Condensate functionalization with microtubule motors directs their nucleation in space and allows manipulating RNA localization.

The localization of RNAs in cells is critical for many cellular processes. Whereas motor-driven transport of ribonucleoprotein (RNP) condensates plays a prominent role in RNA localization in cells, their study remains limited by the scarcity of available tools allowing to manipulate condensates in a spatial manner. To fill this gap, we reconstitute in cellula a minimal RNP transport system based on bioengineered condensates, which were functionalized with kinesins and dynein-like motors, allowing for their positioning at either the cell periphery or centrosomes. This targeting mostly occurs through the active transport of the condensate scaffolds, which leads to localized nucleation of phase-separated condensates. Then, programming the condensates to recruit specific mRNAs is able to shift the localization of these mRNAs toward the cell periphery or the centrosomes. Our method opens novel perspectives for examining the role of RNA localization as a driver of cellular functions.

Evolution is not Uniform Along Coding Sequences.

Amino acids evolve at different speeds within protein sequences, because their functional and structural roles are different. Notably, amino acids located at the surface of proteins are known to evolve more rapidly than those in the core. In particular, amino acids at the N- and C-termini of protein sequences are likely to be more exposed than those at the core of the folded protein due to their location in the peptidic chain, and they are known to be less structured. Because of these reasons, we would expect that amino acids located at protein termini would evolve faster than residues located inside the chain. Here we test this hypothesis and found that amino acids evolve almost twice as fast at protein termini compared with those in the center, hinting at a strong topological bias along the sequence length. We further show that the distribution of solvent-accessible residues and functional domains in proteins readily explain how structural and functional constraints are weaker at their termini, leading to the observed excess of amino acid substitutions. Finally, we show that the specific evolutionary rates at protein termini may have direct consequences, notably misleading in silico methods used to infer sites under positive selection within genes. These results suggest that accounting for positional information should improve evolutionary models.

RNA at the surface of phase-separated condensates impacts their size and number.

Although it is now recognized that specific RNAs and protein families are critical for the biogenesis of ribonucleoprotein (RNP) condensates, how these molecular constituents determine condensate size and morphology is unknown. To circumvent the biochemical complexity of endogenous RNP condensates, the use of programmable tools to reconstitute condensate formation with minimal constituents can be instrumental. Here we report a methodology to form RNA-containing condensates in living cells programmed to specifically recruit a single RNA species. Our bioengineered condensates are made of ArtiGranule scaffolds composed of an orthogonal protein that can bind to a specific heterologously expressed RNA. These scaffolds undergo liquid-liquid phase separation in cells and can be chemically controlled to prevent condensation or to trigger condensate dissolution. We found that the targeted RNAs localize at the condensate surface, either as isolated RNA molecules or as a homogenous corona of RNA molecules around the condensate. The recruitment of RNA changes the material properties of condensates by hardening the condensate body. Moreover, the condensate size scales with RNA surface density; the higher the RNA density is, the smaller and more frequent the condensates are. These results suggest a mechanism based on physical constraints, provided by RNAs at the condensate surface, that limit condensate growth and coalescence.

Inherited deficiency of stress granule ZNFX1 in patients with monocytosis and mycobacterial disease.

Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette-Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.

Mutations in genes encoding regulators of mRNA decapping and translation initiation: links to intellectual disability.

