Repression Of Translation Initiation Research Articles

Synonymous codon usage regulates translation initiation

Nonoptimal synonymous codons repress gene expression, but the underlying mechanisms are poorly understood. We and others have previously shown that nonoptimal codons slow translation elongation speeds and thereby trigger messenger RNA (mRNA) degradation. Nevertheless, transcript levels are often insufficient to explain protein levels, suggesting additional mechanisms by which codon usage regulates gene expression. Using reporters in human and Drosophila cells, we find that transcript levels account for less than half of the variation in protein abundance due to codon usage. This discrepancy is explained by translational differences whereby nonoptimal codons repress translation initiation. Nonoptimal transcripts are also less bound by the translation initiation factors eIF4E and eIF4G1, providing a mechanistic explanation for their reduced initiation rates. Importantly, translational repression can occur without mRNA decay and deadenylation, and it does not depend on the known nonoptimality sensor, CNOT3. Our results reveal a potent mechanism of regulation by codon usage where nonoptimal codons repress further rounds of translation.

4E-BP1-dependent translation in nociceptors controls mechanical hypersensitivity via TRIM32/type I interferon signaling.

Activation of the mechanistic target of rapamycin complex 1 (mTORC1) contributes to the development of chronic pain. However, the specific mechanisms by which mTORC1 causes hypersensitivity remain elusive. The eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is a key mTORC1 downstream effector that represses translation initiation. Here, we show that nociceptor-specific deletion of 4E-BP1, mimicking activation of mTORC1-dependent translation, is sufficient to cause mechanical hypersensitivity. Using translating ribosome affinity purification in nociceptors lacking 4E-BP1, we identified a pronounced translational up-regulation of tripartite motif-containing protein 32 (TRIM32), an E3 ubiquitin ligase that promotes interferon signaling. Down-regulation of TRIM32 in nociceptors or blocking type I interferon signaling reversed the mechanical hypersensitivity in mice lacking 4E-BP1. Furthermore, nociceptor-specific ablation of TRIM32 alleviated mechanical hypersensitivity caused by tissue inflammation. These results show that mTORC1 in nociceptors promotes hypersensitivity via 4E-BP1-dependent up-regulation of TRIM32/interferon signaling and identify TRIM32 as a therapeutic target in inflammatory pain.

Proteostasis regulation through ribosome quality control and no-go-decay.

Cell functionality relies on the existing pool of proteins and their folding into functional conformations. This is achieved through the regulation of protein synthesis, which requires error-free mRNAs and ribosomes. Ribosomes are quality control hubs for mRNAs and proteins. Problems during translation elongation slow down the decoding rate, leading to ribosome halting and the eventual collision with the next ribosome. Collided ribosomes form a specific disome structure recognized and solved by ribosome quality control (RQC) mechanisms. RQC pathways orchestrate the degradation of the problematic mRNA by no-go decay and the truncated nascent peptide, the repression of translation initiation, and the recycling of the stalled ribosomes. All these events maintain protein homeostasis and return valuable ribosomes to translation. As such, cell homeostasis and function are maintained at the mRNA level by preventing the production of aberrant or unnecessary proteins. It is becoming evident that the crosstalk between RQC and the protein homeostasis network is vital for cell function, as the absence of RQC components leads to the activation of stress response and neurodegenerative diseases. Here, we review the molecular events of RQC discovered through well-designed stalling reporters. Given the impact of RQC in proteostasis, we discuss the relevance of identifying endogenous mRNA regulated by RQC and their preservation in stress conditions. This article is categorized under: RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms Translation > Regulation.

19F NMR Untersuchung des Konformationsaustauschs mehrerer Zustände im synthetischen Neomycin-bindenden Riboschalter.

