Translation Rate Research Articles

Error-prone protein synthesis recapitulates early symptoms of Alzheimer disease in aging mice.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

Spatial engineering of E. coli with addressable phase-separated RNAs

Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E.coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.

Vers quel modèle de traduction-terminologie en langues nationales à l’aune d’une éducation bi- plurilingue au Cameroun ?

Towards what model of translation-terminology in national languages in the light of bi-plurilingual education in Cameroon?
 Cameroon, a country characterised by obvious linguistic diversity, recognises the importance of education that is rooted in the culture of the pupils and develops professional or self-employment skills through bi-lingual education. However, the national languages of Cameroon do not have sufficiently elaborate metalanguages to fully convey all the knowledge related to school subjects such as grammar, arithmetic, geometry, etc. They therefore need to be developed and adapted to the needs of the students. They need to be equipped with adequate terminologies to meet the challenge of bi-plurilingual teaching (official languages and national languages), because the production of bi-plurilingual (Cameroonian languages and official languages: French and English) teaching manuals and metalinguistic tools (lexicons, dictionaries, etc.), among others, implies an intense activity of translation-terminology to express certain teaching contents. However, no standard theoretical-practical model has been adopted for translation- terminology involving these languages. This activity thus seems to be left to improvisation. Focusing on recent work in two terminology committees in Cameroon (the ELAN- Afrique committee and the one set up by the IFADEM-RETHE team in Cameroon in 2016), this article outlines some of the salient linguistic problems encountered in translation-terminology activities related to the elaboration of lexicons in national languages, in the perspective of bi-lingual education. These include problems relating to the order of priority in the choice of naming procedures, compliance with the rules of adaptation of borrowings, the length of lexicalised expressions, and the rate of translation of terms. With regard to the choice of naming procedures, certain notions were difficult to agree on and were sometimes the source of lengthy debates within the translation-terminology groups. For example, in view of the high level of abstraction of certain concepts, some members of the terminology committees proposed borrowing as a first recourse, while others, with a purist tendency, considered that procedures employing the internal resources of the target language should be preferred to borrowing, without taking into account the length of the lexemes that would be derived from them. As far as compliance with the rules of adaptation of borrowings is concerned, several terms are not morpho-phonologically adapted, contrary to what is recommended by the cultural approaches. Moreover, several graphemes and consonantal sequences not attested in the alphabet or in the morphophonology of certain languages have been disgracefully taken into account in lexicons. Furthermore, as regards the length of lexicalised expressions, several terms have been translated into excessively long syntagms, or even sentences, whereas several procedures could have allowed their lexicalisation in compliance with the principle of brevity dear to terminology. Finally, some terms, due to their high level of abstraction or technicality, remained untranslated. In addition, the study proposes some theoretical-practical issues in the perspective of a model of translation-terminology of school contents from the official languages into the national languages of Cameroon, by laying the groundwork for an extensive socio-cultural terminological approach that would not only preserve the identity needs of each local community (hence the socio-cultural component of the model), but would also take into account the linguistic and cultural diversity of Cameroon characterised by the other idioms that evolve in this territory with the cultures they convey (hence the extensive character of the model). It is a non-essentialist model in its socio-cultural dimension, which promotes the multidialectal and multilingual character of the territory in translation-terminology activities by encouraging and highlighting the lexical variations that could arise from the different linguistic variants within a single language, as well as the calques and borrowings between Cameroonian languages; this with a view to increasing the possibilities of terminological development.

Human WDR5 promotes breast cancer growth and metastasis via KMT2-independent translation regulation.

Metastatic breast cancer remains a major cause of cancer-related deaths in women, and there are few effective therapies against this advanced disease. Emerging evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. Using an in vivo genetic screen, we identified WDR5 as an actionable epigenetic regulator that is required for metastatic progression in models of triple-negative breast cancer. We found that knockdown of WDR5 in breast cancer cells independently impaired their tumorigenic as well as metastatic capabilities. Mechanistically, WDR5 promotes cell growth by increasing ribosomal gene expression and translation efficiency in a KMT2-independent manner. Consistently, pharmacological inhibition or degradation of WDR5 impedes cellular translation rate and the clonogenic ability of breast cancer cells. Furthermore, a combination of WDR5 targeting with mTOR inhibitors leads to potent suppression of translation and proliferation of breast cancer cells. These results reveal novel therapeutic strategies to treat metastatic breast cancer.

