Translational Switch Research Articles

Synthetic human cell fate regulation by protein-driven RNA switches

Understanding how to control cell fate is crucial in biology, medical science and engineering. In this study, we introduce a method that uses an intracellular protein as a trigger for regulating human cell fate. The ON/OFF translational switches, composed of an intracellular protein L7Ae and its binding RNA motif, regulate the expression of a desired target protein and control two distinct apoptosis pathways in target human cells. Combined use of the switches demonstrates that a specific protein can simultaneously repress and activate the translation of two different mRNAs: one protein achieves both up- and downregulation of two different proteins/pathways. A genome-encoded protein fused to L7Ae controlled apoptosis in both directions (death or survival) depending on its cellular expression. The method has potential for curing cellular defects or improving the intracellular production of useful molecules by bypassing or rewiring intrinsic signal networks.

Enterovirus-Induced miR-141 Contributes to Shutoff of Host Protein Translation by Targeting the Translation Initiation Factor eIF4E

Viruses rely on the host translation machinery to complete their life cycles. Picornaviruses use an internal ribosome entry site to initiate cap-independent protein translation and in parallel host cap-dependent translation is shut off. This process is thought to occur primarily via cleavage of host translation initiation factors eIF4GI and eIF4GII by viral proteases. Here we describe another mechanism whereby miR-141 induced upon enterovirus infection targets the cap-dependent translation initiation factor, eIF4E, for shutoff of host protein synthesis. Knockdown of miR-141 reduces viral propagation, and silencing of eIF4E can completely reverse the inhibitory effect of the miR-141 antagomiR on viral propagation. Ectopic expression of miR-141 promotes the switch from cap-dependent to cap-independent translation. Moreover, we identified a transcription factor, EGR1, which is partly responsible for miR-141 induction in response to enterovirus infection. Our results suggest that upregulation of miR-141 upon enterovirus infection can facilitate viral propagation by expediting the translational switch.

An improved translational switch for long term maintenance of synaptic plasticity

Memory lasts a lifetime, yet the physiological substrate of memory, synaptic contacts, are composed of proteins that have much shorter lifetimes. A physiological analog of memory formation, long-term potentiation (LTP), has a late protein synthesis dependent phase (L-LTP) that can last for many hours in slices, or even days in vivo [1,2]. Our previous studies show that maintenance of L-LTP and memory can be accounted by persistent regulation of on-site synthesis of plasticity-related proteins by a self-sustaining regulation of translation. It has been shown that a αCaMKII-CPEB1 molecular pair can act as a bistable switch with different total amounts of αCaMKII in potentiated and non-potentiated synapses [3]. The molecular interaction model in our previous study comprised αCaMKII which could be in an inactive, active and active and phosphorylated forms together with a translation regulating molecule CPEB1, which can be in an active or inactive form. The model included both degradation and new protein synthesis of αCaMKII. We have showen that this model is bistable [3]. The bistability was caused by interaction of Ca2+-Calmodulin dependent and auto-phosphorylation activation, spontaneous degradation and synthesis loops of αCaMKII. This model could successfully account for maintenance of L-LTP over a long period of time and also proposes an explanation for why application of protein synthesis and αCaMKII inhibitors at induction and maintenance phases of L-LTP result in very different outcomes [3-5] However, the protein synthesis loop in our previous model was very simplistic. Here, we suggest a more detailed model of translation with explicit implementation of mRNA and poly-ribosome concentration in the pre-synaptic spine. We assume that activated CBEB1activates mRNA which then binds preferentially to poly-ribosome, as compared to a non-active mRNA, for αCaMKII synthesis. We show that this system can act as a bistable switch. We also look at the behavior of this system at low poly-ribosome and mRNA concentration levels using stochastic simulations with Gillespie algorithm.

Can a local, PKMξ dependent translational switch account for the maintenance of synaptic plasticity?

