Control Of Protein Translation Research Articles

De novo polyamine synthesis supports metabolic and functional responses in activated murine NK cells.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy.

Conversion of Germ Cells to Somatic Cell Types in C. elegans.

The potential of a cell to produce all types of differentiated cells in an organism is termed totipotency. Totipotency is an essential property of germ cells, which constitute the germline and pass on the parental genetic material to the progeny. The potential of germ cells to give rise to a whole organism has been the subject of intense research for decades and remains important in order to better understand the molecular mechanisms underlying totipotency. A better understanding of the principles of totipotency in germ cells could also help to generate this potential in somatic cell lineages. Strategies such as transcription factor-mediated reprogramming of differentiated cells to stem cell-like states could benefit from this knowledge. Ensuring pluripotency or even totipotency of reprogrammed stem cells are critical improvements for future regenerative medicine applications. The C. elegans germline provides a unique possibility to study molecular mechanisms that maintain totipotency and the germ cell fate with its unique property of giving rise to meiotic cells Studies that focused on these aspects led to the identification of prominent chromatin-repressing factors such as the C. elegans members of the Polycomb Repressive Complex 2 (PRC2). In this review, we summarize different factors that were recently identified, which use molecular mechanisms such as control of protein translation or chromatin repression to ensure maintenance of totipotency and the germline fate. Additionally, we focus on recently identified factors involved in preventing transcription-factor-mediated conversion of germ cells to somatic lineages. These so-called reprogramming barriers have been shown in some instances to be conserved with regard to their function as a cell fate safeguarding factor in mammals. Overall, continued studies assessing the different aspects of molecular pathways involved in maintaining the germ cell fate in C. elegans may provide more insight into cell fate safeguarding mechanisms also in other species.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Integrative Network Biology Framework Elucidates Molecular Mechanisms of SARS-CoV-2 Pathogenesis.

SummaryCOVID-19 (coronavirus disease 2019) is a respiratory illness caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although the pathophysiology of this virus is complex and largely unknown, we employed a network-biology-fueled approach and integrated transcriptome data pertaining to lung epithelial cells with human interactome to generate Calu-3-specific human-SARS-CoV-2 interactome (CSI). Topological clustering and pathway enrichment analysis show that SARS-CoV-2 targets central nodes of the host-viral network, which participate in core functional pathways. Network centrality analyses discover 33 high-value SARS-CoV-2 targets, which are possibly involved in viral entry, proliferation, and survival to establish infection and facilitate disease progression. Our probabilistic modeling framework elucidates critical regulatory circuitry and molecular events pertinent to COVID-19, particularly the host-modifying responses and cytokine storm. Overall, our network-centric analyses reveal novel molecular components, uncover structural and functional modules, and provide molecular insights into the pathogenicity of SARS-CoV-2 that may help foster effective therapeutic design.

System-Wide Analysis of Protein Acetylation and Ubiquitination Reveals a Diversified Regulation in Human Cancer Cells.

Post-translational modifications are known to be widely involved in the regulation of various biological processes, through the extensive diversification of each protein function at the cellular network level. In order to unveil the system-wide function of the protein lysine modification in cancer cell signaling, we performed global acetylation and ubiquitination proteome analyses of human cancer cells, based on high-resolution nanoflow liquid chromatography–tandem mass spectrometry, in combination with the efficient biochemical enrichment of target modified peptides. Our large-scale proteomic analysis enabled us to identify more than 5000 kinds of ubiquitinated sites and 1600 kinds of acetylated sites, from representative human cancer cell lines, leading to the identification of approximately 900 novel lysine modification sites in total. Very interestingly, 236 lysine residues derived from 141 proteins were found to be modified with both ubiquitination and acetylation. As a consequence of the subsequent motif extraction analyses, glutamic acid (E) was found to be highly enriched at the position (−1) for the lysine acetylation sites, whereas the same amino acid was relatively dispersed along the neighboring residues of the lysine ubiquitination sites. Our pathway analysis also indicated that the protein translational control pathways, such as the eukaryotic initiation factor 2 (EIF2) and the ubiquitin signaling pathways, were highly enriched in both of the acetylation and ubiquitination proteome data at the network level. This report provides the first integrative description of the protein acetylation and ubiquitination-oriented systematic regulation in human cancer cells.