AbstractDer synthetische Neomycin‐bindende Riboschalter interagiert mit seinem Liganden Neomycin sowie mit den verwandten Antibiotika Ribostamycin und Paromomycin. Die Bindung dieser Aminoglykoside induziert sehr ähnliche Grundzustandsstrukturen in der RNA, allerdings kann nur Neomycin die Initiierung der Translation effizient unterdrücken. Der molekulare Ursprung dieser Unterschiede wurde auf Unterschiede in der Dynamik der Ligand‐Riboschalter‐Komplexe zurückgeführt. In diesem Artikel kombinieren wir fünf komplementäre fluorbasierte NMR‐Methoden, um die Dynamik der drei Riboschalter‐Komplexe im Sekunden‐ bis Mikrosekundenbereich genau zu quantifizieren. Unsere Daten offenbaren komplexe Austauschprozesse mit bis zu vier strukturell unterschiedlichen Zuständen. Wir interpretieren unsere Ergebnisse in einem Modell, das ein Zusammenspiel zwischen verschiedenen chemischen Gruppen in den Antibiotika und spezifischen Basen im Riboschalter zeigt. Allgemeiner unterstreichen unsere Daten das Potenzial von 19F NMR‐Methoden, komplexe Austauschprozesse mit mehreren angeregten Zuständen zu charakterisieren.

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19 F NMR.

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19 F NMR methods to characterize complex exchange processes with multiple excited states.

IRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation.

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.

CsrA-Mediated Translational Activation of ymdA Expression in Escherichia coli.

The sequence-specific RNA-binding protein CsrA is the central component of the conserved global regulatory Csr system. In Escherichia coli, CsrA regulates many cellular processes, including biofilm formation, motility, carbon metabolism, iron homeostasis, and stress responses. Such regulation often involves translational repression by CsrA binding to an mRNA target, thereby inhibiting ribosome binding. While CsrA also extensively activates gene expression, no detailed mechanism for CsrA-mediated translational activation has been demonstrated. An integrated transcriptomic study identified ymdA as having the strongest CsrA-mediated activation across the E. coli transcriptome. Here, we determined that CsrA activates ymdA expression posttranscriptionally. Gel mobility shift, footprint, toeprint, and in vitro coupled transcription-translation assays identified two CsrA binding sites in the leader region of the ymdA transcript that are critical for translational activation. Reporter fusion assays confirmed that CsrA activates ymdA expression at the posttranscriptional level in vivo Furthermore, loss of binding at either of the two CsrA binding sites abolished CsrA-dependent activation. mRNA half-life studies revealed that CsrA also contributes to stabilization of ymdA mRNA. RNA structure prediction revealed an RNA hairpin upstream of the ymdA start codon that sequesters the Shine-Dalgarno (SD) sequence, which would inhibit ribosome binding. This hairpin also contains one of the two critical CsrA binding sites, with the other site located just upstream. Our results demonstrate that bound CsrA destabilizes the SD-sequestering hairpin such that the ribosome can bind and initiate translation. Since YmdA represses biofilm formation, CsrA-mediated activation of ymdA expression may repress biofilm formation under certain conditions.IMPORTANCE The Csr system of E. coli controls gene expression and physiology on a global scale. CsrA protein, the central component of this system, represses translation initiation of numerous genes by binding to target transcripts, thereby competing with ribosome binding. Variations of this mechanism are so common that CsrA is sometimes called a translational repressor. Although CsrA-mediated activation mechanisms have been elucidated in which bound CsrA inhibits RNA degradation, no translation activation mechanism has been defined. Here, we demonstrate that CsrA binding to two sites in the 5' untranslated leader of ymdA mRNA activates translation by destabilizing a structure that otherwise prevents ribosome binding. The extensive role of CsrA in activating gene expression suggests the common occurrence of similar activation mechanisms.

A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.

Conserved mRNA-granule component Scd6 targets Dhh1 to repress translation initiation and activates Dcp2-mediated mRNA decay in vivo.

Scd6 protein family members are evolutionarily conserved components of translationally silent mRNA granules. Yeast Scd6 interacts with Dcp2 and Dhh1, respectively a subunit and a regulator of the mRNA decapping enzyme, and also associates with translation initiation factor eIF4G to inhibit translation in cell extracts. However, the role of Scd6 in mRNA turnover and translational repression in vivo is unclear. We demonstrate that tethering Scd6 to a GFP reporter mRNA reduces mRNA abundance via Dcp2 and suppresses reporter mRNA translation via Dhh1. Thus, in a dcp2Δ mutant, tethered Scd6 reduces GFP protein expression with little effect on mRNA abundance, whereas tethered Scd6 has no impact on GFP protein or mRNA expression in a dcp2Δ dhh1Δ double mutant. The conserved LSm domain of Scd6 is required for translational repression and mRNA turnover by tethered Scd6. Both functions are enhanced in a ccr4Δ mutant, suggesting that the deadenylase function of Ccr4-Not complex interferes with a more efficient repression pathway enlisted by Scd6. Ribosome profiling and RNA-Seq analysis of scd6Δ and dhh1Δ mutants suggests that Scd6 cooperates with Dhh1 in translational repression and turnover of particular native mRNAs, with both processes dependent on Dcp2. Our results suggest that Scd6 can (i) recruit Dhh1 to confer translational repression and (ii) activate mRNA decapping by Dcp2 with attendant degradation of specific mRNAs in vivo, in a manner dependent on the Scd6 LSm domain and modulated by Ccr4.