Memory can last a human lifetime, yet at the synaptic level it is encoded by proteins that turnover at rates that are much faster than the lifetime of memory. A critical open question in the neuroscience of learning and memory is how memory and its cellular basis synaptic plasticity can be maintained for longer than its molecular substrates. Recently it has been shown that the activity of a brain specific kinase, PKMξ, is necessary for the maintenance but not the induction of synaptic plasticity. It is also known that synthesis of new proteins is necessary for inducing long lasting synaptic plasticity and memory. We postulate that a molecular network that includes activation, translation and degradation of PKMξ can be a bi-stable molecular switch, that can account for the maintenance of long-term synaptic plasticity and memory. We formulate a mathematical model of such a molecular network, and show that it can be bi-stable and that it accounts for many of the experimental observations regarding the activation, synthesis and inhibition of PKMξ. Our model also makes testable experimental predictions.

Deregulation of oncogene-induced senescence and p53 translational control in X-linked dyskeratosis congenita

Defects in ribosome biogenesis and function are present in a growing list of human syndromes associated with cancer susceptibility. One example is X-linked dyskeratosis congenita (X-DC) in which the DKC1 gene, encoding for an enzyme that modifies ribosomal RNA, is found to be mutated. How ribosome dysfunction leads to cancer remains poorly understood. A critical cellular response that counteracts cellular transformation is oncogene-induced senescence (OIS). Here, we show that during OIS, a switch between cap- and internal ribosome entry site (IRES)-dependent translation occurs. During this switch, an IRES element positioned in the 5'untranslated region of p53 is engaged and facilitates p53 translation. We further show that in DKC1(m) cells, p53 IRES-dependent translation is impaired during OIS ex vivo and on DNA damage in vivo. This defect in p53 translation perturbs the cellular response that counteracts oncogenic insult. We extend these findings to X-DC human patient cells in which similar impairments in p53 IRES-dependent translation are observed. Importantly, re-introduction of wild-type DKC1 restores p53 expression in these cells. These results provide insight into the basis for cancer susceptibility in human syndromes associated with ribosome dysfunction.

Biomolecular engineering of intracellular switches in eukaryotes

Tools to selectively and reversibly control gene expression are useful to study and model cellular functions. When optimized, these cellular switches can turn a protein's function "on" and "off" based on cues designated by the researcher. These cues include small molecules, drugs, hormones, and even temperature variations. Here we review three distinct areas in gene expression that are commonly targeted when designing cellular switches. Transcriptional switches target gene expression at the level of mRNA polymerization, with examples including the tetracycline gene induction system as well as nuclear receptors. Translational switches target the process of turning the mRNA signal into protein, with examples including riboswitches and RNA interference. Post-translational switches control how proteins interact with one another to attenuate or relay signals. Examples of post-translational modification include dimerization and intein splicing. In general, the delay times between switch and effect decreases from transcription to translation to post-translation; furthermore, the fastest switches may offer the most elegant opportunities to influence and study cell behavior. We discuss the pros and cons of these strategies, which directly influence their usefulness to study and implement drug targeting at the tissue and cellular level.

Design of an overhead crane for the ITER NB cell remote handling maintenance operations

In the neutral beam cell of ITER all the maintenance operations on the neutral beam components (BLC's) must be performed by an overhead crane of large payload capability (30–50 tonnes). A crane system is presented consisting of a monorail, a carriage, and a lifting mechanism. The monorail must give access to the BLC's in the beam line vessel, the front components connecting the NB vessel with the Tokamak, and a storage area at the north end of the NB cell. Rail switching points are required at the intersections between radial and toroidal branches. A translational switching mechanism is proposed. The crane carriage consists of two independent sub-carriages, each composed of four wheels. A set of four secondary wheels attached to the main carriage prevents the crane tilting due to the CoG misalignment of some loads. The elevation system proposed consists of an electromechanical crane of four independent drums and 50 tonnes payload. In parallel with the crane design, a logistics and space availability study has been carried out, leading to the detection of clearance or transport problems that could be taken into account in the final crane design.

Scheme for doubling the number of wings in hyperchaotic attractors

The construction methods for designing multi-wing attractors are rarely mentioned little. With the application of coordinate translation, mirror mapping and hysteresis switching to an existing five-dimensional hyperchaotic system, we can double the number of wings and a four-wing attractor is realized. If similar operations have been applied to the system n-1 times, a 2n-wing attractor can be constructed. A simple circuit implementation was designed for doubling the number of wings in attractors. On the basis of preserving the hyperchaotic property of resulting system, the complexity of attractor is enhanced and it is more suitable for use in secure encryption communication.