タンパク合成と翻訳制御に関するEMBOワークショップ（ドイツ）参加報告

タンパク合成と翻訳制御に関するEMBOワークショップ（ドイツ）参加報告

Mechanical Regulation of Protein Translation in the Cardiovascular System.

The cardiovascular system can sense and adapt to changes in mechanical stimuli by remodeling the physical properties of the heart and blood vessels in order to maintain homeostasis. Imbalances in mechanical forces and/or impaired sensing are now not only implicated but are, in some cases, considered to be drivers for the development and progression of cardiovascular disease. There is now growing evidence to highlight the role of mechanical forces in the regulation of protein translation pathways. The canonical mechanism of protein synthesis typically involves transcription and translation. Protein translation occurs globally throughout the cell to maintain general function but localized protein synthesis allows for precise spatiotemporal control of protein translation. This Review will cover studies on the role of biomechanical stress -induced translational control in the heart (often in the context of physiological and pathological hypertrophy). We will also discuss the much less studied effects of mechanical forces in regulating protein translation in the vasculature. Understanding how the mechanical environment influences protein translational mechanisms in the cardiovascular system, will help to inform disease pathogenesis and potential areas of therapeutic intervention.

FLI1 promotes protein translation via the transcriptional regulation of MKNK1 expression.

The disruption of protein translation machinery is a common feature of cancer initiation and progression, and drugs that target protein translation offer new avenues for therapy. The translation initiation factor, eukaryotic initiation factor 4E (eIF4E), is induced in a number of cancer cell lines and is one such candidate for therapeutic intervention. Friend leukemia integration 1 (FLI1) is a potent oncogenic transcription factor that promotes various types of cancer by promoting several hallmarks of cancer progression. FLI1 has recently been implicated in protein translation through yet unknown mechanisms. This study identified a positive association between FLI1 expression and mitogen-activated protein kinase (MAPK)-interacting serine/threonine kinase1 (MKNK1), the immediate upstream regulator of the eIF4E initiation factor. The short hairpin RNA (shRNA)-mediated silencing or overexpression of FLI1 in leukemic cell lines downregulated or upregulated MKNK1 expression, respectively. Promoter analysis identified a potent FLI1 binding site in the regulatory region of the MKNK1 promoter. In transient transfection experiments, FLI1 increased MKNK1 promoter activity, which was blocked by mutating the FLI1 binding site. FLI1 specifically affected the expression of MKNK1, but not that of MKNK2. The siRNA-mediated downregulation of MKNK1 downregulated the expression of survivin (BIRC5) and significantly suppressed cell proliferation in culture. FLI1 inhibitory compounds were shown to downregulate this oncogene through the suppression of MAPK/extracellular-regulated kinase (ERK) signaling and the subsequent activation of miR-145, leading to a lower MKNK1 expression and the suppression of leukemic growth. These results uncover a critical role for FLI1 in the control of protein translation and the importance of targeting its function and downstream mediators, such as MKNK1, for cancer therapy.

Structural basis for the dimerization of Gemin5 and its role in protein recruitment and translation control.

In all organisms, a selected type of proteins accomplishes critical roles in cellular processes that govern gene expression. The multifunctional protein Gemin5 cooperates in translation control and ribosome binding, besides acting as the RNA-binding protein of the survival of motor neuron (SMN) complex. While these functions reside on distinct domains located at each end of the protein, the structure and function of the middle region remained unknown. Here, we solved the crystal structure of an extended tetratricopeptide (TPR)-like domain in human Gemin5 that self-assembles into a previously unknown canoe-shaped dimer. We further show that the dimerization module is functional in living cells driving the interaction between the viral-induced cleavage fragment p85 and the full-length Gemin5, which anchors splicing and translation members. Disruption of the dimerization surface by a point mutation in the TPR-like domain prevents this interaction and also abrogates translation enhancement induced by p85. The characterization of this unanticipated dimerization domain provides the structural basis for a role of the middle region of Gemin5 as a central hub for protein-protein interactions.