Translational Repression of the RpoS Antiadapter IraD by CsrA Is Mediated via Translational Coupling to a Short Upstream Open Reading Frame.

ABSTRACTCsrA is a global regulatory RNA binding protein that has important roles in regulating carbon metabolism, motility, biofilm formation, and numerous other cellular processes. IraD functions as an antiadapter protein that inhibits RssB-mediated degradation of RpoS, the general stress response and stationary-phase sigma factor of Escherichia coli. Here we identified a novel mechanism in which CsrA represses iraD translation via translational coupling. Expression studies with quantitative reverse transcriptase PCR, Western blotting, and lacZ fusions demonstrated that CsrA represses iraD expression. Gel mobility shift, footprint, and toeprint studies identified four CsrA binding sites in the iraD leader transcript, all of which are far upstream of the iraD ribosome binding site. Computational modeling and RNA structure mapping identified an RNA structure that sequesters the iraD Shine-Dalgarno (SD) sequence. Three open reading frames (ORFs), all of which are translated, were identified in the iraD leader region. Two of these ORFs do not affect iraD expression. However, the translation initiation region of the third ORF contains three of the CsrA binding sites, one of which overlaps its SD sequence. Furthermore, the ORF stop codon overlaps the iraD start codon, a sequence arrangement indicative of translational coupling. In vivo expression and in vitro translation studies with wild-type and mutant reporter fusions demonstrated that bound CsrA directly represses translation initiation of this ORF. We further established that CsrA-dependent repression of iraD translation occurs entirely via translational coupling with this ORF, leading to accelerated iraD mRNA decay.

Differentiation of human keratinocytes requires translational control by the eIF2 kinase GCN2

In the human epidermis, differentiation of keratinocytes is essential to form a barrier to protect against a variety of environmental insults. This differentiation process can become dysregulated in many skin diseases, including psoriasis, eczema, and non‐melanoma skin cancer. Although changes in transcriptional networks during keratinocyte differentiation are well characterized, little is known about the role of translational control. A major mechanism of translational control involves phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2‐P). In response to environmental stresses, induced eIF2‐P represses translation initiation, allowing cells to conserve resources and trigger cytoprotective gene expression. In addition to repression of global protein synthesis, eIF2‐P induces preferential translation of specific mRNAs that promote stress adaptation, including the transcription factors ATF4 and CHOP. Since keratinocytes undergo a massive change in morphology and gene expression during differentiation, we hypothesized that translational control plays a major role. To investigate this hypothesis we measured both genome‐wide and gene‐specific levels of translation. We observed a global reduction in translation initiation as measured by polysome profiling during differentiation in both monolayer and 3D organotypic keratinocyte cultures. Expression of eIF2‐P, ATF4, and CHOP was limited to suprabasal (differentiated) keratinocytes in vivo as visualized histologically in human skin tissue. Keratinocyte differentiation in vitro induced eIF2‐P to levels similar to that seen following endoplasmic reticulum stress, but eIF2‐P alone was not sufficient to induce differentiation. Of interest, the epidermal differentiation transcript involucrin (IVL) was preferentially bound to large polysomes following differentiation, indicative of preferential translation, which was similar to that seen for ATF4 and CHOP. Inhibition of eIF2‐P blocked both general and gene‐specific translational control during differentiation. Loss of either eIF2‐P or the upstream protein kinase GCN2 abrogated both IVL protein and mRNA expression. Strikingly, loss of GCN2 altered epidermal tissue formation in a 3D organotypic model as characterized by disorganized keratinocyte layers and a decreased number of cornified cells. These results provide novel evidence that a whole‐cell repression of protein synthesis alongside gene‐specific translational control occur during normal epidermal differentiation. These findings suggest that translational control through GCN2/eIF2‐P is important for proper epidermal stratification, likely though translational regulation of differentiation genes such as IVL. The GCN2/eIF2‐P pathway in keratinocyte biology provides for novel targets for intervention in dermatological diseases characterized by abnormal differentiation.Support or Funding InformationRuth L. Kirschstein National Research Service Award (1F31ES026517 to AEC)