Translational switch for long‐term maintenance of synaptic plasticity

Memory can last a lifetime, yet synaptic contacts that contribute to the storage of memory are composed of proteins that have much shorter lifetimes. A physiological model of memory formation, long-term potentiation (LTP), has a late protein-synthesis-dependent phase (L-LTP) that can last for many hours in slices or even for days in vivo. Could the activity-dependent synthesis of new proteins account for the persistence of L-LTP and memory? Here, we examine the proposal that a self-sustaining regulation of translation can form a bistable switch that can persistently regulate the on-site synthesis of plasticity-related proteins. We show that an αCaMKII–CPEB1 molecular pair can operate as a bistable switch. Our results imply that L-LTP should produce an increase in the total amount of αCaMKII at potentiated synapses. This study also proposes an explanation for why the application of protein synthesis and αCaMKII inhibitors at the induction and maintenance phases of L-LTP result in very different outcomes.

Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of multiple components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, Pol III, and rDNA1,2. An outstanding question is whether and how increasing the cellular protein synthesis capacity can affect the multi-step process leading to cancer. We utilized ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in Eμ–Myc/+ transgenic mice to normal levels and show that in this context Myc's oncogenic potential is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a novel paradigm that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation employed to regulate the expression of selective mRNAs. We show that an aberrant increase in cap-dependent translation downstream Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site (IRES)-dependent translation required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic-specific expression of the endogenous IRES-dependent form of Cdk11 (p58-PITSLRE)3-5, which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in Eμ–Myc/+ mice. When accurate translational control is re-established in Eμ–Myc/+ mice, genome instability is suppressed. Our findings reveal how perturbations in translational control provide a highly specific outcome on gene expression, genome stability, and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post-genomic level.

Translation and Code Switching in the Lyrics of Bilingual Popular Songs

This paper examines the ways in which translation and code switching may be exploited in the creation of song lyrics featuring more than one language and points out some contrasts between the functions they fulfil in such songs and the ways they are exploited elsewhere. The discussion is based on illustrations drawn from a variety of sources, ranging from Western pop to North African rai music. The strategies identified include using translation to either replace or reduplicate the source material, rewriting with varying degrees of divergence from the original, juxtaposing components from different languages, and composing directly in a code switching variety. It is argued that in such lyrics translation and code switching often serve to produce very similar effects. Both may be used as affirmations of identity, as in-group markers, as stylistic devices, as a means of opening up the lyrics to outsiders or of producing effects such as alienation and exclusion. The effects achieved in particular cases are related to such variables as the identity of the performer/persona, the background of the audience and the specific content and theme of the song.

Trans-Translation is Involved in the CcpA-Dependent Tagging and Degradation of TreP in Bacillus subtilis

TreP [trehalose-permease (phosphotransferase system (PTS) trehalose-specific enzyme IIBC component)] is one of the target proteins of tmRNA-mediated trans-translation in Bacillus subtilis [Fujihara et al. (2002) Detection of tmRNA-mediated trans-translation products in Bacillus subtilis. Genes Cells, 7, 343-350]. The TreP synthesis is subject to CcpA-dependent carbon catabolite repression (CCR), and the treP gene contains catabolite-responsive element (cre) sequence, a binding site of repressor protein CcpA, in the coding region. Here, we demonstrated that the tmRNA-tagging of TreP occurs depending on the gene for CcpA. In the presence of CcpA, the transcription of treP mRNA terminates at 8-9 nucleotides upstream of the 5'-edge of the internal cre sequence, and translational switch to the tag-sequence occurs at the 101st amino-acid (asparagine) position from N-terminus of TreP. The results show that trans-translation reaction is involved in the tagging and degradation of the N-terminal TreP fragment produced by truncated mRNA, which is a product of transcriptional roadblock by CcpA binding to the cre sequence in the internal coding region.