Ywhae/14-3-3ε Expression Promotes Translation Initiation and Protein Synthesis in Multiple Myeloma Regulating Proteasome Load and Contributing to Proteasome Inhibitor Sensitivity/Resistance

Dysregulated translational control and high protein turnover is a feature of multiple myeloma (MM) cells; as a result, proteasome inhibitors (PI), triggering a misfolded protein stress and death response, have been extremely successfully in MM therapy. But despite success of PIs and other novel therapeutic approaches, majority of patients progress; thus, investigating the mechanisms driving the drug sensitivity and/or resistance is important. We here report a novel chaperone protein 14-3-3ε with significant impact on MM cell pathobiology and patient outcome. 14-3-3 proteins are a family of master regulators of intracellular signaling that influence several cellular processes through binding to specific serine/threonine-phosphorylated residues on a diverse array of cellular proteins. With known interaction of 14-3-3 proteins with several functionally diverse molecules and well recognized chaperon function, we sought to evaluate their role in impacting proteasome activity. We compared the sensitivity of 14 MM cell lines to two proteasome inhibitors, Bortezomib (BTZ) and Carfilzomib (CFZ) with expression of all 14-3-3 proteins at both mRNA and protein levels. We compared the measured drug activity and individual 14-3-3 gene expression across all cell lines by Pearson correlation coefficients and observed that the expression level of only YWHAE (coding gene for the isoform 14-3-3ε) showed a significant negative correlation with both BTZ and CFZ response. We therefore evaluated its expression in primary MM cells and found its lower expression to be associated with poor outcome in MM patients receiving a bortezomib (Btz)-based therapy. To elucidate the underlying molecular mechanism responsible for 14-3-3ε contribution to PIs sensitivity, we performed integrated analysis of protein interactome and transcriptomic changes following 14-3-3ε gene modulation. These studies showed that 14-3-3ε impacts mTORC1 signaling in MM cells by binding to serine-phosphorylated residues on mTOR and its upstream negative regulator TSC2, resulting in mTORC1 activation. Conversely, depletion of 14-3-3ε inhibits TSC2 phosphorylation, causing a subsequent inhibition of mTORC1 signaling. One major conserved function of mTORC1 is to promote mRNA translation and therefore protein synthesis via activation of S6 kinase 1 (S6K1) as well as inhibition of the eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP). We have therefore assessed the impact of 14-3-3ε on translational efficiency in MM cells. In the viable MM cell population, 14-3-3ε depletion caused up to 50% reduction of protein synthesis in three MM cell lines, whereas 14-3-3ε overexpression in the KMS20 cell line or addback in YWHAE-KO cells (H929 and KMS11) resulted in a marked upregulation of protein synthesis. As MM is characterized by excess production of monoclonal immunoglobulins, we evaluated impact of 14-3-3ε perturbation on intracellular and secretion light chains production. We observed a significant decrease in the intracellular abundance and secretion of the light chains with 14-3-3ε KD in all MM cell lines tested. These observations were corroborated by gain-of-function studies where ectopic overexpression of 14-3-3ε in MM cells was associated with increased protein load (and an enhanced sensitivity to PIs) in vitro as well as in vivo. Importantly, we confirmed a significant correlation between 14-3-3ε expression, PIs sensitivity and protein load (evaluated as M protein production) in primary MM cells from two independent datasets. Moreover, MM patients with del17p, where 14-3-3ε is located, have lower 14-3-3ε expression and decreased monoclonal protein level, providing an explanation for inability of BTZ to overcome high-risk feature associated with del17p. In conclusion, we here report for the first time a unique function for 14-3-3ε in modulating the sensitivity to PIs through regulation of protein synthesis and M protein load in MM cells. Altogether these observations suggest that 14-3-3ε is a predictor of clinical outcome and may serve as a potential target to modulate PIs sensitivity in MM. Disclosures Anderson: Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder ; Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board. Munshi:Takeda: Consultancy; Takeda: Consultancy; Oncopep: Consultancy; Abbvie: Consultancy; Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Oncopep: Consultancy; Adaptive: Consultancy; Janssen: Consultancy; Celgene: Consultancy; Janssen: Consultancy.