Nuclear Matrix Protein 4 Is a Novel Regulator of Ribosome Biogenesis and Controls the Unfolded Protein Response via Repression of Gadd34 Expression

The unfolded protein response (UPR) maintains protein homeostasis by governing the processing capacity of the endoplasmic reticulum (ER) to manage ER client loads; however, key regulators within the UPR remain to be identified. Activation of the UPR sensor PERK (EIFAK3/PEK) results in the phosphorylation of the α subunit of eIF2 (eIF2α-P), which represses translation initiation and reduces influx of newly synthesized proteins into the overloaded ER. As part of this adaptive response, eIF2α-P also induces a feedback mechanism through enhanced transcriptional and translational expression of Gadd34 (Ppp1r15A),which targets type 1 protein phosphatase for dephosphorylation of eIF2α-P to restore protein synthesis. Here we describe a novel mechanism by which Gadd34 expression is regulated through the activity of the zinc finger transcription factor NMP4 (ZNF384, CIZ). NMP4 functions to suppress bone anabolism, and we suggest that this occurs due to decreased protein synthesis of factors involved in bone formation through NMP4-mediated dampening of Gadd34 and c-Myc expression. Loss of Nmp4 resulted in an increase in c-Myc and Gadd34 expression that facilitated enhanced ribosome biogenesis and global protein synthesis. Importantly, protein synthesis was sustained during pharmacological induction of the UPR through a mechanism suggested to involve GADD34-mediated dephosphorylation of eIF2α-P. Sustained protein synthesis sensitized cells to pharmacological induction of the UPR, and the observed decrease in cell viability was restored upon inhibition of GADD34 activity. We conclude that NMP4 is a key regulator of ribosome biogenesis and the UPR, which together play a central role in determining cell viability during endoplasmic reticulum stress.

Dynamics of Translation of Single mRNA Molecules In Vivo

SummaryRegulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5′ UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.

Evidence That Base-pairing Interaction between Intron and mRNA Leader Sequences Inhibits Initiation of HAC1 mRNA Translation in Yeast

The Hac1 transcription factor in yeast up-regulates a collection of genes that control protein homeostasis. Base-pairing interactions between sequences in the intron and the 5'-untranslated region (5' UTR) of the HAC1 mRNA represses Hac1 protein production under basal conditions, whereas cytoplasmic splicing of the intron by the Ire1 kinase-endonuclease, activated under endoplasmic reticulum stress conditions, relieves the inhibition and enables Hac1 synthesis. Using a random mutational screen as well as site-directed mutagenesis, we identify point mutations within the 5' UTR-intron interaction site that derepress translation of the unspliced HAC1 mRNA. We also show that insertion of an in-frame AUG start codon upstream of the interaction site releases the translational block, demonstrating that an elongating ribosome can disrupt the interaction. Moreover, overexpression of translation initiation factor eIF4A, a helicase, enhances production of Hac1 from an mRNA containing an upstream AUG start codon at the beginning of the base-paired region. These results suggest that the major block of translation occurs at the initiation stage. Supporting this interpretation, the point mutations that enhanced Hac1 production resulted in an increased percentage of the HAC1 mRNA associating with polysomes versus free ribosomal subunits. Thus, our results provide evidence that the 5' UTR-intron interaction represses translation initiation on the unspliced HAC1 mRNA.

