The kinesin KIF1C transports APC-dependent mRNAs to cell protrusions.

RNA localization and local translation are important for numerous cellular functions. In mammals, a class of mRNAs localize to cytoplasmic protrusions in an APC-dependent manner, with roles during cell migration. Here, we investigated this localization mechanism. We found that the KIF1C motor interacts with APC-dependent mRNAs and is required for their localization. Live cell imaging revealed rapid, active transport of single mRNAs over long distances that requires both microtubules and KIF1C. Two-color imaging directly revealed single mRNAs transported by single KIF1C motors, with the 3'UTR being sufficient to trigger KIF1C-dependent RNA transport and localization. Moreover, KIF1C remained associated with peripheral, multimeric RNA clusters and was required for their formation. These results reveal a widespread RNA transport pathway in mammalian cells, in which the KIF1C motor has a dual role in transporting RNAs and clustering them within cytoplasmic protrusions. Interestingly, KIF1C also transports its own mRNA, suggesting a possible feedback loop acting at the level of mRNA transport.

A choreography of centrosomal mRNAs reveals a conserved localization mechanism involving active polysome transport.

Local translation allows for a spatial control of gene expression. Here, we use high-throughput smFISH to screen centrosomal protein-coding genes, and we describe 8 human mRNAs accumulating at centrosomes. These mRNAs localize at different stages during cell cycle with a remarkable choreography, indicating a finely regulated translational program at centrosomes. Interestingly, drug treatments and reporter analyses reveal a common translation-dependent localization mechanism requiring the nascent protein. Using ASPM and NUMA1 as models, single mRNA and polysome imaging reveals active movements of endogenous polysomes towards the centrosome at the onset of mitosis, when these mRNAs start localizing. ASPM polysomes associate with microtubules and localize by either motor-driven transport or microtubule pulling. Remarkably, the Drosophila orthologs of the human centrosomal mRNAs also localize to centrosomes and also require translation. These data identify a conserved family of centrosomal mRNAs that localize by active polysome transport mediated by nascent proteins.

Apolipoprotein C-III and cardiovascular diseases: when genetics meet molecular pathologies.

Cardiovascular diseases (CVD) have overtaken infectious diseases and are currently the world's top killer. A quite strong linkage between this type of ailments and elevated plasma levels of triglycerides (TG) has been always noticed. Notably, this risk factor is mired in deep confusion, since its role in atherosclerosis is uncertain. One of the explanations that aim to decipher this persistent enigma was provided by apolipoprotein C-III (apoC-III), a small protein historically recognized as an important regulator of TG metabolism. Preeminently, hundreds of studies have been carried out in order to explore the APOC3 genetic background, as well as to establish a correlation between its variants and dyslipidemia-related disorders, pointing to an earnest predictive power for future outcomes. Among several polymorphisms reported within the APOC3, the SstI site in its 3'-untranslated region (3'-UTR) was the most consistently and robustly associated with an increased CVD risk. As more genetic data supporting its importance in cardiovascular events aggregate, it was declared, correspondingly, that apoC-III exerts various atherogenic effects, either by intervening in the function and catabolism of many lipoproteins, or by inducing endothelial inflammation and smooth muscle cells (SMC) proliferation. This review was designed to shed the light on the structural and functional aspects of the APOC3 gene, the existing association between its SstI polymorphism and CVD, and the specific molecular mechanisms that underlie apoC-III pathological implications. In addition, the translation of all these gathered knowledges into preventive and therapeutic benefits will be detailed too.

Anticancer Activity of Thymoquinone Cubic Phase Nanoparticles Against Human Breast Cancer: Formulation, Cytotoxicity and Subcellular Localization.