Translational switch for long term maintenance of synaptic plasticity

Memory can last a lifetime, yet synaptic contacts that contribute to the storage of memory are composed of proteins that have much shorter lifetimes. A physiological model of memory formation, long-term potentiation (LTP), has a late protein-synthesis-dependent phase (L-LTP) that can last for many hours in slices or even for days in vivo. Could the activity-dependent synthesis of new proteins account for the persistence of L-LTP and memory? Here, we examine the proposal that a selfsustaining regulation of translation can form a bistable switch that can persistently regulate the onsite synthesis of plasticity-related proteins. We show that an aCaMKII–CPEB1 molecular pair can operate as a bistable switch. Our results imply that L-LTP should produce an increase in the total amount of aCaMKII at potentiated synapses. This study also proposes an explanation for why the application of protein synthesis and aCaMKII inhibitors at the induction and maintenance phases of L-LTP result in very different outcomes.

Spatial Regulation of nanos Is Required for Its Function in Dendrite Morphogenesis

Spatial control of mRNA translation can generate cellular asymmetries and functional specialization of polarized cells like neurons. A requirement for the translational repressor Nanos (Nos) in the Drosophila larval peripheral nervous system (PNS) implicates translational control in dendrite morphogenesis [1]. Nos was first identified by its requirement in the posterior of the early embryo for abdomen formation [2]. Nos synthesis is targeted to the posterior pole of the oocyte and early embryo through translational repression of unlocalized nos mRNA coupled with translational activation of nos mRNA localized at the posterior pole [3, 4]. Abolishment of nos localization prevents abdominal development, whereas translational derepression of unlocalized nos mRNA suppresses head/thorax development, emphasizing the importance of spatial regulation of nos mRNA [3, 5]. Loss and overexpression of Nos affect dendrite branching complexity in class IV dendritic arborization (da) neurons, suggesting that nos also might be regulated in these larval sensory neurons [1]. Here, we show that localization and translational control of nos mRNA are essential for da neuron morphogenesis. RNA-protein interactions that regulate nos translation in the oocyte and early embryo also regulate nos in the PNS. Live imaging of nos mRNA shows that the cis-acting signal responsible for posterior localization in the oocyte/embryo mediates localization to the processes of class IV da neurons but suggests a different transport mechanism. Targeting of nos mRNA to the processes of da neurons may reflect a local requirement for Nos protein in dendritic translational control.

A Hypoxia-Controlled Cap-Dependent to Cap-Independent Translation Switch in Breast Cancer

Translational regulation is critical in cancer development and progression. Translation sustains tumor growth and development of a tumor vasculature, a process known as angiogenesis, which is activated by hypoxia. Here we first demonstrate that a majority of large advanced breast cancers overexpress translation regulatory protein 4E-BP1 and initiation factor eIF4G. Using model animal and cell studies, we then show that overexpressed 4E-BP1 and eIF4G orchestrate a hypoxia-activated switch from cap-dependent to cap-independent mRNA translation that promotes increased tumor angiogenesis and growth at the level of selective mRNA translation. Elevated levels of 4E-BP1 trigger hypoxia inhibition of cap-dependent mRNA translation at high-oxygen levels and, with eIF4G, increase selective translation of mRNAs containing internal ribosome entry sites (IRESs) that include key proangiogenic, hypoxia, and survival mRNAs. The switch from cap-dependent to cap-independent mRNA translation facilitates tumor angiogenesis and hypoxia responses in animal models.

Going for baroque at the Escherichia coli K1 cell surface

Phase variation is usually thought of as the stochastic switching between alternatively expressed ('on') and unexpressed ('off') phenotypic states. However, coupling synthesis of a monotonous homopolysaccharide to a mechanism of random but incomplete chemical modification produces almost infinite structural variation. Potentially limitless variability implies that evolution can produce highly ornate or extravagant flourishes reminiscent of the baroque style. Here, we describe an analysis of capsular polysialic acid form variation in Escherichia coli K1, demonstrating that the large number of variant structures is controlled by a single contingency locus. The mechanism for generating maximum structural diversity from maximal genetic parsimony is conferred by a simple translational switch carried on a K1-specific prophage.