Metabolic Re-Programming in Notch-Activated T-ALL By mTOR Inhibitor AZD2014 Combined with L-Asparaginase

Adult T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis associated with resistance and rapid relapses, necessitating novel therapeutic strategies. Malignant T cells require high concentrations of nutrients to sustain their increased rates of proliferation, and frequent activation of mTOR signaling, a central regulator of cell metabolism, has been shown in a high-risk subgroup of T-ALL with constitutive Notch activation (Chan, Blood, 2007;110,278).In this study, we analyzed the metabolic response mechanisms of Notch-activated aggressive T-ALL to the ATP-competitive mTOR inhibitor AZD2014 and to the combination of AZD2014 with a conventional chemotherapeutic agent, L-asparaginase (ASNase). To investigate the molecular modifications of key cellular metabolic processes induced by these agents, we undertook a comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS, Agilent Technologies) for metabolic profiling.We first assessed the anti-proliferative effects of the combination of AZD2014 and ASNase in CUTLL1, a T-ALL cell line with a Notch gain-of-function mutation. The medianinhibitory concentrations of AZD-2014 and ASNase were 250 nM and 0.005 U/mL, respectively (MTT assay, 48 hours). The combination of AZD2014 (100 nM) and ASNase (0.001 U/mL) caused apoptosis induction with increase in the % of sub-G1 fraction (Control: 5.5±1.8, AZD-2014: 9.5±2.1, ASNase: 10.9 ±1.5, AZD-2014/ASNase: 17.9 ±4.9; p<0.05). Inhibition of mTOR kinase activity by AZD2014 resulted in downregulation of mTORC1 substrates phospho-(p-)S6K (Ser240-244) and p-4EBP1 (Thr37/46) and mTORC2 target p-Akt (Ser473) and induced autophagy, as shown by the conversion of the autophagosomal marker LC3-I to LC-II. AZD2014 further impaired the expression of oncogenic transcription factor c-Myc, an intermediary between Notch and mTOR signaling. In turn, ASNase downregulated c-Myc expression without inhibiting mTOR signaling.We next investigated the metabolic changes in CUTLL1 cells after AZD2014 (100 nM) and/or ASNase (0.001 U/mL) treatment (24 hours). CE-MS analysis demonstrated differences in 66 polar metabolites (p<0.05). As expected, ASNase downregulated L-asparagine monohydrate levels. Although AZD2014 did not change the level of L-asparagine monohydrate by itself, it significantly enhanced its depletion by ASNase. The AZD2014 and ASNase combination also impaired aspartate synthesis. In the TCA cycle, the AZD2014 / ASNase combination significantly downregulated gluconeogenesis by decreasing malate and fructose-6-phosphate and inhibited fatty acid synthesis with citrate depletion. In nucleotide metabolism, the combination treatment suppressed guanine and hypoxanthine levels, which led to decreases in uric acid. Finally, glutamate metabolism was markedly downregulated by depleting intracellular glutamine. All these changes were less prominent in CUTLL1 cells treated with single-agent AZD2014 or ASNase.These results indicate that AZD2014 effectively facilitates the ASNase-induced blockade of glycolysis/gluconeogenesis and fatty acid synthesis and represses asparate and glutamate metabolism in Notch-activated T-ALL cells. The multiple molecular alterations resulting from mTOR blockade by AZD2014, such as repression of translation initiation through potent inhibition of S6K and 4EBP1, induction of autophagy, and inhibition of c-Myc, may further impair the cellular metabolism altered by ASNase in aggressive T-ALL. In conclusion, metabolic re-programming by the combination of AZD2014 and L-asparaginase in Notch-activated T-ALL provides avenues for novel rationally designed combination regimens in ALL. DisclosuresNo relevant conflicts of interest to declare.

MicroRNAs Trigger Dissociation of eIF4AI and eIF4AII from Target mRNAs in Humans

In animals, key functions of microRNA-induced silencing complex (miRISC) are translational repression and deadenylation followed by mRNA decay. While miRISC represses translation initiation, it is poorly understood how miRISC exerts this function. Here we assessed the effect of miRISC on synergistic recruitment of translation initiation factors to target mRNAs by using direct biochemical assays. We show that miRISC promotes eIF4AI and eIF4AII release from target mRNAs prior to dissociation of eIF4E and eIF4G in a deadenylation-independent manner. Strikingly, miRISC-induced release of eIF4AI and eIF4AII from target mRNAs and miRISC-induced inhibition of cap-dependent translation can both be counteracted by the RNA-binding protein HuD via a direct interaction of HuD with eIF4A. Furthermore, the pharmacological eIF4A inhibitor silvestrol, which locks eIF4A on mRNAs, conferred resistance to miRNA-mediated translational repression. In summary, we propose that both eIF4AI and eIF4AII are functionally important targets in miRISC-mediated translation control.