INTRODUCTION: Triple negative breast cancer is an aggressive disorder which accounts for at least 15% of breast cancer diagnosis and a high percentage of breast cancer morbidity, hence intensive research efforts are focused on the development of effective therapies to overcome the disease. Thymoquinone (TQ), the bioactive constituent of Nigella sativa, exhibits anticancer activity, yet its translation to the clinic is hindered by its poor bioavailability and lack of quantification method in blood and tissues. To overcome these limitations, cubosomes were utilized for the encapsulation and delivery of this anticancer molecule. METHODS: Thymoquinone loaded cubosomes were prepared through the emulsification homogenization method. The physicochemical characteristics, including particle size, zeta potential, morphology and entrapment efficiency, were studied. Moreover, the in vitro antitumor activity was tested on breast cancer cell lines (MCF-7 and MDA-MB-231) and compared to non-tumorigenic cell line (MCF-10A). Subcellular localization, cellular uptake and apoptotic effects of the formulations were assessed. RESULTS: The results revealed that the TQ loaded cubosomal formulation exhibited a mean particle size of 98.0 ± 4.10 nm with narrow unimodal distribution. The high entrapment efficiency (96.60 ± 3.58%) and zeta potential (31.50 ±4.20 mV) conceived the effectiveness of this nanosystem for TQ encapsulation. Cell viability in both breast cancer cell lines demonstrated a dose-dependent decrease in response to treatment with free TQ or TQ-loaded cubosomes, with enhanced antitumor activity upon treating with the latter formulation. A significant increase in apoptotic bodies and cleaved caspase 3 was observed upon treatment of MDA-MB-231 cells with either TQ or TQ-loaded cubosomes. Localization and trafficking studies unveiled that cubosomes accumulate in the cytoplasm of the studied breast cancer cell lines. DISCUSSION: Our results show that thymoquinone encapsulation in cubosomal nanoparticles provides a promising anticancer drug delivery system with the ability to label, detect and subsequently trace it within the human cells.

A Dual Protein-mRNA Localization Screen Reveals Compartmentalized Translation and Widespread Co-translational RNA Targeting.

Local translation allows spatial control of gene expression. Here, we performed a dual protein-mRNA localization screen, using smFISH on 523 human cell lines expressing GFP-tagged genes. 32 mRNAs displayed specific cytoplasmic localizations with local translation at unexpected locations, including cytoplasmic protrusions, cell edges, endosomes, Golgi, the nuclear envelope, and centrosomes, the latter being cell-cycle-dependent. Automated classification of mRNA localization patterns revealed a high degree of intercellular heterogeneity. Surprisingly, mRNA localization frequently required ongoing translation, indicating widespread co-translational RNA targeting. Interestingly, while P-body accumulation was frequent (15 mRNAs), four mRNAs accumulated in foci that were distinct structures. These foci lacked the mature protein, but nascent polypeptide imaging showed that they were specialized translation factories. For ß-catenin, foci formation was regulated by Wnt, relied on APC-dependent polysome aggregation, and led to nascent protein degradation. Thus, translation factories uniquely regulate nascent protein metabolism and create a fine granular compartmentalization of translation.

A novel multifaceted approach for wound healing: Optimization and in vivo evaluation of spray dried tadalafil loaded pro-nanoliposomal powder.

The topical delivery of nanotherapeutics at the injury site for skin regeneration has received increasing attention as a strategy for wound treatment. This study aimed to investigate the preparation of spray dried tadalafil loaded pro-nanoliposomes powder as a novel system to accelerate wound healing process. The optimization was carried out employing 32 factorial design based on phospholipid and cholesterol concentrations. The physicochemical characterizations, in vitro cellular assessment and in vivo performance were evaluated. The results obtained pointed out that phospholipid concentration presented a positive effect on the entrapment efficacy and particle size, while cholesterol hindered the entrapment efficacy yet presented a prominent influence on particle size. Moreover, the optimized formulation showed a sustained release, high zeta potential and uniform spherical particles indicating entrapment of tadalafil in its amorphous state as demonstrated by FTIR and XPRD results. Cell viability and in vitro scratch assay demonstrated no cytotoxicity on human fibroblast cell lines and the ability of the drug and optimized formulation to promote cell migration. In vivo wound healing studies revealed significantly higher wound closure rates for areas treated with optimized loaded-formulation (65.95±6.47%) compared to unloaded formulation (29.78±9.65%), free drug (38.87±11.44%) and sham group (10.22±5.11%). In the in vivo study, histopathological specimens supported the previous results with presentation of cascade of healing elements via the angiogenetic activity of tadalafil. These outcomes provide an insight of a novel and emerging therapeutic drug system for wound treatment in clinical practice.

