Evolutionarily divergent mTOR remodels translatome for tissue regeneration.

An outstanding mystery in biology is why some species, such as the axolotl, can regenerate tissues whereas mammals cannot1. Here, we demonstrate that rapid activation of protein synthesis is a unique feature of the injury response critical for limb regeneration in the axolotl (Ambystoma mexicanum). By applying polysome sequencing, we identify hundreds of transcripts, including antioxidants and ribosome components that are selectively activated at the level of translation from pre-existing messenger RNAs in response to injury. By contrast, protein synthesis is not activated in response to non-regenerative digit amputation in the mouse. We identify the mTORC1 pathway as a key upstream signal that mediates tissue regeneration and translational control in the axolotl. We discover unique expansions in mTOR protein sequence among urodele amphibians. By engineering an axolotl mTOR (axmTOR) in human cells, we show that these changes create a hypersensitive kinase that allows axolotls to maintain this pathway in a highly labile state primed for rapid activation. This change renders axolotl mTOR more sensitive to nutrient sensing, and inhibition of amino acid transport is sufficient to inhibit tissue regeneration. Together, these findings highlight the unanticipated impact of the translatome on orchestrating the early steps of wound healing in a highly regenerative species and provide a missing link in our understanding of vertebrate regenerative potential.

PHGDH and cancer: new job for an old enzyme!

How do cancer cells bolster their energy metabolism under conditions of stress? Recent work by Shu et al (2022) unveils a novel, non-canonical function of the de novo serine synthesis pathway enzyme phosphoglycerate dehydrogenase (PHGDH) as a regulator of mitochondrial translation and tumor progression in liver cancer.

N-myc-Mediated Translation Control Is a Therapeutic Vulnerability in Medulloblastoma.

Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (∼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic. SIGNIFICANCE: Translatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.

Behavioral and hormonal effects of prenatal and maternal separation stresses in postpartum rats.

The aim of this study was to evaluate the hormone and anxiety levels of rat dams who were exposed to prenatal and maternal separation stress paradigms. Sprague-Dawley rat dams were divided into Prenatal Stress (PS), Maternal Separation (MS), Prenatal Stress and Maternal Separation (PS+MS), and Control (C) groups. All animals were subjected to the open field test on the 21st postnatal day. Same-day blood samples were obtained from the tail vein in order to examine corticotrophin-releasing hormone (CRH), estradiol, oxytocin, epinephrine, norepinephrine, prolactin, progesterone, brain-derived neurotrophic factor (BDNF), endorphin, and vasopressin levels of animals via enzyme-linked immunosorbent assay (ELISA). Oxytocin levels were the highest in the control group and the lowest in the MS group. CRH levels in the MS group were significantly higher than in the PS group (p < .05). Intriguingly, the BDNF level was the lowest in the control and highest in the MS group. While there was a strong correlation in the CRH, vasopressin, BDNF levels in the control group, various relations were observed in the stress groups. Stressed animals exhibited several behavioral anomalies including decreased fear responses such as freezing, enhanced duration, and increased number of entries into the central zone of the open field test apparatus. PS dams exhibited reductions in estradiol and norepinephrine levels relative to control or MS dams.

Environmental enrichment as a strategy: Attenuates the anxiety and memory impairment in social isolation stress.

BACKGROUND: Social isolation (SI) early in life produces behavioral and cognitive abnormalities. On the contrary, environmental enrichment (EE) offers beneficial effects on brain plasticity and development. This study was designed to examine how EE affects memory functions, anxiety level, and the expression levels of memory/anxiety-related genes such as NR2A, NR2B, BDNF, and cFos in the hippocampus of socially isolated rats. MATERIALS AND METHODS: Wistar albino male rats (n = 40) were separated into the five groups: Standard cage (SC), SI, EE, SI + SC, and SI + EE group. For each group, eight rats were housed, either grouped or isolated, in a standard or 3-week EE, respectively. Morris water maze test (MWMT) was used for measuring the learning and memory function. Elevated plus maze (EPM) and open field (OF) were used for the evaluation of anxiety behavior. Blood corticosterone level was evaluated by the ELISA method. The expression levels of genes were measured by the RT-PCR method. RESULTS: Results showed that EE increased memory performance in the SI group (p < 0.05). SI caused anxiety while EE improved anxiety behavior (p < 0.05). There was no significant difference between the groups in the OF test. Corticosterone levels did not change between groups. BDNF expression level was downregulated in EE and SI + SC compared with the SC group (respectively; p = 0.012; p = 0.011). NR2A, NR2B, and cFos expression levels did not change between groups significantly. CONCLUSIONS: SI impaired memory performance while EE has beneficial effects on memory in socially isolated rats. EE alone was insufficient to cause alterations in the memory performance. The therapeutic effects of EE became strengthened while applied together with stress protocol. Together with improving the effectiveness of memory function, EE has the potential to decrease anxiety behavior. EE seemed to be the reason for decreasing in BDNF.