Application of Discriminative Training Algorithm Based on the Improved Gaussian Mixture Model in English Translation Evaluation

English translation, also a kind of language conversion, refers to the activities of expressing each other in English to express another language or another language to express English. This paper aimed to evaluate and research methods of English translation based on the improved Gaussian mixture model (GMM). The paper proposed a simple evaluation and analysis of English translation using a discriminative training algorithm. In the experimental part, on the one hand, taking students, teachers, and translators as case study objects, through a questionnaire survey, it can be known that the number of students who scored more than 80 points in the English translation test is increasing every year, from 11.30% in the first year to 21.13% in the fourth year, an increase of nearly 10%. On the other hand, it can be seen from the speech translation experiment that the correct translation rate was 46.18% by using intelligent technology to distinguish translation. The experimental results showed that the discriminative training algorithm under the improved GMM algorithm is effective in the research of English translation evaluation.

Modelling Neurological Diseases in Large Animals: Criteria for Model Selection and Clinical Assessment.

Issue: The impact of neurological disorders is recognised globally, with one in six people affected in their lifetime and few treatments to slow or halt disease progression. This is due in part to the increasing ageing population, and is confounded by the high failure rate of translation from rodent-derived therapeutics to clinically effective human neurological interventions. Improved translation is demonstrated using higher order mammals with more complex/comparable neuroanatomy. These animals effectually span this translational disparity and increase confidence in factors including routes of administration/dosing and ability to scale, such that potential therapeutics will have successful outcomes when moving to patients. Coupled with advancements in genetic engineering to produce genetically tailored models, livestock are increasingly being used to bridge this translational gap. Approach: In order to aid in standardising characterisation of such models, we provide comprehensive neurological assessment protocols designed to inform on neuroanatomical dysfunction and/or lesion(s) for large animal species. We also describe the applicability of these exams in different large animals to help provide a better understanding of the practicalities of cross species neurological disease modelling. Recommendation: We would encourage the use of these assessments as a reference framework to help standardise neurological clinical scoring of large animal models.

Design and Implementation of Interactive English Translation System in Internet of Things Auxiliary Information Processing

Information technology has penetrated into all aspects of human life. Nowadays, with the rapid development of science and technology, information technology has gradually become the cornerstone of the development of other technologies. The Internet of Things is an important part of the new generation of information technology. Language is the medium of communication between people. Driven by economic globalization and the development of the Internet, information is growing rapidly, and there are more and more exchanges and exchanges between countries. The emergence of high-efficiency and high-economic machine translation solves these difficulties, and the interactive English translation system is the current research hotspot, which is intended to improve the output translation quality of the English translation system. The main work of this paper is to analyze the existing interactive machine translation technology, especially the interactive machine translation based on a phrase model, using the Internet of Things as a knowledge source. According to the characteristics of segment analysis and human-computer interaction mechanism, from the network, a wealth of information are available from open sources. In this paper, on the basis of the segment analysis, the human-machine cooperation translation strategy of human-machine cooperation with complementary human-machine advantages was discussed, and the system designed was verified. It is proved that the system has high performance in improving the accuracy and recall rate of machine English translation. Compared with the existing English translation system, the accuracy has improved by more than 20% in the case of fewer iterations, and in the case of 90 iterations, the accuracy can improve by 100%.

Single amino-acid mutation in a Drosoph ila melanogaster ribosomal protein: An insight in uL11 transcriptional activity.