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.

A computational framework to study sub-cellular RNA localization.

RNA localization is a crucial process for cellular function and can be quantitatively studied by single molecule FISH (smFISH). Here, we present an integrated analysis framework to analyze sub-cellular RNA localization. Using simulated images, we design and validate a set of features describing different RNA localization patterns including polarized distribution, accumulation in cell extensions or foci, at the cell membrane or nuclear envelope. These features are largely invariant to RNA levels, work in multiple cell lines, and can measure localization strength in perturbation experiments. Most importantly, they allow classification by supervised and unsupervised learning at unprecedented accuracy. We successfully validate our approach on representative experimental data. This analysis reveals a surprisingly high degree of localization heterogeneity at the single cell level, indicating a dynamic and plastic nature of RNA localization.

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.

Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells.

Translation is an essential step in gene expression. In this study, we used an improved SunTag system to label nascent proteins and image translation of single messenger ribonucleoproteins (mRNPs) in human cells. Using a dedicated reporter RNA, we observe that translation of single mRNPs stochastically turns on and off while they diffuse through the cytoplasm. We further measure a ribosome density of 1.3 per kilobase and an elongation rate of 13-18 amino acids per second. Tagging the endogenous POLR2A gene revealed similar elongation rates and ribosomal densities and that nearly all messenger RNAs (mRNAs) are engaged in translation. Remarkably, tagging of the heavy chain of dynein 1 (DYNC1H1) shows this mRNA accumulates in foci containing three to seven RNA molecules. These foci are translation sites and thus represent specialized translation factories. We also observe that DYNC1H1 polysomes are actively transported by motors, which may deliver the mature protein at appropriate cellular locations. The SunTag should be broadly applicable to study translational regulation in live single cells.

smiFISH and FISH-quant - a flexible single RNA detection approach with super-resolution capability.

Single molecule FISH (smFISH) allows studying transcription and RNA localization by imaging individual mRNAs in single cells. We present smiFISH (single molecule inexpensive FISH), an easy to use and flexible RNA visualization and quantification approach that uses unlabelled primary probes and a fluorescently labelled secondary detector oligonucleotide. The gene-specific probes are unlabelled and can therefore be synthesized at low cost, thus allowing to use more probes per mRNA resulting in a substantial increase in detection efficiency. smiFISH is also flexible since differently labelled secondary detector probes can be used with the same primary probes. We demonstrate that this flexibility allows multicolor labelling without the need to synthesize new probe sets. We further demonstrate that the use of a specific acrydite detector oligonucleotide allows smiFISH to be combined with expansion microscopy, enabling the resolution of transcripts in 3D below the diffraction limit on a standard microscope. Lastly, we provide improved, fully automated software tools from probe-design to quantitative analysis of smFISH images. In short, we provide a complete workflow to obtain automatically counts of individual RNA molecules in single cells.

A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting.

Live-cell imaging has revealed unexpected features of gene expression. Here using improved single-molecule RNA microscopy, we show that synthesis of HIV-1 RNA is achieved by groups of closely spaced polymerases, termed convoys, as opposed to single isolated enzymes. Convoys arise by a Mediator-dependent reinitiation mechanism, which generates a transient but rapid succession of polymerases initiating and escaping the promoter. During elongation, polymerases are spaced by few hundred nucleotides, and physical modelling suggests that DNA torsional stress may maintain polymerase spacing. We additionally observe that the HIV-1 promoter displays stochastic fluctuations on two time scales, which we refer to as multi-scale bursting. Each time scale is regulated independently: Mediator controls minute-scale fluctuation (convoys), while TBP-TATA-box interaction controls sub-hour fluctuations (long permissive/non-permissive periods). A cellular promoter also produces polymerase convoys and displays multi-scale bursting. We propose that slow, TBP-dependent fluctuations are important for phenotypic variability of single cells.