Localization of a TORC1-eIF4F translation complex during CD8+ T cell activation drives divergent cell fate.

Activated CD8+ T lymphocytes differentiate into heterogeneous subsets. Using super-resolution imaging, we found that prior to the first division, dynein-dependent vesicular transport polarized active TORC1 toward the microtubule-organizing center (MTOC) at the proximal pole. This active TORC1 was physically associated with active eIF4F, required for the translation of c-myc mRNA. As a consequence, c-myc-translating polysomes polarized toward the cellular pole proximal to the immune synapse, resulting in localized c-myc translation. Upon division, the TORC1-eIF4A complex preferentially sorted to the proximal daughter cell, facilitating asymmetric c-Myc synthesis. Transient disruption of eIF4A activity at first division skewed long-term cell fate trajectories to memory-like function. Using a genetic barcoding approach, we found that first-division sister cells often displayed differences in transcriptional profiles that largely correlated with c-Myc and TORC1 target genes. Our findings provide mechanistic insights as to how distinct T cell fate trajectories can be established during the first division.

Protein synthesis control in cancer: selectivity and therapeutic targeting.

Translational control of mRNAs is a point of convergence for many oncogenic signals through which cancer cells tune protein expression in tumorigenesis. Cancer cells rely on translational control to appropriately adapt to limited resources while maintaining cell growth and survival, which creates a selective therapeutic window compared to non-transformed cells. In this review, we first discuss how cancer cells modulate the translational machinery to rapidly and selectively synthesize proteins in response to internal oncogenic demands and external factors in the tumor microenvironment. We highlight the clinical potential of compounds that target different translation factors as anti-cancer therapies. Next, we detail how RNA sequence and structural elements interface with the translational machinery and RNA-binding proteins to coordinate the translation of specific pro-survival and pro-growth programs. Finally, we provide an overview of the current and emerging technologies that can be used to illuminate the mechanisms of selective translational control in cancer cells as well as within the microenvironment.

Revealing molecular pathways for cancer cell fitness through a genetic screen of the cancer translatome.

The major cap-binding protein eukaryotic translation initiation factor 4E (eIF4E), an ancient protein required for translation of all eukaryotic genomes, is a surprising yet potent oncogenic driver. The genetic interactions that maintain the oncogenic activity of this key translation factor remain unknown. In this study, we carry out a genome-wide CRISPRi screen wherein we identify more than 600 genetic interactions that sustain eIF4E oncogenic activity. Our data show that eIF4E controls the translation of Tfeb, a key executer of the autophagy response. This autophagy survival response is triggered by mitochondrial proteotoxic stress, which allows cancer cell survival. Our screen also reveals a functional interaction between eIF4E and a single anti-apoptotic factor, Bcl-xL, in tumor growth. Furthermore, we show that eIF4E and the exon-junction complex (EJC), which is involved in many steps of RNA metabolism, interact to control the migratory properties of cancer cells. Overall, we uncover several cancer-specific vulnerabilities that provide further resolution of the cancer translatome.

Stress caused by environmental effects on the birth process and some of the labor hormones at rats: ideal birth environment and hormones.