MicroRNAs Block Assembly of eIF4F Translation Initiation Complex in Drosophila

miRNAs silence their complementary target mRNAs by translational repression as well as by poly(A) shortening and mRNA decay. In Drosophila, miRNAs are typically incorporated into Argonaute1 (Ago1) to form the effector complex called RNA-induced silencing complex (RISC). Ago1-RISC associates with a scaffold protein GW182, which recruits additional silencing factors. We have previously shown that miRNAs repress translation initiation by blocking formation of the 48S and 80S ribosomal complexes. However, it remains unclear how ribosome recruitment is impeded. Here, we examined the assembly of translation initiation factors on the target mRNA under repression. We show that Ago1-RISC induces dissociation of eIF4A, a DEAD-box RNA helicase, from the target mRNA without affecting 5' cap recognition by eIF4E in a manner independent of GW182. In contrast, direct tethering of GW182 promotes dissociation of both eIF4E and eIF4A. We propose that miRNAs act to block the assembly of the eIF4F complex during translation initiation.

Molecular Insights into microRNA-Mediated Translational Repression in Plants

microRNAs (miRNAs) bind Argonaute proteins in order to form RNA-induced silencing complexes (RISCs) that can silence the expression of complementary mRNAs. Plant miRNAs can mediate the cleavage of their target mRNAs as well as the repression of their translation. Here, by using an in vitro system prepared from plant culture cells, we biochemically dissect the mechanisms by which Arabidopsis thaliana ARGONAUTE1 RISC (AtAGO1-RISC) silences its mRNA targets. We find that AtAGO1-RISC has the ability to repress translation initiation without promoting deadenylation or mRNA decay. Strikingly, AtAGO1-RISC bound in the 5' untranslated region or the open reading frame can sterically block the recruitment or movement of ribosomes. These silencing effects require more extensive base pairing to the target site in comparison to typical animal miRNAs. Our data provide mechanistic insights into miRNA-mediated translational repression in plants.