The ribosomal protein uL11 is located at the basis of the ribosome P-stalk and plays a paramount role in translational efficiency. In addition, no mutant for uL11 is available suggesting that this gene is haplo-insufficient as many other Ribosomal Protein Genes (RPGs). We have previously shown that overexpression of Drosophila melanogaster uL11 enhances the transcription of many RPGs and Ribosomal Biogenesis genes (RiBis) suggesting that uL11 might globally regulate the level of translation through its transcriptional activity. Moreover, uL11 trimethylated on lysine 3 (uL11K3me3) interacts with the chromodomain of the Enhancer of Polycomb and Trithorax Corto, and both proteins co-localize with RNA Polymerase II at many sites on polytene chromosomes. These data have led to the hypothesis that the N-terminal end of uL11, and more particularly the trimethylation of lysine 3, supports the extra-ribosomal activity of uL11 in transcription. To address this question, we mutated the lysine 3 codon using a CRISPR/Cas9 strategy and obtained several lysine 3 mutants. We describe here the first mutants of D. melanogaster uL11. Unexpectedly, the uL11K3A mutant, in which the lysine 3 codon is replaced by an alanine, displays a genuine Minute phenotype known to be characteristic of RPG deletions (longer development, low fertility, high lethality, thin and short bristles) whereas the uL11K3Y mutant, in which the lysine 3 codon is replaced by a tyrosine, is unaffected. In agreement, the rate of translation decreases in uL11K3A but not in uL11K3Y. Co-immunoprecipitation experiments show that the interaction between uL11 and the Corto chromodomain is impaired by both mutations. However, Histone Association Assays indicate that the mutant proteins still bind chromatin. RNA-seq analyses from wing imaginal discs show that Corto represses RPG expression whereas very few genes are deregulated in uL11 mutants. We propose that Corto, by repressing RPG expression, ensures that all ribosomal proteins are present at the correct stoichiometry, and that uL11 fine-tunes its transcriptional regulation of RPGs.

Codon Usage and mRNA Stability are Translational Determinants of Cellular Response to Canonical Ferroptosis Inducers

Ferroptosis is a non-apoptotic cell death mechanism characterized by the generation of lipid peroxides. While many effectors in the ferroptosis pathway have been mapped, its epitranscriptional regulation is not yet fully understood. Ferroptosis can be induced via system xCT inhibition (Class I) or GPX4 inhibition (Class II). Previous works have revealed important differences in cellular response to different ferroptosis inducers. Importantly, blocking mRNA transcription or translation appears to protect cells against Class I ferroptosis inducing agents but not Class II. In this work, we examined the impact of blocking transcription (via Actinomycin D) or translation (via Cycloheximide) on Erastin (Class I) or RSL3 (Class II) induced ferroptosis. Blocking transcription or translation protected cells against Erastin but was detrimental against RSL3. Cycloheximide led to increased levels of GSH alone or when co-treated with Erastin via the activation of the reverse transsulfuration pathway. RNA sequencing analysis revealed early activation of a strong alternative splice program before observed changes in transcription. mRNA stability analysis revealed divergent mRNA stability changes in cellular response to Erastin or RSL3. Importantly, codon optimality biases were drastically different in either condition. Our data also implicated translation repression and rate as an important determinant of the cellular response to ferroptosis inducers. Given that mRNA stability and codon usage can be influenced via the tRNA epitranscriptome, we evaluated the role of a tRNA modifying enzyme in ferroptosis stress response. Alkbh1, a tRNA demethylase, led to translation repression and increased the resistance to Erastin but made cells more sensitive to RSL3.

Multiple Roles of m6A RNA Modification in Translational Regulation in Cancer.

Despite its discovery in the early 1970s, m6A modification within mRNA molecules has only powerfully entered the oncology field in recent years. This chemical modification can control all aspects of the maturation of mRNAs, both in the nucleus and in the cytoplasm. Thus, the alteration in expression levels of writers, erasers, and readers may significantly contribute to the alteration of gene expression observed in cancer. In particular, the activation of oncogenic pathways can lead to an alteration of the global rate of mRNA translation or the selective translation of specific mRNAs. In both cases, m6A can play an important role. In this review, we highlight the role of m6A in the regulation of translation by focusing on regulatory mechanisms and cancer-related functions of this novel but still controversial field.

CRISPR-RNAa: targeted activation of translation using dCas13 fusions to translation initiation factors.