OBJECTIVE: The aim of this study was to compare the effect of environmental conditions on the birth hormones and the labor of rats. MATERIALS AND METHODS: This Study is animal experiment. A total of of 18 pregnant Sprague Dawley rats were divided into control, stress and enriched groups. Animals in the stress group were exposed to unexpected variable stress paradigm three times a day during the third trimester of their pregnancies. Whereas animals raised in the enriched environment were kept in larger cages equipped with various toys. They were subjected to open field test for 5 minutes in the last trimester. Blood samples were taken from the tail vein at the beginning of birth, and 10 parameters (including corticotropin-releasing hormone, oxytocin, endorphin, epinephrine, norepinephrine, prolactin, estrogen, progesterone, vasopressin, and brain-derived neurotrophic factor) involved in labor were assessed. Kruskal Wallis, Mann Whitney U, and Spearman's rho correlation analysis were used to compare data. RESULTS: Interactions of hormones were significantly different among the groups. While hormonal interactions in the control group were similar to the physiological parameters, other groups displayed various results. There were significant (p < .05) differences in the values of corticotropin-releasing hormone (CRH) and vasopressin hormone levels. In the open Field test, standing distribution scores of animals displayed differences among control, stress and enriched environment groups (p < .05). CONCLUSION: These results showed that labor environment diversely affects physiology aspects of birth. It is known that many factors such as procedures in a hospital environment, birth environment, noise, and birth position affect the hormones at birth. Therefore, the birth environment, either at home or at the hospital, needs to be well-organized accordingly.

HELZ directly interacts with CCR4-NOT and causes decay of bound mRNAs.

Eukaryotic superfamily (SF) 1 helicases have been implicated in various aspects of RNA metabolism, including transcription, processing, translation, and degradation. Nevertheless, until now, most human SF1 helicases remain poorly understood. Here, we have functionally and biochemically characterized the role of a putative SF1 helicase termed "helicase with zinc-finger," or HELZ. We discovered that HELZ associates with various mRNA decay factors, including components of the carbon catabolite repressor 4-negative on TATA box (CCR4-NOT) deadenylase complex in human and Drosophila melanogaster cells. The interaction between HELZ and the CCR4-NOT complex is direct and mediated by extended low-complexity regions in the C-terminal part of the protein. We further reveal that HELZ requires the deadenylase complex to mediate translational repression and decapping-dependent mRNA decay. Finally, transcriptome-wide analysis of Helz-null cells suggests that HELZ has a role in the regulation of the expression of genes associated with the development of the nervous system.

A rare presentation of meralgia paraesthetica in limb girdle muscular dystrophy.

A 44-year-old female with paraesthesia and pain on the left anterolateral thigh who had been diagnosed with limb-girdle muscular dystrophy by electromyography and muscle biopsy is presented. Neurological examination revealed atrophy of the proximal muscles of both shoulders, plus pseudo hypertrophy of both calves. Electromyography exhibited a myopathic pattern. Musculoskeletal ultrasound imaging demonstrated a fusiform nerve swelling below the inguinal ligament suggesting lateral femoral cutaneous nerve compression, consistent with meralgia paraesthetica. Treatment with a perineural injection of betamethasone dipropionate and betamethasone sodium phosphate combination, and prilocaine-HCl, under ultrasound guidance, was performed. Symptoms resolved within 6 h. At 3-week follow-up, the patient was asymptomatic, and there was no paraesthesia or pain on examination. In this case, ultrasound-guided perineural injection of the lateral femoral cutaneous nerve with local anaesthetics and steroid served both diagnostic and therapeutic functions.

A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin.

Human (Hs) Roquin1 and Roquin2 are RNA-binding proteins that promote mRNA target degradation through the recruitment of the CCR4-NOT deadenylase complex and are implicated in the prevention of autoimmunity. Roquin1 recruits CCR4-NOT via a C-terminal region that is not conserved in Roquin2 or in invertebrate Roquin. Here we show that Roquin2 and Drosophila melanogaster (Dm) Roquin also interact with the CCR4-NOT complex through their C-terminal regions. The C-terminal region of Dm Roquin contains multiple motifs that mediate CCR4-NOT binding. One motif binds to the CAF40 subunit of the CCR4-NOT complex. The crystal structure of the Dm Roquin CAF40-binding motif (CBM) bound to CAF40 reveals that the CBM adopts an α-helical conformation upon binding to a conserved surface of CAF40. Thus, despite the lack of sequence conservation, the C-terminal regions of Roquin proteins act as an effector domain that represses the expression of mRNA targets via recruitment of the CCR4-NOT complex.