Could an Impairment in Local Translation of mRNAs in Glia be Contributing to Pathogenesis in ALS?

One of the key pathways implicated in amyotrophic lateral sclerosis (ALS) pathogenesis is abnormal RNA processing. Studies to date have focussed on defects in RNA stability, splicing, and translation, but this review article will focus on the largely overlooked RNA processing mechanism of RNA trafficking, with particular emphasis on the importance of glia. In the central nervous system (CNS), oligodendrocytes can extend processes to myelinate and metabolically support up to 50 axons and astrocytes can extend processes to cover up to 100,000 synapses, all with differing local functional requirements. Furthermore, many of the proteins required in these processes are large, aggregation-prone proteins which would be difficult to transport in their fully translated, terminally-folded state. This, therefore, highlights a critical requirement in these cells for local control of protein translation, which is achieved through specific trafficking of mRNAs to each process and local translation therein. Given that a large number of RNA-binding proteins have been implicated in ALS, and RNA-binding proteins are essential for trafficking mRNAs from the nucleus to glial processes for local translation, RNA misprocessing in glial cells is a likely source of cellular dysfunction in ALS. To date, neurons have been the focus of ALS research, but an intrinsic deficit in glia, namely astrocytes and oligodendrocytes, could have an additive effect on declining neuronal function in ALS. This review article aims to highlight the key evidence that supports the contention that RNA trafficking deficits in astrocytes and oligodendrocytes may contribute to in ALS.

Eukaryotic initiation factor 4A2 promotes experimental metastasis and oxaliplatin resistance in colorectal cancer

BackgroundDeregulation of protein translation control is a hallmark of cancers. Eukaryotic initiation factor 4A2 (EIF4A2) is required for mRNA binding to ribosome and plays an important role in translation initiation. However, little is known about its functions in colorectal cancer (CRC).MethodsAnalysis of CRC transcriptome data from TCGA identified that EIF4A2 was associated with poor prognosis. Immunohistochemistry study of EIF4A2 was carried out in 297 paired colorectal tumor and adjacent normal tissue samples. In vitro and in vivo cell-biological assays were performed to study the biological functions of EIF4A2 on experimental metastasis and sensitivity to oxaliplatin treatment. Bioinformatic prediction, chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assay were carried out to unveil the transcription factor of EIF4A2 regulation.ResultsEIF4A2 Expression is significantly higher in colorectal tumors. Multivariate analysis suggests EIF4A2 as an independent predictor of overall, disease-free and progression-free survival. Dysfunction of EIF4A2 by genetic knock-down or small-molecule inhibitor silvestrol dramatically inhibited CRC invasion and migration, sphere formation and enhanced sensitivity to oxaliplatin treatment in vitro and in vivo. Notably, EIF4A2 knock-down also suppressed lung metastasis in vivo. qRT-PCR and immunoblotting analyses identified c-Myc as a downstream target and effector of EIF4A2. ChIP and dual-luciferase reporter assays validated the bioinformatical prediction of ZNF143 as a specific transcription factor of EIF4A2.ConclusionsEIF4A2 promotes experimental metastasis and oxaliplatin resistance in CRC. Silvestrol inhibits tumor growth and has synergistic effects with oxaliplatin to induce apoptosis in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models.

Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses.

The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.

Theophylline-inducible riboswitch accurately regulates protein expression at low level in Escherichia coli.

Fine-tuning of enzyme expression at low levels is an important challenge for metabolic engineers. Here, theophylline-inducible riboswitch for translational regulation was evaluated. The background expression, translation rate, and time delay for its induction was reported. To evaluate the effect of the amount of mRNA on its translation rate, transcription of the riboswitch RNA with red fluorescent protein (RFP) was controlled by the lac system with addition of isopropyl β-D-1-thiogalactopyranoside in Escherichia coli. Regardless of the amount of riboswitch mRNA, the translation of RFP was completely suppressed without theophylline during both growth and stationary phases. Furthermore, a strong positive correlation between theophylline concentration (0 to 1mM) and specific RFP production rate was observed. The specific RFP production rate with the riboswitch was approximately 2.3% of that without the riboswitch. Furthermore, 60min of time delay for RFP expression was observed after adding theophylline during the stationary phase. Theophylline-inducible riboswitch precisely controls protein translation at low expression levels with significantly low background expression. It can emerge as a powerful tool for fine tuning of enzyme expression.