Tools for synthetically controlling gene expression are a cornerstone of genetic engineering. CRISPRi and CRISPRa technologies have been applied extensively for programmable modulation of gene transcription, but there are few such tools for targeted modulation of protein translation rates. Here, we employ CRISPR-Cas13 as a programmable activator of translation. We develop a novel variant of the catalytically-deactivated Cas13d enzyme dCasRx by fusing it to translation initiation factor IF3. We demonstrate dCasRx-IF3’s ability to enhance expression 21.3-fold above dCasRx when both are targeted to the start of the 5′ untranslated region of mRNA encoding red fluorescent protein in Escherichia coli. Activation of translation is location-dependent, and we show dCasRx-IF3 represses translation when targeted to the ribosomal binding site, rather than enhancing it. We provide evidence that dCasRx-IF3 targeting enhances mRNA stability relative to dCasRx, providing mechanistic insights into how this new tool functions to enhance gene expression. We also demonstrate targeted upregulation of native LacZ 2.6-fold, showing dCasRx-IF3’s ability to enhance expression of endogenous genes. dCasRx-IF3 requires no additional host modification to influence gene expression. This work outlines a novel approach, CRISPR-RNAa, for post-transcriptional control of translation to activate gene expression.

Risk Factors for Failure of Closed Forearm Fracture Reduction in the Pediatric Emergency Department.

This study aimed to explore risk factors for failure of forearm fracture closed reduction in the pediatric emergency department (ED) and to suggest indications for initial surgery. This retrospective cohort study included all patients aged 0 to 18 years who presented to our pediatric ED with an extraarticular forearm fracture treated with closed reduction between May 2017 and April 2021. We explored risk factors for procedural failure, defined as a need for surgical intervention within 6 weeks of the closed reduction attempt. Of 375 patients (median age 8.1 years, 294 [78.2%] boys), 44 (11.7%) patients sustained a reduction failure, of whom 42 (95.5%) had both radius and ulna fractures. Of the 259 patients with fractures of both bones, the following parameters were independent predictors for reduction failure: refracture (adjusted odds ratio [aOR] 17.6, P < 0.001), open fracture (aOR 10.1, P = 0.007), midshaft fracture (aOR 2.6, P = 0.004), radial translation rate 37% and higher in either plane (aOR 5.1, P = 0.004), and age of 10 years and older (aOR 2.9, P = 0.01). Most pediatric forearm fractures can be successfully managed by closed reduction in the ED. Two-bone fractures had the strongest association with reduction failure. Refracture, open fracture, midshaft location, initial radius bone translation of 37% and higher (and not initial angulation), and patient age of 10 years and older are independent risk factors for reduction failure in two-bone fractures. We propose a risk score for reduction failure that can serve as a decision-making tool.

Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus.

In microbiological research, it is important to understand the time course of each step in a pathogen’s lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3–4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5′ UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.

Salt‐Detached Strike‐Slip Faulting, Outer Kwanza Basin, Offshore Angola

AbstractWe here use a 3D seismic reflection dataset from the Outer Kwanza Basin, offshore Angola to examine the structure and growth of salt‐detached strike‐slip faults. The faults occur in four, up to 13.8 km‐long, NE‐trending arrays that are physically linked by restraining bends and releasing stepovers, and which presently overlie Aptian salt and base‐salt relief related to pre‐salt faulting. We suggest that these faults formed to accommodate along‐margin variations in the rate and magnitude of differential seaward translation and salt diapirism, which commenced in the Early Cretaceous. We illustrate that the arrays grew by tip propagation of isolated fault segments, some of which linked during the Albian‐Cenomanian (i.e., 113–100.5 Ma, or the initial 11%–13% of their deformation history). Some arrays then reached their near‐final length within the subsequent ca. 77 Ma (or the next 69%–81% of their deformation history), while others attained this later, during the subsequent ca. 18 Ma (i.e., after 95% of their deformation history). During this time, the segments formed and then breached releasing and restraining stepovers, with the arrays as a whole growing by alternating periods of lengthening and throw accumulation, punctuated by phases of inactivity. Our results also show that scatter in the D‐L scaling of strike‐slip faults reflects the propagation, interaction, and linkage of individual segments.

RNA-Binding Proteins in the Regulation of Adipogenesis and Adipose Function.