Effects of Combined Bonded Maxillary Expansion and Face Mask on Dental Arch Length in Patients with Skeletal Class III Malocclusions.

OBJECTIVE: To evaluate the effects of combined rapid maxillary expansion (RME) and face mask (FM) therapy during the mixed dentition period on the dental arch length in patients with skeletal Class III malocclusion. STUDY DESIGN: We evaluated pre- and post-treatment orthodontic models of 52 patients (25 girls, 27 boys) aged 8-12 years with skeletal Class III malocclusion(ANB<0) accompanied by maxillary transverse deficiency and retrognatism treated by bonded RME-FM therapy for a mean duration of 8 months. Palatal rugae, the cusp tips of permanent first molars, deciduous molars/permanent premolars, deciduous canines and the incisal edges of permanent central incisors were marked on orthodontic models, which were then photocopied. Inter-molar, inter-premolar and inter-canine widths; the arch length; the arch depth and molar and incisor sagittal movements were measured on these photocopies. Statistical comparisons were made using paired t-tests. RESULTS: Inter-molar, inter-premolar and inter-canine widths and the arch length showed significant increases after treatment, while the arch depth showed a significant decrease (p<0.001 for all). CONCLUSIONS: With the study limitations, our results suggest that combined RME-FM therapy increases the arch length in the mixed dentition of patients with skeletal Class III malocclusion.

miRISC and the CCR4-NOT complex silence mRNA targets independently of 43S ribosomal scanning.

miRNAs associate with Argonaute (AGO) proteins to silence the expression of mRNA targets by inhibiting translation and promoting deadenylation, decapping, and mRNA degradation. A current model for silencing suggests that AGOs mediate these effects through the sequential recruitment of GW182 proteins, the CCR4-NOT deadenylase complex and the translational repressor and decapping activator DDX6. An alternative model posits that AGOs repress translation by interfering with eIF4A function during 43S ribosomal scanning and that this mechanism is independent of GW182 and the CCR4-NOT complex in Drosophila melanogaster Here, we show that miRNAs, AGOs, GW182, the CCR4-NOT complex, and DDX6/Me31B repress and degrade polyadenylated mRNA targets that are translated via scanning-independent mechanisms in both human and Dm cells. This and additional observations indicate a common mechanism used by these proteins and miRNAs to mediate silencing. This mechanism does not require eIF4A function during ribosomal scanning.

A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.

CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.

Structure and assembly of the NOT module of the human CCR4-NOT complex.

The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets.

GW182 proteins cause PABP dissociation from silenced miRNA targets in the absence of deadenylation.

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)-binding protein (PABP) and the CCR4-NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. Here, we demonstrate that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4-NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. Our findings indicate that the recruitment of the CCR4-NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation.

The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets.

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases.

Autophagy-related gene, TdAtg8, in wild emmer wheat plays a role in drought and osmotic stress response.

An autophagy-related gene Atg8 was cloned for the first time from wild emmer wheat, named as TdAtg8, and its role on autophagy under abiotic stress conditions was investigated. Examination of TdAtg8 expression patterns indicated that Atg8 expression was strongly upregulated under drought stress, especially in the roots when compared to leaves. LysoTracker(®) red marker, utilized to observe autophagosomes, revealed that autophagy is constitutively active in Triticum dicoccoides. Moreover, autophagy was determined to be induced in plants exposed to osmotic stress when compared to plants grown under normal conditions. Functional studies were executed in yeast to confirm that the TdATG8 protein is functional, and showed that the TdAtg8 gene complements the atg8∆::kan MX yeast mutant strain grown under nitrogen deficiency. For further functional analysis, TdATG8 protein was expressed in yeast and analyzed using Western immunoblotting. Atg8-silenced plants were exposed to drought stress and chlorophyll and malondialdehyde (MDA) content measurements demonstrated that Atg8 plays a key role on drought stress tolerance. In addition, Atg8-silenced plants exposed to osmotic stress were found to have decreased Atg8 expression level in comparison to controls. Hence, Atg8 is a positive regulator in osmotic and drought stress response.