Genetic association and functional characterization of MCPH1 gene variation in bipolar disorder and schizophrenia.

A rare microcephalin 1 gene (MCPH1) variant rs61749465A>G (p.Asp61Gly) with prior evidence for association with schizophrenia (p = 3.78 × 10-7 ) was tested for association in 2,300 bipolar disorder (BPD) participants, 1,930 SCZ participants and 1,820 normal comparison subjects. We report evidence for association of rs61749465A>G with BPD (p = 0.0009). rs61749465 is located in the N-terminal of the BRCT1 domain of MCPH1. Bioinformatic analysis predicted the Asp61Gly substitution to be damaging to MCPH1 function. A second MCPH1 BRCT1 domain variant (rs199422124C>G; p.Thr27Arg), reported to cause autosomal recessive microcephaly, was not detected in the participants tested here. We sought to characterize the functional effects of these variants on MCPH1 function. Cell count assays indicated that rs199422124 allele G had a greater impact on cell survival compared to the G allele of rs61749465. Gene expression analysis combined with gene network and pathway analysis indicated that rs61749465 allele G may impact protein translation and cell cycle control. The evidence for association between rs61749465A>G and psychosis in both BPD and SCZ warrants further replication. Likewise, the data from the functional analyses point to molecular mechanisms that may underlie the proposed MCPH1 mediated risk of psychosis and pathogenesis in autosomal recessive microcephaly require additional experimental validation.

Measuring Amber Initiator tRNA Orthogonality in a Genomically Recoded Organism

Using engineered initiator tRNA for precise control of protein translation within cells has great promise within future orthogonal translation systems to decouple housekeeping protein metabolism from that of engineered genetic systems. Previously, E.coli strain C321.ΔA. exp lacking all UAG stop codons was created, freeing this "amber" stop codon for other purposes. An engineered "amber initiator" tRNACUAfMet that activates translation at UAG codons is available, but little is known about this tRNA's orthogonality. Here, we combine for the first time the amber initiator tRNACUAfMet in C321.ΔA. exp and measure its cellular effects. We found that the tRNACUAfMet expression resulted in a nearly 200-fold increase in fluorescent reporter expression with a unimodal population distribution and no apparent cellular fitness defects. Proteomic analysis revealed upregulated ribosome-associated, tRNA degradation, and amino acid biosynthetic proteins, with no evidence for off-target translation initiation. In contrast to previous work, we show that UAG-initiated proteins carry N-terminal methionine, but have no evidence for glutamine. Together, our results identify beneficial features of using the amber initiator tRNACUAfMet to control gene expression while also revealing fundamental challenges to using engineered initiator tRNAs as the basis for orthogonal translation initiation systems.

EZH2 cooperates with gain-of-function p53 mutants to promote cancer growth and metastasis.

In light of the increasing number of identified cancer-driven gain-of-function (GOF) mutants of p53, it is important to define a common mechanism to systematically target several mutants, rather than developing strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation-sequencing (RIP-seq), we identified the Polycomb-group histone methyltransferase EZH2 as a p53 mRNA-binding protein. EZH2 bound to an internal ribosome entry site (IRES) in the 5'UTR of p53 mRNA and enhanced p53 protein translation in a methyltransferase-independent manner. EZH2 augmented p53 GOF mutant-mediated cancer growth and metastasis by increasing protein levels of mutant p53. EZH2 overexpression was associated with worsened outcome selectively in patients with p53-mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant-mediated cancer growth. Our findings reveal a non-methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants.

Neurite-Enriched MicroRNA-218 Stimulates Translation of the GluA2 Subunit and Increases Excitatory Synaptic Strength.