The obesity epidemic represents a critical public health issue worldwide, as it is a vital risk factor for many diseases, including type 2 diabetes (T2D) and cardiovascular disease. Obesity is a complex disease involving excessive fat accumulation. Proper adipose tissue accumulation and function are highly transcriptional and regulated by many genes. Recent studies have discovered that post-transcriptional regulation, mainly mediated by RNA-binding proteins (RBPs), also plays a crucial role. In the lifetime of RNA, it is bound by various RBPs that determine every step of RNA metabolism, from RNA processing to alternative splicing, nucleus export, rate of translation, and finally decay. In humans, it is predicted that RBPs account for more than 10% of proteins based on the presence of RNA-binding domains. However, only very few RBPs have been studied in adipose tissue. The primary aim of this paper is to provide an overview of RBPs in adipogenesis and adipose function. Specifically, the following best-characterized RBPs will be discussed, including HuR, PSPC1, Sam68, RBM4, Ybx1, Ybx2, IGF2BP2, and KSRP. Characterization of these proteins will increase our understanding of the regulatory mechanisms of RBPs in adipogenesis and provide clues for the etiology and pathology of adipose-tissue-related diseases.

Geneticin reduces mRNA stability.

Messenger RNA (mRNA) translation can lead to higher rates of mRNA decay, suggesting the ribosome plays a role in mRNA destruction. Furthermore, mRNA features, such as codon identities, which are directly probed by the ribosome, correlate with mRNA decay rates. Many amino acids are encoded by synonymous codons, some of which are decoded by more abundant tRNAs leading to more optimal translation and increased mRNA stability. Variable translation rates for synonymous codons can lead to ribosomal collisions as ribosomes transit regions with suboptimal codons, and ribosomal collisions can promote mRNA decay. In addition to different translation rates, the presence of certain codons can also lead to higher or lower rates of amino acid misincorporation which could potentially lead to protein misfolding if a substituted amino acid fails to make critical contacts in a structure. Here, we test whether Geneticin-G418, an aminoglycoside antibiotic known to promote amino acid misincorporation-affects mRNA stability. We observe that G418 decreases firefly luciferase mRNA stability in an in vitro translation system and also reduces mRNA stability in mouse embryonic stem cells (mESCs). G418-sensitive mRNAs are enriched for certain optimal codons that contain G or C in the wobble position, arguing that G418 blunts the stabilizing effects of codon optimality.

In silico integrative analysis of multi-omics reveals regulatory layers for diurnal gene expression in mouse liver.

Diurnal oscillation persists throughout the body and plays an essential role in maintaining physiological homeostasis. Disruption of diurnal rhythm contributes to many diseases including type 2 diabetes. The regulatory mechanism of the transcription-translation feedback loop (TTFL) of core clock genes is well-established, while a systematic study across all regulatory layers of gene expression, including gene transcription, RNA translation, and DNA binding protein (DBP) activities, is still lacking. We comprehensively bioinformatics analyzed the rhythmicity of gene transcription, mature RNA abundance, protein abundance and DBP activity using publicly available omic-datasets from mouse livers. We found that the core clock genes, Bmal1 and Rev-erbα, persistently retained rhythmicity in all stages, which supported the essential rhythmic function along with the TTFL. Interestingly, there were many layer-specific rhythmic genes playing layer-specific rhythmic functions. The systematic analysis of gene transcription rate, RNA translation efficiency, and post-translation modification of DBP were incorporated to determine the potential mechanisms for layer-specific rhythmic genes. We observed the gene with rhythmic expression in both mature RNA and protein layers were largely due to relatively consistent translation rate. In addition, rhythmic translation rate induced the rhythms of protein whose mature RNA levels were not rhythmic. Further analysis revealed a phosphorylation-mediated and an enhancer RNA-mediated cycling regulation between the corresponding layers. This study presents a global view of the oscillating genes in multiple layers via a systematical analysis and indicates the complexity of regulatory mechanisms across different layers for further functional study.

Signal Peptide Features Determining the Substrate Specificities of Targeting and Translocation Components in Human ER Protein Import.