GCN2 and TOR converge on aging

Coordinated responses in all tissues upon nutrient stress ensure the survival of an organism in periods of food unavailability. Two evolutionarily conserved, nutrient-sensing signaling pathways that promote stress adaptation following starvation are the general amino acid control pathway that activates GCN2 kinase and the target of rapamycin (TOR) kinase pathway [1]. In response to amino acid deprivation GCN2 is activated, upon binding of uncharged tRNAs, whereas TOR is inhibited through regulation of its localization. In both cases the outcome is reduction of global protein synthesis, albeit different mechanisms are involved. GCN2 directly phosphorylates the eukaryotic initiation factor 2α (eIF2α), whereas inactivation of TOR dephosphorylates the ribosomal protein S6 kinase (S6K) and the eukaryotic translation initiation factor 4E (eIF4)-binding protein (4E-BP). The result is repression of translation initiation of most mRNAs, accompanied by favored translation of specific mRNAs. Translational control of gene expression in response to nutrient and other environmental stresses contributes to stress management via energy saving and selective synthesis of stress-responsive proteins [2]. These effects might be also responsible for lifespan extension through down-regulation of mRNA translation. Inhibition of TOR signaling, by genetic or pharmacological means, has been associated with treatment of several diseases and increased lifespan in many organisms, including humans [3]. Similarly, a robust nutritional intervention that slows aging and its related pathology in diverse species is dietary restriction (DR), the effects of which, at the molecular level, are attributed largely to TOR inhibition. In consistence, deficiency of downstream targets of TOR such as S6K or translation factors/regulators extends lifespan in model systems [4]. Also, under conditions of impaired TOR, the activation of autophagy, a catabolic process that enhances degradation and recycling of damaged cellular components during ageing, contributes to longevity. Interestingly, regulation of autophagy in response to nutrient starvation involves GCN2 signaling, in yeast and mammalian cells [5]. Despite that both GCN2 and TOR can sense nutrient deprivation and regulate protein synthesis and autophagy, the impact of GCN2 signaling in longevity and its connection to TOR is not clear. Evidence in mice indicates that reduced amino acid levels activate GCN-2 and can suppress TOR activity on its targets S6K and 4E-BP. In our recent work [6], we showed that GCN-2 in Caenorhabditis elegans can influence the lifespan of nutrient-sensitized worms, through regulation of TOR signaling. Deletion of gcn-2 had no effect on growth and lifespan of wild-type animals under normal conditions but reduced their lifespan under amino acid limitation, as this was recapitulated through RNAi-mediated silencing of aminoacyl-tRNA synthetases genes. Moreover, disruption of gcn-2 decreased the long-life of eat-2 mutants (a genetic model of DR) and of TOR-defective worms. Longevity conferred by inactivation of the downstream target of TOR rsks-1/S6K, but not of ife-2/eIF4E, was also affected by GCN-2 loss. In C. elegans, the TOR-S6K signaling antagonizes a forkhead transcription factor, PHA-4/FoxA, with roles in early development, survival of first stage (L1) larvae under starvation and DR-mediated longevity of adults [7, 8]. We revealed that GCN-2 was required for the induction of pha-4 under conditions of amino acid deprivation or TOR inactivation. Consequently, the expression of known PHA-4 target genes, involved in stress defense and autophagy, was induced in a GCN-2-dependent manner thus contributing to stress survival and longevity of worms under nutrient or other stress (Figure ​(Figure11). Figure 1 Model depicting the function of GCN-2 in response to nutrient or environmental stress and its impact on survival and longevity of C. elegans. Although the ways in which PHA-4 regulates lifespan are not well-defined they may be relevant to the mechanisms used by L1 larvae to survive starvation [8]. PHA-4 has a broad role in regulation of gene expression but can preferentially bind to specific set of genes under distinct developmental or environmental conditions. Among the candidate PHA-4 targets in starved larvae are stress-responsive and autophagic genes, some of which are PHA-4-regulated in dietary restricted adults, suggesting common mechanisms. Intriguingly, genes involved in metabolic processes such as many nuclear hormone receptors (NHRs) and multiple regulators of sterol and fatty-acid metabolism constitute candidate targets of PHA-4 in starved larvae. Lipid metabolism and fat storage occurs mostly in the intestine of worms, where PHA-4 is induced upon DR, and has been linked to lifespan regulation. Thus, alterations in metabolic pathways in specific tissues, beyond cellular protection, could contribute to longevity induced by TOR inactivation during nutrient stress, but their dependence on GCN-2 remains to be addressed. Also, important questions remain with respect to the interplay between the GCN-2 and other stress signaling pathways that modulate lifespan under such conditions. Elucidation of the above is of great interest, as certain amino acid-restricted diets in rodents extend lifespan, improve their metabolic traits and confer protection from various age-related diseases.

An engineered small RNA-mediated genetic switch based on a ribozyme expression platform

An important requirement for achieving many goals of synthetic biology is the availability of a large repertoire of reprogrammable genetic switches and appropriate transmitter molecules. In addition to engineering genetic switches, the interconnection of individual switches becomes increasingly important for the construction of more complex genetic networks. In particular, RNA-based switches of gene expression have become a powerful tool to post-transcriptionally program genetic circuits. RNAs used for regulatory purposes have the advantage to transmit, sense, process and execute information. We have recently used the hammerhead ribozyme to control translation initiation in a small molecule-dependent fashion. In addition, riboregulators have been constructed in which a small RNA acts as transmitter molecule to control translation of a target mRNA. In this study, we combine both concepts and redesign the hammerhead ribozyme to sense small trans-acting RNAs (taRNAs) as input molecules resulting in repression of translation initiation in Escherichia coli. Importantly, our ribozyme-based expression platform is compatible with previously reported artificial taRNAs, which were reported to act as inducers of gene expression. In addition, we provide several insights into key requirements of riboregulatory systems, including the influences of varying transcriptional induction of the taRNA and mRNA transcripts, 5′-processing of taRNAs, as well as altering the secondary structure of the taRNA. In conclusion, we introduce an RNA-responsive ribozyme-based expression system to the field of artificial riboregulators that can serve as reprogrammable platform for engineering higher-order genetic circuits.