Intellectual disability (ID) affects at least 1% of the population, and typically presents in the first few years of life. ID is characterized by impairments in cognition and adaptive behavior and is often accompanied by further delays in language and motor skills, as seen in many neurodevelopmental disorders (NDD). Recent widespread high-throughput approaches that utilize whole-exome sequencing or whole-genome sequencing have allowed for a considerable increase in the identification of these pathogenic variants in monogenic forms of ID. Notwithstanding this progress, the molecular and cellular consequences of the identified mutations remain mostly unknown. This is particularly important as the associated protein dysfunctions are the prerequisite to the identification of targets for novel drugs of these rare disorders. Recent Next-Generation sequencing-based studies have further established that mutations in genes encoding proteins involved in RNA metabolism are a major cause of NDD. Here, we review recent studies linking germline mutations in genes encoding factors mediating mRNA decay and regulators of translation, namely DCPS, EDC3, DDX6 helicase and ID. These RNA-binding proteins have well-established roles in mRNA decapping and/or translational repression, and the mutations abrogate their ability to remove 5' caps from mRNA, diminish their interactions with cofactors and stabilize sub-sets of transcripts. Additional genes encoding RNA helicases with roles in translation including DDX3X and DHX30 have also been linked to NDD. Given the speed in the acquisition, analysis and sharing of sequencing data, and the importance of post-transcriptional regulation for brain development, we anticipate mutations in more such factors being identified and functionally characterized.

GC content shapes mRNA storage and decay in human cells.

mRNA translation and decay appear often intimately linked although the rules of this interplay are poorly understood. In this study, we combined our recent P-body transcriptome with transcriptomes obtained following silencing of broadly acting mRNA decay and repression factors, and with available CLIP and related data. This revealed the central role of GC content in mRNA fate, in terms of P-body localization, mRNA translation and mRNA stability: P-bodies contain mostly AU-rich mRNAs, which have a particular codon usage associated with a low protein yield; AU-rich and GC-rich transcripts tend to follow distinct decay pathways; and the targets of sequence-specific RBPs and miRNAs are also biased in terms of GC content. Altogether, these results suggest an integrated view of post-transcriptional control in human cells where most translation regulation is dedicated to inefficiently translated AU-rich mRNAs, whereas control at the level of 5' decay applies to optimally translated GC-rich mRNAs.

Rare De Novo Missense Variants in RNA Helicase DDX6 Cause Intellectual Disability and Dysmorphic Features and Lead to P-Body Defects and RNA Dysregulation.

The human RNA helicase DDX6 is an essential component of membrane-less organelles called processing bodies (PBs). PBs are involved in mRNA metabolic processes including translational repression via coordinated storage of mRNAs. Previous studies in human cell lines have implicated altered DDX6 in molecular and cellular dysfunction, but clinical consequences and pathogenesis in humans have yet to be described. Here, we report the identification of five rare de novo missense variants in DDX6 in probands presenting with intellectual disability, developmental delay, and similar dysmorphic features including telecanthus, epicanthus, arched eyebrows, and low-set ears. All five missense variants (p.His372Arg, p.Arg373Gln, p.Cys390Arg, p.Thr391Ile, and p.Thr391Pro) are located in two conserved motifs of the RecA-2 domain of DDX6 involved in RNA binding, helicase activity, and protein-partner binding. We use functional studies to demonstrate that the first variants identified (p.Arg373Gln and p.Cys390Arg) cause significant defects in PB assembly in primary fibroblast and model human cell lines. These variants' interactions with several protein partners were also disrupted in immunoprecipitation assays. Further investigation via complementation assays included the additional variants p.Thr391Ile and p.Thr391Pro, both of which, similarly to p.Arg373Gln and p.Cys390Arg, demonstrated significant defects in P-body assembly. Complementing these molecular findings, modeling of the variants on solved protein structures showed distinct spatial clustering near known protein binding regions. Collectively, our clinical and molecular data describe a neurodevelopmental syndrome associated with pathogenic missense variants in DDX6. Additionally, we suggest DDX6 join the DExD/H-box genes DDX3X and DHX30 in an emerging class of neurodevelopmental disorders involving RNA helicases.

RNA is a critical element for the sizing and the composition of phase-separated RNA-protein condensates.