In human cells, approximately 30% of all polypeptides enter the secretory pathway at the level of the endoplasmic reticulum (ER). This process involves cleavable amino-terminal signal peptides (SPs) or more or less amino-terminal transmembrane helices (TMHs), which serve as targeting determinants, at the level of the precursor polypeptides and a multitude of cytosolic and ER proteins, which facilitate their ER import. Alone or in combination SPs and TMHs guarantee the initial ER targeting as well as the subsequent membrane integration or translocation. Cytosolic SRP and SR, its receptor in the ER membrane, mediate cotranslational targeting of most nascent precursor polypeptide chains to the polypeptide-conducting Sec61 complex in the ER membrane. Alternatively, fully-synthesized precursor polypeptides and certain nascent precursor polypeptides are targeted to the ER membrane by either the PEX-, SND-, or TRC-pathway. Although these targeting pathways may have overlapping functions, the question arises how relevant this is under cellular conditions and which features of SPs and precursor polypeptides determine preference for a certain pathway. Irrespective of their targeting pathway(s), most precursor polypeptides are integrated into or translocated across the ER membrane via the Sec61 channel. For some precursor polypeptides specific Sec61 interaction partners have to support the gating of the channel to the open state, again raising the question why and when this is the case. Recent progress shed light on the client spectrum and specificities of some auxiliary components, including Sec62/Sec63, TRAM1 protein, and TRAP. To address the question which precursors use a certain pathway or component in intact human cells, i.e., under conditions of fast translation rates and molecular crowding, in the presence of competing precursors, different targeting organelles, and relevant stoichiometries of the involved components, siRNA-mediated depletion of single targeting or transport components in HeLa cells was combined with label-free quantitative proteomics and differential protein abundance analysis. Here, we present a summary of the experimental approach as well as the resulting differential protein abundance analyses and discuss their mechanistic implications in light of the available structural data.

Function of Cajal Bodies in Nuclear RNA Retention in A. thaliana Leaves Subjected to Hypoxia.

Retention of RNA in the nucleus precisely regulates the time and rate of translation and controls transcriptional bursts that can generate profound variability in mRNA levels among identical cells in tissues. In this study, we investigated the function of Cajal bodies (CBs) in RNA retention in A. thaliana leaf nuclei during hypoxia stress was investigated. It was observed that in ncb-1 mutants with a complete absence of CBs, the accumulation of poly(A+) RNA in the leaf nuclei was lower than that in wt under stress. Moreover, unlike in root cells, CBs store less RNA, and RNA retention in the nuclei is much less intense. Our results reveal that the function of CBs in the accumulation of RNA in nuclei under stress depends on the plant organ. Additionally, in ncb-1, retention of introns of mRNA RPB1 (largest subunit of RNA polymerase II) mRNA was observed. However, this isoform is highly accumulated in the nucleus. It thus follows that intron retention in transcripts is more important than CBs for the accumulation of RNA in nuclei. Accumulated mRNAs with introns in the nucleus could escape transcript degradation by NMD (nonsense-mediated mRNA decay). From non-fully spliced mRNAs in ncb-1 nuclei, whose levels increase during hypoxia, introns are removed during reoxygenation. Then, the mRNA is transferred to the cytoplasm, and the RPB1 protein is translated. Despite the accumulation of isoforms in nuclei with retention of introns in reoxygenation, ncb-1 coped much worse with long hypoxia, and manifested faster yellowing and shrinkage of leaves.

Designing of an Efficient Whole-Cell Biocatalyst System for Converting L-Lysine Into Cis-3-Hydroxypipecolic Acid.

Cis-3-hydroxypipecolic acid (cis-3-HyPip), a key structural component of tetrapeptide antibiotic GE81112, which has attracted substantial attention for its broad antimicrobial properties and unique ability to inhibit bacterial translation initiation. In this study, a combined strategy to increase the productivity of cis-3-HyPip was investigated. First, combinatorial optimization of the ribosomal binding site (RBS) sequence was performed to tune the gene expression translation rates of the pathway enzymes. Next, in order to reduce the addition of the co-substrate α-ketoglutarate (2-OG), the major engineering strategy was to reconstitute the tricarboxylic acid (TCA) cycle of Escherichia coli to force the metabolic flux to go through GetF catalyzed reaction for 2-OG to succinate conversion, a series of engineered strains were constructed by the deletion of the relevant genes. In addition, the metabolic flux (gltA and icd) was improved and glucose concentrations were optimized to enhance the supply and catalytic efficiency of continuous 2-OG supply powered by glucose. Finally, under optimal conditions, the cis-3-HyPip titer of the best strain catalysis reached 33 mM, which was remarkably higher than previously reported.