry site (IRES)-dependent translation of EMCV protein. Altogether, our work discovered host SFPQ as a new target to inhibit EMCV replication and infection.

Critical Role for Saccharomyces cerevisiae Asc1p in Translational Initiation at Elevated Temperatures.

The eukaryotic ribosomal protein RACK1/Asc1p is localized to the mRNA exit channel of the 40S subunit but lacks a defined role in mRNA translation. Saccharomyces cerevisiae deficient in ASC1 exhibit temperature-sensitive growth. Using this null mutant, potential roles for Asc1p in translation and ribosome biogenesis are evaluated. At the restrictive temperature the asc1Δ null mutant has reduced polyribosomes. To test the role of Asc1p in ribosome stability, cryo-EM is used to examine the structure of 80S ribosomes in an asc1Δ yeast deletion mutant at both the permissive and nonpermissive temperatures. CryoEM indicates that loss of Asc1p does not severely disrupt formation of this complex structure. No defect is found in rRNA processing in the asc1Δ null mutant. A proteomic approach is applied to survey the effect of Asc1p loss on the global translation of yeast proteins. At the nonpermissive temperature, the asc1Δ mutant has reduced levels of ribosomal proteins and other factors critical for translation. Collectively, these results are consistent with recent observations suggesting that Asc1p is important for ribosome occupancy of short mRNAs. The results show the Asc1 ribosomal protein is critical in translation during heat stress.

Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2

Disordered extensions at the termini and short internal insertions distinguish eukaryotic ribosomal proteins (r-proteins) from their anucleated archaeal counterparts. Here, we report an NMR structure of such a eukaryotic-specific segment (ESS) in the r-protein eS26 in complex with the escortin Tsr2. The structure reveals how ESS attracts Tsr2 specifically to importin:eS26 complexes entering the nucleus in order to trigger non-canonical RanGTP-independent disassembly. Tsr2 then sequesters the released eS26 and prevents rebinding to the importin, providing an alternative allosteric mechanism to terminate the process of nuclear import. Notably, a Diamond–Blackfan anemia-associated Tsr2 mutant protein is impaired in binding to ESS, unveiling a critical role for this interaction in human hematopoiesis. We propose that eS26-ESS and Tsr2 are components of a nuclear sorting system that co-evolved with the emergence of the nucleocytoplasmic barrier and transport carriers.

Disassembly of yeast 80S ribosomes into subunits is a concerted action of ribosome-assisted folding of denatured protein

It has been shown by several groups that ribosome can assist folding of denatured protein in vitro and the process is conserved across the species. Domain V of large ribosomal rRNA which occupies the intersubunit side of the large subunit was identified as the key player responsible for chaperoning the folding process. Thus, it is conceivable that denatured protein needs to access the intersubunit space of the ribosome in order to get folded.In this study, we have investigated the mechanism of release of the protein from the eukaryotic ribosome following reactivation. We have observed significant splitting of yeast 80S ribosome when incubated with the denatured BCAII protein. Energy-free disassembly mechanism functions in low Mg+2 ion concentration for prokaryotic ribosomes. Eukaryotic ribosomes do not show significant splitting even at low Mg+2 ion concentration. In this respect, denatured protein-induced disassembly of eukaryotic ribosome without the involvement of any external energy source is intriguing.For prokaryotic ribosomes, it was reported that the denatured protein induces ribosome splitting into subunits in order to access domain V-rRNA. In contrast, our results suggest an alternative mechanism for eukaryotic ribosomal rRNA-mediated protein folding and subsequent separation of the subunits by which release of the activated-protein occurs.

Spliced leader RNA trans-splicing discovered in copepods

Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.

Biological insights into the expression of translation initiation factors from recombinant CHOK1SV cell lines and their relationship to enhanced productivity.

Translation initiation is on the critical pathway for the production of monoclonal antibodies (mAbs) by mammalian cells. Formation of a closed loop structure comprised of mRNA, a number of eukaryotic initiation factors (eIFs) and ribosomal proteins has been proposed to aid re-initiation of translation and therefore increase global translational efficiency. We have determined mRNA and protein levels of the key components of the closed loop, eIFs (eIF3a, eIF3b, eIF3c, eIF3h, eIF3i and eIF4G1), poly(A)-binding protein (PABP) 1 and PABP-interacting protein 1 (PAIP1), across a panel of 30 recombinant mAb-producing GS-CHOK1SV cell lines with a broad range of growth characteristics and production levels of a model recombinant mAb. We have used a multi-level statistical approach to investigate the relationship between key performance indicators (cell growth and recombinant antibody productivity) and the intracellular amounts of target translation initiation factor proteins and the mRNAs encoding them. We show that high-producing cell lines maintain amounts of the translation initiation factors involved in the formation of the closed loop mRNA, maintaining these proteins at appropriate levels to deliver enhanced recombinant protein production. We then utilize knowledge of the amounts of these factors to build predictive models for and use cluster analysis to identify, high-producing cell lines. The present study therefore defines the translation initiation factor amounts that are associated with highly productive recombinant GS-CHOK1SV cell lines that may be targets for screening highly productive cell lines or to engineer new host cell lines with the potential for enhanced recombinant antibody productivity.