Liquid-liquid phase separation is thought to be a key organizing principle in eukaryotic cells to generate highly concentrated dynamic assemblies, such as the RNP granules. Numerous in vitro approaches have validated this model, yet a missing aspect is to take into consideration the complex molecular mixture and promiscuous interactions found in vivo. Here we report the versatile scaffold ArtiG to generate concentration-dependent RNA-protein condensates within living cells, as a bottom-up approach to study the impact of co-segregated endogenous components on phase separation. We demonstrate that intracellular RNA seeds the nucleation of the condensates, as it provides molecular cues to locally coordinate the formation of endogenous high-order RNP assemblies. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context.

Pat1 RNA-binding proteins: Multitasking shuttling proteins.

Post-transcriptional regulation of gene expression is largely achieved at the level of splicing in the nucleus, and translation and mRNA decay in the cytosol. While the regulation may be global, through the direct inhibition of central factors, such as the spliceosome, translation initiation factors and mRNA decay enzymes, in many instances transcripts bearing specific sequences or particular features are regulated by RNA-binding factors which mobilize or impede recruitment of these machineries. This review focuses on the Pat1 family of RNA-binding proteins, conserved from yeast to man, that enhance the removal of the 5' cap by the decapping enzyme Dcp1/2, leading to mRNA decay and also have roles in translational repression. Like Dcp1/2, other decapping coactivators, including DDX6 and Edc3, and translational repressor proteins, Pat1 proteins are enriched in cytoplasmic P-bodies, which have a principal role in mRNA storage. They also concentrate in nuclear Cajal-bodies and splicing speckles and in man, impact splice site choice in some pre-mRNAs. Pivotal to these functions is the association of Pat1 proteins with distinct heptameric Lsm complexes: the cytosolic Pat1/Lsm1-7 complex mediates mRNA decay and the nuclear Pat1/Lsm2-8 complex alternative splicing. This dual role of human Pat1b illustrates the power of paralogous complexes to impact distinct processes in separate compartments. The review highlights our recent findings that Pat1b mediates the decay of AU-rich mRNAs, which are particularly enriched in P-bodies, unlike the decapping activator DDX6, which acts on GC-rich mRNAs, that tend to be excluded from P-bodies, and discuss the implications for mRNA decay pathways. This article is categorized under: RNA Turnover and Surveillance > Regulation of RNA Stability RNRNA Processing > Splicing Regulation/Alternative Splicing Translation > Translation Regulation.

Re-viewing the 3D Organization of mRNPs.

In this issue of Molecular Cell, using leading-edge technologies, Metkar et al. (2018) and Adivarahan et al. (2018) revisit the spatial organization of mRNPs, showing that they form flexible rod-like structures prior to translation that decompact during translation while the closed-loop conformation is rarely observed.

Tau/DDX6 interaction increases microRNA activity.

Tauopathies, such as Alzheimer's disease, are characterized by intracellular aggregates of insoluble Tau proteins. Originally described as a microtubule binding protein, recent studies demonstrated additional physiological roles for Tau. The fact that a single protein can regulate multiple cellular functions has posed challenge in terms of understanding mechanistic cues behind the pathology. Here, we used tandem-affinity purification methodology coupled to mass spectrometry to identify novel interaction partners. We found that Tau interacts with DDX6, a DEAD box RNA helicase involved in translation repression and mRNA decay as well as in the miRNA pathway. Our results demonstrate that Tau increases the silencing activity of the miRNA let-7a, miR-21 and miR-124 through DDX6. Importantly, Tau mutations (P301S, P301L) found in the inherited tauopathies, frontotemporal dementia and parkinsonism linked to chromosome 17, disrupt Tau/DDX6 interaction and impair gene silencing by let-7a. Altogether, these data demonstrated a new unexpected role for Tau in regulating miRNA activity.

P-Bodies: Cytosolic Droplets for Coordinated mRNA Storage.

