Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo.

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

Glucose availability dictates the export of the soluble and prion forms of Sup35p via periplasmic or extracellular vesicles.

The yeast [PSI+ ] prion originates from the self-perpetuating transmissible aggregates of the translation termination factor Sup35p. We previously showed that infectious Sup35p particles are exported outside the cells via extracellular vesicles (EV). This finding suggested a function for EV in the vertical and horizontal transmission of yeast prions. Here we report a significant export of Sup35p within periplasmic vesicles (PV) upon glucose starvation. We show that PV are up to three orders of magnitude more abundant than EV. However, PV and EV are different in terms of size and protein content, and their export is oppositely regulated by glucose availability in the growth medium. Overall, our work suggests that the export of prion particles to both the periplasm and the extracellular space needs to be considered to address the physiological consequences of vesicle-mediated yeast prions trafficking.

Translation Termination and Ribosome Recycling in Eukaryotes.

Termination of mRNA translation occurs when a stop codon enters the A site of the ribosome, and in eukaryotes is mediated by release factors eRF1 and eRF3, which form a ternary eRF1/eRF3-guanosine triphosphate (GTP) complex. eRF1 recognizes the stop codon, and after hydrolysis of GTP by eRF3, mediates release of the nascent peptide. The post-termination complex is then disassembled, enabling its constituents to participate in further rounds of translation. Ribosome recycling involves splitting of the 80S ribosome by the ATP-binding cassette protein ABCE1 to release the 60S subunit. Subsequent dissociation of deacylated transfer RNA (tRNA) and messenger RNA (mRNA) from the 40S subunit may be mediated by initiation factors (priming the 40S subunit for initiation), by ligatin (eIF2D) or by density-regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT1). These events may be subverted by suppression of termination (yielding carboxy-terminally extended read-through polypeptides) or by interruption of recycling, leading to reinitiation of translation near the stop codon.

Atomic mutagenesis of stop codon nucleotides reveals the chemical prerequisites for release factor-mediated peptide release.

Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.

Quantitative Study of the Oligomerization of Yeast Prion Sup35NM Proteins.

The fibrillation of misfolded neurodegenerative disease-related proteins has been extensively studied over the past few decades, but the initial oligomerization has been rarely examined even though some recent evidence indicated that small protein oligomers are more neurotoxic than long protein fibers. It is rather difficult to study the initial oligomerization (nucleation) because most experimental methods, such as the ß-sheet-related fluorescence assay and X-ray diffraction, are unable to detect small structureless protein oligomers. In this study, we have successfully developed a method to link a short thermally sensitive poly(N-isopropylacrylamide) (PNIPAM) chain to a model protein, Sup35NM, at a specific 31st residue site (Sup35NM-31m-PNIPAM) via the efficient thiol-ene Michael addition reaction. The oligomerization was studied by a combination of laser light scattering, the thioflavin T assay, and transmission electron microscopy. We found that the lag phase of Sup35NM was delayed from 12 to >24 h under the physiological condition after the PNIPAM linkage. The oligomerization and fibrillation constants decreased from 5.0 × 10-3 to 1.5 × 10-3 h-1 and from 3.0 × 10-2 to 1.8 × 10-2 µM-1 h-1, respectively, presumably because of the steric hindrance introduced by the PNIPAM chain. Moreover, after initiating the oligomerization, we found that the oligomer distribution in the first 6 h repeatedly and quantitatively follows the Smoluchowski coagulation model. Our study paves the way for controllably and quantitatively studying the oligomerization kinetics of amyloidogenic proteins. In addition, by investigating the effects of different small molecules on the oligomerization kinetics, we should be able to screen potential drugs to slow the development of neurodegenerative diseases.

Reassigning stop codons via translation termination: How a few eukaryotes broke the dogma.