Local control of protein translation is a fundamental process for the regulation of synaptic plasticity. It has been demonstrated that the local protein synthesis occurring in axons and dendrites can be shaped by numerous mechanisms, including miRNA-mediated regulation. However, several aspects underlying this regulatory process have not been elucidated yet. Here, we analyze the differential miRNA profile in cell bodies and neurites of primary hippocampal neurons and find an enrichment of the precursor and mature forms of miR-218 in the neuritic projections. We show that miR-218 abundance is regulated during hippocampal development and by chronic silencing or activation of neuronal network. Overexpression and knockdown of miR-218 demonstrated that miR-218 targets the mRNA encoding the GluA2 subunit of AMPA receptors and modulates its expression. At the functional level, miR-218 overexpression increases glutamatergic synaptic transmission at both single neuron and network levels. Our data demonstrate that miR-218 may play a key role in the regulation of AMPA-mediated excitatory transmission and in the homeostatic regulation of synaptic plasticity.

Ribosome specialization and its potential role in the control of protein translation and skeletal muscle size.

The ribosome is typically viewed as a supramolecular complex with constitutive and invariant capacity in mediating translation of mRNA into protein. This view has been challenged by recent research revealing that ribosome composition could be heterogeneous, and this heterogeneity leads to functional ribosome specialization. This review presents the idea that ribosome heterogeneity results from changes in its various components, including variations in ribosomal protein (RP) composition, posttranslational modifications of RPs, changes in ribosomal-associated proteins, alternative forms of rRNA, and posttranscriptional modifications of rRNAs. Ribosome heterogeneity could be orchestrated at several levels and may depend on numerous factors, such as the subcellular location, cell type, tissue specificity, the development state, cell state, ribosome biogenesis, RP turnover, physiological stimuli, and circadian rhythm. Ribosome specialization represents a completely new concept for the regulation of gene expression. Specialized ribosomes could modulate several aspects of translational control, such as mRNA translation selectivity, translation initiation, translational fidelity, and translation elongation. Recent research indicates that the expression of Rpl3 is markedly increased, while that of Rpl3l is highly reduced during mouse skeletal muscle hypertrophy. Moreover, Rpl3l overexpression impairs the growth and myogenic fusion of myotubes. Although the function of Rpl3 and Rpl3l in the ribosome remains to be clarified, these findings suggest that ribosome specialization may be potentially involved in the control of protein translation and skeletal muscle size. Limited data concerning ribosome specialization are currently available in skeletal muscle. Future investigations have the potential to delineate the function of specialized ribosomes in skeletal muscle.

In vivo transcriptome changes of human white blood cells in response to vitamin D

In the vitamin D intervention study VitDbol (NCT02063334) blood samples were drawn directly before an oral bolus (2000 μg vitamin D3) and 24 h later. The focus of phase II of VitDbol was the transcriptome-wide analysis of the effects of vitamin D gene expression in human peripheral blood mononuclear cells (PBMCs). All five participants responded in an individual fashion to the bolus by increases in serum levels of the vitamin D metabolites 25-hydroxyvitamin D3 (25(OH)D3) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3). RNA sequencing identified 15.040 commonly expressed genes in PBMCs, 702 (4,7%) of which were significantly (p < 0,05) affected by the vitamin D3 bolus. KEGG pathway analysis suggested that these genes are involved in general protein translation, monocyte differentiation and cellular growth control. Previously published transcriptome-wide studies in comparable cell systems confirmed 234 of the 702 vitamin D target genes, leaving many genes, such as HLA-A and HLA-C, as novel discoveries. Interestingly, in vivo stimulated PBMCs of this study showed a larger number of common vitamin D target genes with the monocytic cell line THP-1 than with in vitro stimulated PBMCs. The expression pattern of vitamin D target genes differed significantly between individuals and the average expression change can serve as a marker for vitamin D responsiveness. In conclusion, this study demonstrates that under in vivo conditions changes in 25(OH)D3 and 1,25(OH)2D3 serum concentrations alter the expression of more than 700 vitamin D target genes in human leukocytes.