P-bodies (PBs) are cytosolic RNP granules that are conserved among eukaryotic organisms. In the past few years, major progress has been made in understanding the biochemical and biophysical mechanisms that lead to their formation. However, whether they play a role in mRNA storage or decay remains actively debated. P-bodies were recently isolated from human cells by a novel fluorescence-activated particle sorting (FAPS) approach that enabled the characterization of their protein and RNA content, providing new insights into their function. Together with recent innovative imaging studies, these new data show that mammalian PBs are primarily involved not in RNA decay but rather in the coordinated storage of mRNAs encoding regulatory functions. These small cytoplasmic droplets could thus be important for cell adaptation to the environment.

P-Body Purification Reveals the Condensation of Repressed mRNA Regulons.

Within cells, soluble RNPs can switch states to coassemble and condense into liquid or solid bodies. Although these phase transitions have been reconstituted in vitro, for endogenous bodies the diversity of the components, the specificity of the interaction networks, and the function of the coassemblies remain to be characterized. Here, by developing a fluorescence-activated particle sorting (FAPS) method to purify cytosolic processing bodies (P-bodies) from human epithelial cells, we identified hundreds of proteins and thousands of mRNAs that structure a dense network of interactions, separating P-body from non-P-body RNPs. mRNAs segregating into P-bodies are translationally repressed, but not decayed, and this repression explains part of the poor genome-wide correlation between RNA and protein abundance. P-bodies condense thousands of mRNAs that strikingly encode regulatory processes. Thus, we uncovered how P-bodies, by condensing and segregating repressed mRNAs, provide a physical substrate for the coordinated regulation of posttranscriptional mRNA regulons.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes.

Pat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5' to 3' mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3' UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

Early postpartum gastric band slippage after bariatric surgery in an adolescent obese girl.

We report here a case of a rarely described complication of laparoscopic adjustable gastric banding (LAGB), slippage during the postpartum period, after LAGB had been performed in an adolescent obese girl. The LAGB had been placed after one year of clinical survey initiated at the age of 16. Maximal pre-operative body mass index (BMI) was 48.5 kg.m(-2) and obesity was associated with insulin resistance. Before pregnancy, there was a loss of 17 Kg (final BMI = 41.5 kg.m(-2)) and a resolution of insulin resistance. The patient became pregnant 21 months after LAGB, and whole pregnancy and delivery were uneventful for both mother and fetus. Six weeks after delivery, the patient suddenly complained for total food intolerance, due to a band slippage, leading to removal of the band. Slippage is now a rare complication of LAGB, but can happen during pregnancy and the postpartum period as well.

The DDX6-4E-T interaction mediates translational repression and P-body assembly.

4E-Transporter binds eIF4E via its consensus sequence YXXXXLΦ, shared with eIF4G, and is a nucleocytoplasmic shuttling protein found enriched in P-(rocessing) bodies. 4E-T inhibits general protein synthesis by reducing available eIF4E levels. Recently, we showed that 4E-T bound to mRNA however represses its translation in an eIF4E-independent manner, and contributes to silencing of mRNAs targeted by miRNAs. Here, we address further the mechanism of translational repression by 4E-T by first identifying and delineating the interacting sites of its major partners by mass spectrometry and western blotting, including DDX6, UNR, unrip, PAT1B, LSM14A and CNOT4. Furthermore, we document novel binding between 4E-T partners including UNR-CNOT4 and unrip-LSM14A, altogether suggesting 4E-T nucleates a complex network of RNA-binding protein interactions. In functional assays, we demonstrate that joint deletion of two short conserved motifs that bind UNR and DDX6 relieves repression of 4E-T-bound mRNA, in part reliant on the 4E-T-DDX6-CNOT1 axis. We also show that the DDX6-4E-T interaction mediates miRNA-dependent translational repression and de novo P-body assembly, implying that translational repression and formation of new P-bodies are coupled processes. Altogether these findings considerably extend our understanding of the role of 4E-T in gene regulation, important in development and neurogenesis.

Laparoscopic adjustable gastric banding in adolescents: Results at two years including psychosocial aspects.