The genetic code determines how amino acids are encoded within mRNA. It is universal among the vast majority of organisms, although several exceptions are known. Variant genetic codes are found in ciliates, mitochondria, and numerous other organisms. All revealed genetic codes (standard and variant) have at least one codon encoding a translation stop signal. However, recently two new genetic codes with a reassignment of all three stop codons were revealed in studies examining the protozoa transcriptomes. Here, we discuss this finding and the recent studies of variant genetic codes in eukaryotes. We consider the possible molecular mechanisms allowing the use of certain codons as sense and stop signals simultaneously. The results obtained by studying these amazing organisms represent a new and exciting insight into the mechanism of stop codon decoding in eukaryotes. Also see the video abstract here.

Genome-wide pathway analysis in attention-deficit/hyperactivity disorder.

This study aimed to (1) to identify candidate single-nucleotide polymorphisms (SNPs) and mechanisms of attention-deficit/hyperactivity disorder (ADHD) and (2) to generate SNP-to-gene-to-pathway hypotheses. An ADHD genome-wide association study (GWAS) dataset that included 428,074 SNPs in 924 trios (2,758 individuals) of European descent was used in this study. The Identify candidate Causal SNPs and Pathways (ICSNPathway) analysis was applied to the GWAS dataset. ICSNPathway analysis identified 11 candidate SNPs, 6 genes, and 6 pathways, which provided 6 hypothetical biological mechanisms. The strongest hypothetical biological mechanism was that rs2532502 alters the role of CD27 in the context of the pathways of positive regulation of nucleocytoplasmic transport [nominal p < 0.001; false discovery rate (FDR) = 0.028]. The second strongest mechanism was the rs1820204, rs1052571, rs1052576 → CASP9 → mitochondrial pathway (nominal p < 0.001; FDR = 0.032). The third mechanism was the rs1801516 → ATM → CD25 pathway (nominal p < 0.001; FDR = 0.034). By applying the ICSNPathway analysis to the ADHD GWAS data, 11 candidate SNPs, 6 genes that included CD27, CASP9, ATM, CD12orf65, OXER1, and ACRY, and 6 pathways were identified that may contribute to ADHD susceptibility.

Biology and genetics of prions causing neurodegeneration.

Prions are proteins that acquire alternative conformations that become self-propagating. Transformation of proteins into prions is generally accompanied by an increase in ß-sheet structure and a propensity to aggregate into oligomers. Some prions are beneficial and perform cellular functions, whereas others cause neurodegeneration. In mammals, more than a dozen proteins that become prions have been identified, and a similar number has been found in fungi. In both mammals and fungi, variations in the prion conformation encipher the biological properties of distinct prion strains. Increasing evidence argues that prions cause many neurodegenerative diseases (NDs), including Alzheimer's, Parkinson's, Creutzfeldt-Jakob, and Lou Gehrig's diseases, as well as the tauopathies. The majority of NDs are sporadic, and 10% to 20% are inherited. The late onset of heritable NDs, like their sporadic counterparts, may reflect the stochastic nature of prion formation; the pathogenesis of such illnesses seems to require prion accumulation to exceed some critical threshold before neurological dysfunction manifests.

Polypeptide chain release factor eRF3 is a novel molecular partner of survivin.

The eukaryotic class II polypeptide chain release factor (eRF3) is an eRF1- and ribosome-dependent GTPase involved in translation termination of protein biosynthesis. eRF3 is a multifunctional protein that is also involved in chromosomal segregation and cytokinesis during mitosis. Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is involved in the organisation of spindle and cell apoptosis. Interaction between survivin and eRF3a-F3 or eRF3b, encoded by the GSPT1 and GSPT2 genes, respectively, was confirmed using yeast two-hybrid (Y2H) and pull-down assays in vitro, and co-immunoprecipitation in vivo. The domains involved in the formation of the survivin-eRF3s complex have been identified. The sites on survivin that interact with eRF3 are located in the baculovirus IAP repeat domain (residues 65-76), which forms a beta-strand structure with an overall negative charge. The sites on eRF3 that interact with survivin were localised to the N-terminal domain(NTD; residues 131-200). Cell localisation experiments indicate that both factors are in the nucleus, suggesting that they cooperatively function in nuclear processes.