Mobile contingency locus controlling Escherichia coli K1 polysialic acid capsule acetylation

Escherichia coli K1 is part of a reservoir of adherent, invasive facultative pathogens responsible for a wide range of human and animal disease including sepsis, meningitis, urinary tract infection and inflammatory bowel syndrome. A prominent virulence factor in these diseases is the polysialic acid capsular polysaccharide (K1 antigen), which is encoded by the kps/neu accretion domain inserted near pheV at 67 map units. Some E. coli K1 strains undergo form (phase) variation involving loss or gain of O-acetyl esters at carbon positions 7 or 9 of the individual sialic acid residues of the polysialic acid chains. Acetylation is catalysed by the receptor-modifying acetyl coenzyme-A-dependent O-acetyltransferase encoded by neuO, a phase variable locus mapping near the integrase gene of the K1-specific prophage, CUS-3, which is inserted in argW at 53.1 map units. As the first E. coli contingency locus shown to operate by a translational switch, further investigation of neuO should provide a better understanding of the invasive K1 pathotype. Minimal estimates of morbidity and economic costs associated with human infections caused by extraintestinal pathogenic E. coli strains such as K1 indicate at least 6.5 million cases with attendant medical costs exceeding 2.5 billion US dollars annually in the United States alone.

Glorund, a Drosophila hnRNP F/H Homolog, Is an Ovarian Repressor of nanos Translation

Patterning of the anterior-posterior body axis of the Drosophila embryo requires production of Nanos protein selectively in the posterior. Spatially restricted Nanos synthesis is accomplished by translational repression of unlocalized nanos mRNA together with translational activation of posteriorly localized nanos. Repression of unlocalized nanos mRNA is mediated by a bipartite translational control element (TCE) in its 3' untranslated region. TCE stem-loop II functions during embryogenesis, through its interaction with the Smaug repressor. Stem-loop III represses unlocalized nanos mRNA during oogenesis, but trans-acting factors that carry out this function have remained elusive. Here we identify a Drosophila hnRNP, Glorund, that interacts specifically with stem-loop III. We establish that the ability of the TCE to repress translation in vivo reflects its ability to bind Glorund in vitro. These data, together with the analysis of a glorund null mutant, reveal a specific role for an hnRNP in repression of nanos translation during oogenesis.

Stepping Transfer Messenger RNA through the Ribosome

tmRNA (transfer messenger RNA) is a unique molecule used by all bacteria to rescue stalled ribosomes and to mark unfinished peptides with a specific degradation signal. tmRNA is recruited by arrested ribosomes in which it facilitates the translational switch from cellular mRNA to the mRNA part of tmRNA. Small protein B (SmpB) is a key partner for the trans-translation activity of tmRNA both in vivo and in vitro. It was shown that SmpB acts at the initiation step of the trans-translation process by facilitating tmRNA aminoacylation and binding to the ribosome. Little is known about the subsequent steps of trans-translation. Here we demonstrated the first example of an investigation of tmRNA.ribosome complexes at different stages of trans-translation. Our results show that the structural element at the position of tmRNA pseudoknot 3 remains intact during the translation of the mRNA module of tmRNA and that it is localized on the surface of the ribosome. At least one SmpB molecule remains bound to a ribosome.tmRNA complex isolated from the cell when translation is blocked at different positions within the mRNA part of tmRNA.

Uncoupling of RNAi from active translation in mammalian cells

Small inhibitory RNAs (siRNAs) are produced from longer RNA duplexes by the RNAse III family member Dicer. The siRNAs function as sequence-specific guides for RNA cleavage or translational inhibition. The precise mechanism by which siRNAs direct the RNA-induced silencing complex (RISC) to find the complementary target mRNA remains a mystery. Some biochemical evidence connects RNAi with translation making attractive the hypothesis that RISC is coupled with the translational apparatus for scanning mRNAs. Such coupling would facilitate rapid alignment of the siRNA antisense with the complementary target sequence. To test this hypothesis we took advantage of a well-characterized translational switch afforded by the ferritin IRE-IRP to analyze RNAi mediated cleavage of a target mRNA in the presence and absence of translation. Our results demonstrate that neither active translation nor unidirectional scanning is required for siRNA mediated target degradation. Our findings demonstrate that nontranslated mRNAs are highly susceptible to RNAi, and blocking scanning from both the 5' and 3' ends of an mRNA does not impede RNAi. Interestingly, RNAi is about threefold more active in the absence of translation.