BACKGROUND/PURPOSE: Obesity now affects 3%-4% of the pediatric population and contributes to the increase in cardiac mortality in adulthood. Bariatric surgery is the best treatment for weight loss and the obesity-associated comorbidities in adults. We report here our experience of laparoscopic adjustable gastric banding (LAGB) in adolescents. METHODS: The medical charts of the first 16 patients operated on in our center were reviewed. Data were compiled concerning weight loss, physical and biological comorbidities, health-related quality of life (QOL) and surgical complications before surgery and during 24months of follow-up. RESULTS: The maximal pre-operative median body mass index was 43.0kg·m(-2), decreasing to 33.0kg·m(-2) at 2years post-LAGB, which corresponded to a 49.2% excess body weight loss (p<0.001). Most comorbidities (glucose intolerance, hypertension and sleep apnea) resolved within the first year post-LAGB and QOL was improved on the PedsQL™ scales. No severe surgical complications were noted, with only three re-interventions for device failure (2) or band removal (1). CONCLUSION: LAGB is well tolerated in adolescents and shows a beneficial impact on weight loss and obesity-related comorbidities. Associated with global management, it may have a positive impact on patients' QOL and social and psychological status.

Estimation of Recent and Ancient Inbreeding in a Small Endogamous Tunisian Community Through Genomic Runs of Homozygosity.

Runs of homozygosity (ROHs) are extended genomic regions of homozygous genotypes that record populations' mating patterns in the past. We performed microarray genotyping on 15 individuals from a small isolated Tunisian community. We estimated the individual and population genome-wide level of homozygosity from data on ROH above 0.5 Mb in length. We found a high average number of ROH per individual (48.2). The smallest ROH category (0.5-1.49 Mb) represents 0.93% of the whole genome, while medium-size (1.5-4.99 Mb) and long-size ROH (≥5 Mb) cover 1.18% and 0.95%, respectively. We found that genealogical individual inbreeding coefficients (Fped ) based on three- to four-generation pedigrees are not reliable indicators of the current proportion of genome-wide homozygosity inferred from ROH (FROH ) either for 0.5 or 1.5 Mb ROH length thresholds, while identity-by-descent sharing is a function of shared coancestry. This study emphasizes the effect of reproductive isolation and a prolonged practice of consanguinity that limits the genetic heterogeneity. It also provides evidence of both recent and ancient parental relatedness contribution to the current level of genome-wide homozygosity in the studied population. These findings may be useful for evaluation of long-term effects of inbreeding on human health and for future applications of ROHs in identifying recessive susceptibility genes.

Comparative ultrastructure of CRM1-Nucleolar bodies (CNoBs), Intranucleolar bodies (INBs) and hybrid PML/p62 bodies uncovers new facets of nuclear body dynamic and diversity.

In order to gain insights on the nuclear organization in mammalian cells, we characterized ultrastructurally nuclear bodies (NBs) previously described as fluorescent foci. Using high resolution immunoelectron microscopy (I-EM), we provide evidence that CNoBs (CRM1-Nucleolar bodies) and INBs (Intranucleolar bodies) are distinct genuine nucleolar structures in untreated HeLa cells. INBs are fibrillar and concentrate the post-translational modifiers SUMO1 and SUMO-2/3 as strongly as PML bodies. In contrast, the smallest CRM1-labeled CNoBs are vitreous, preferentially located at the periphery of the nucleolus and, intricately linked to the chromatin network. Upon blockage of the CRM1-dependent nuclear export by leptomycin B (LMB), CNoBs disappear while p62/SQSTM1-containing fibrillar nuclear bodies are induced. These p62 bodies are enriched in ubiquitinated proteins. They progressively associate with PML bodies to form hybrid bodies of which PML decorates the periphery while p62/SQSTM1 is centrally-located. Our study is expanding the repertoire of nuclear bodies; revealing a previously unrecognized composite nucleolar landscape and a new mode of interactions between ubiquitous (PML) and stress-induced (p62) nuclear bodies, resulting in the formation of hybrid bodies.