Biological role of the two overlapping poly(A)-binding protein interacting motifs 2 (PAM2) of eukaryotic releasing factor eRF3 in mRNA decay.

Eukaryotic releasing factor GSPT/eRF3 mediates translation termination-coupled mRNA decay via interaction with a cytosolic poly(A)-binding protein (PABPC1). A region of eRF3 containing two overlapping PAM2 (PABPC1-interacting motif 2) motifs is assumed to bind to the PABC domain of PABPC1, on the poly(A) tail of mRNA. PAM2 motifs are also found in the major deadenylases Caf1-Ccr4 and Pan2-Pan3, whose activities are enhanced upon PABPC1 binding to these motifs. Their deadenylase activities are regulated by eRF3, in which two overlapping PAM2 motifs competitively prevent interaction with PABPC1. However, it is unclear how these overlapping motifs recognize PABC and regulate deadenylase activity in a translation termination-coupled manner. We used a dominant-negative approach to demonstrate that the N-terminal PAM2 motif is critical for eRF3 binding to PABPC1 and that both motifs are required for function. Isothermal titration calorimetry (ITC) and NMR analyses revealed that the interaction is in equilibrium between the two PAM2-PABC complexes, where only one of the two overlapping PAM2 motifs is PABC-bound and the other is PABC-unbound and partially accessible to the other PABC. Based on these results, we proposed a biological role for the overlapping PAM2 motifs in the regulation of deadenylase accessibility to PABPC1 at the 3' end of poly(A).

Translation termination efficiency modulates ATF4 response by regulating ATF4 mRNA translation at 5' short ORFs.

The activating transcription factor 4 (ATF4) promotes transcriptional upregulation of specific target genes in response to cellular stress. ATF4 expression is regulated at the translational level by two short open reading frames (uORFs) in its 5'-untranslated region (5'-UTR). Here, we describe a mechanism regulating ATF4 expression in translation termination-deficient human cells. Using microarray analysis of total RNA and polysome-associated mRNAs, we show that depletion of the eucaryotic release factor 3a (eRF3a) induces upregulation of ATF4 and of ATF4 target genes. We show that eRF3a depletion modifies ATF4 translational control at regulatory uORFs increasing ATF4 ORF translation. Finally, we show that the increase of REDD1 expression, one of the upregulated targets of ATF4, is responsible for the mTOR pathway inhibition in eRF3a-depleted cells. Our results shed light on the molecular mechanisms connecting eRF3a depletion to mammalian target of rapamycin (mTOR) pathway inhibition and give an example of ATF4 activation that bypasses the signal transduction cascade leading to the phosphorylation of eIF2α. We propose that in mammals, in which the 5'-UTR regulatory elements of ATF4 mRNA are strictly conserved, variations in translation termination efficiency allow the modulation of the ATF4 response.

[Allele-specific therapy: suppression of nonsense mutations by readthrough inducers]. / Thérapie spécifique d'allèle - Suppression de mutations non-sens par des inducteurs de « translecture ¼

Ten percent of human hereditary diseases are linked to nonsense mutations (premature termination codon). These mutations lead to premature translation termination, trigger the synthesis of a truncated protein and possibly lead to mRNA degradation by the NMD pathway (nonsense mediated mRNA decay). For the past ten years, therapeutic strategies have emerged which attempt to use molecules that facilitate tRNA incorporation at premature stop codon (readthrough), thus allowing for the synthesis of a full length protein. Molecules currently used for this approach are mostly aminoglycoside antibiotics (gentamicin, amikacin…) that bind the decoding center of the ribosome. This therapeutic approach has been studied for various genetic diseases including Duchenne muscular dystrophy (DMD) and cystic fibrosis. The feasibility of this approach depends on induced readthrough level, mRNA quantity, re-expressed protein functionality and characteristics of each disease.

Opposing effects of glutamine and asparagine govern prion formation by intrinsically disordered proteins.

Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here, we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N richness promotes assembly of benign self-templating amyloids; Q richness promotes formation of toxic nonamyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs.

Optical trapping with high forces reveals unexpected behaviors of prion fibrils.

Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we describe methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A crucial advance was to use the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces before fibril rupture. Experiments with selective amyloid-disrupting agents and mutations demonstrated that such discontinuities were caused by the unfolding of individual subdomains. Thus, our results reveal unusually strong noncovalent intermolecular contacts that maintain fibril integrity even when individual monomers partially unfold and extend fibril length.

Epigenetics in the extreme: prions and the inheritance of environmentally acquired traits.

Prions are an unusual form of epigenetics: Their stable inheritance and complex phenotypes come about through protein folding rather than nucleic acid-associated changes. With intimate ties to protein homeostasis and a remarkable sensitivity to stress, prions are a robust mechanism that links environmental extremes with the acquisition and inheritance of new traits.

Growth temperature-dependent contributions of response regulators, σB, PrfA, and motility factors to Listeria monocytogenes invasion of Caco-2 cells.

Foodborne pathogens encounter rapidly changing environmental conditions during transmission, including exposure to temperatures below 37°C. The goal of this study was to develop a better understanding of the effects of growth temperatures and temperature shifts on regulation of invasion phenotypes and invasion-associated genes in Listeria monocytogenes. We specifically characterized the effects of L. monocytogenes growth at different temperatures (30°C vs. 37°C) on (i) the contributions to Caco-2 invasion of different regulators (including σ(B), PrfA, and 14 response regulators [RRs]) and invasion proteins (i.e., InlA and FlaA), and on (ii) gadA, plcA, inlA, and flaA transcript levels and their regulation. Overall, Caco-2 invasion efficiency was higher for L. monocytogenes grown at 30°C than for bacteria grown at 37°C (p = 0.0051 for the effect of temperature on invasion efficiency; analysis of variance); the increased invasion efficiency of the parent strain 10403S (serotype 1/2a) observed after growth at 30°C persisted for 2.5 h exposure to 37°C. For L. monocytogenes grown at 30°C, the motility RRs DegU and CheY and σ(B), but not PrfA, significantly contributed to Caco-2 invasion efficiency. For L. monocytogenes grown at 37°C, none of the 14 RRs tested significantly contributed to Caco-2 invasion, whereas σ(B) and PrfA contributed synergistically to invasion efficiency. At both growth temperatures there was significant synergism between the contributions to invasion of FlaA and InlA; this synergism was more pronounced after growth at 30°C than at 37°C. Our data show that growth temperature affects invasion efficiency and regulation of virulence-associated genes in L. monocytogenes. These data support increasing evidence that a number of environmental conditions can modulate virulence-associated phenotypes of foodborne bacterial pathogens, including L. monocytogenes.

The chitinolytic activity of Listeria monocytogenes EGD is regulated by carbohydrates but also by the virulence regulator PrfA.

Chitin, an insoluble polymer of N-acetyl-D-glucosamine (GlcNAc), is one of the most abundant carbohydrate polymers in marine and terrestrial environments. Chitin hydrolysis by Listeria monocytogenes depends on two chitinase-encoding genes, chiA and chiB, and the aim of this study was to investigate their regulation. Chitin induces the expression of both chitinases in late exponential growth phase, and chiA but not chiB is furthermore induced by the monomer GlcNAc. Furthermore, their expression is subjected to catabolite control. Chitinases expressed by bacterial pathogens have proven to be important not only for nutrient acquisition and environmental survival but also for infecting animals and humans. Interestingly, the central L. monocytogenes virulence gene regulator, PrfA, is required for the chitinolytic phenotype, as chitinase activity was significantly reduced in prfA mutant cells compared to its level in wild-type cells. In agreement with this, Northern blot analysis showed that the amounts of chiA and chiB transcripts upon induction by chitin were significantly lower in the prfA mutant than in the wild type. The chitinolytic activity and chiA and chiB expression were reduced in the absence of the sigB gene, indicating that σ(B) is also important for the production of chitinases. The chiA, chiB, and chiA chiB mutants were not impaired for in vitro adhesion and invasion in epithelial cell lines, but the chiA chiB double mutant showed less survival ability in a chitin-enriched medium. The regulation of chitinolytic activity in L. monocytogenes is complex, and taken together, the results indicate that the biological role of this activity may not be limited to the external environment.

Terminating human mitochondrial protein synthesis: a shift in our thinking.

Until recently, human mitochondria were regarded as unusual as they appeared to employ four stop codons to terminate translation. In addition to the UAA/UAG of the universal genetic code, two arginine triplets (AGA/AGG) had been re-assigned as termination signals. This posed the conundrum of what factor was responsible for recognizing these triplets to promote translation termination? Recent data indicates that in fact no protein is required to recognize AGA/AGG. Indeed, it is the absence of any cognate factor, tRNA or polypeptide that is important. On encountering either of these 'hungry' codons at the end of an open reading frame, instead of requiring a novel or modified release factor, human mitoribosomes employ -1 frameshifting to reposition a standard UAG codon in the A-site, indicating that only the universal UAA and UAG are used as stop codons. This renders a single mitochondrial release factor, mtRF1a, previously shown to be capable of terminating 11 of the 13 open reading frames encoded by the mitochondrial genome, to be sufficient to release all nascent human mitochondrial gene products from the mitoribosome.

Identification of a cellular factor that modulates HIV-1 programmed ribosomal frameshifting.

Programmed -1 ribosomal frameshifting (PRF) is a distinctive mode of gene expression utilized by some viruses, including human immunodeficiency virus type 1 (HIV-1), to produce multiple proteins from a single mRNA. -1 PRF induces a subset of elongating ribosomes to shift their translational reading frame by 1 base in the 5' direction. The appropriate ratio of Gag to Gag-Pol synthesis is tightly regulated by the PRF signal which promotes ribosomes to shift frame, and even small changes in PRF efficiency, either up or down, have significant inhibitory effects upon virus production, making PRF essential for HIV-1 replication. Although little has been reported about the cellular factors that modulate HIV-1 PRF, the cis-acting elements regulating PRF have been extensively investigated, and the PRF signal of HIV-1 was shown to include a slippery site and frameshift stimulatory signal. Recently, a genome-wide screen performed to identify cellular factors that affect HIV-1 replication demonstrated that down-regulation of eukaryotic release factor 1 (eRF1) inhibited HIV-1 replication. Because of the eRF1 role in translation, we hypothesized that eRF1 is important for HIV-1 PRF. Using a dual luciferase reporter system harboring a HIV-1 PRF signal, results showed that depletion or inhibition of eRF1 enhanced PRF in yeast, rabbit reticulocyte lysates, and mammalian cells. Consistent with the eRF1 role in modulating HIV PRF, depleting eRF1 increased the Gag-Pol to Gag ratio in cells infected with replication-competent virus. The increase in PRF was independent of a proximal termination codon and did not result from increased ribosomal pausing at the slippery site. This is the first time that a cellular factor has been identified which can promote HIV-1 PRF and highlights HIV-1 PRF as essential for replication and an important but under exploited antiviral drug target.

Amyloid oligomers: diffuse oligomer-based transmission of yeast prions.

Prions are infectious proteins, in which self-propagating amyloid conformations of proteins are transmitted. The budding yeast Saccharomyces cerevisiae, one of the best-studied model eukaryotes, also has prions, and thus provides a tractable model system with which to understand the mechanisms of prion phenomena. The yeast prions are protein-based heritable elements, such as [PSI(+)], in which aggregates of prion proteins are transmitted to daughter cells in a non-Mendelian manner. Although the genetic approaches preceded the yeast prion studies, recent investigations of the dynamic aspects of the prion proteins have unraveled the molecular mechanisms by which prions are propagated and transmitted. In particular, several lines of evidence have revealed that the oligomeric species of prion proteins dispersed in the cytoplasm are critical for the transmission. This review summarizes the topics on the transmissible entities of yeast prions, focusing mainly on the Sup35 protein in [PSI(+)].