Altered RNA Editing in Atopic Dermatitis Highlights the Role of Double-Stranded RNA for Immune Surveillance.

Atopic dermatitis (AD) is associated with dysregulated type 1 IFN‒mediated responses, in parallel with the dominant type 2 inflammation. However, the pathophysiology of this dysregulation is largely unknown. Adenosine-to-inosine RNA editing plays a critical role in immune regulation by preventing double-stranded RNA recognition by MDA5 and IFN activation. We studied global adenosine-to-inosine editing in AD to elucidate the role played by altered editing in the pathophysiology of this disease. Analysis of three RNA-sequencing datasets of AD skin samples revealed reduced levels of adenosine-to-inosine RNA editing in AD. This reduction was seen globally throughout Alu repeats as well as in coding genes and in specific pre-mRNA loci expected to create long double-stranded RNA, the main substrate of MDA5 leading to type I IFN activation. Consistently, IFN signature genes were upregulated. In contrast, global editing was not altered in systemic lupus erythematosus and systemic sclerosis, despite IFN activation. Our results indicate that altered editing leading to impairment of the innate immune response may be involved in the pathogenesis of AD. Possibly, it may be relevant for additional autoimmune and inflammatory diseases.

Primary Melanoma miRNA Trafficking Induces Lymphangiogenesis.

Melanoma, the deadliest cutaneous tumor, initiates within the epidermis; during progression, cells invade into the dermis and become metastatic through the lymphatic and blood system. Before melanoma cell invasion into the dermis, an increased density of dermal lymphatic vessels is observed, generated by a mechanism which is not fully understood. In this study, we show that, while at the primary epidermal stage (in situ), melanoma cells secrete extracellular vesicles termed melanosomes, which are uptaken by dermal lymphatic cells, leading to transcriptional and phenotypic pro-lymphangiogenic changes. Mechanistically, melanoma-derived melanosomes traffic mature let-7i to lymphatic endothelial cells, which mediate pro-lymphangiogenic phenotypic changes by the induction of type I IFN signaling. Furthermore, transcriptome analysis upon treatment with melanosomes or let-7i reveals the enhancement of IFI6 expression in lymphatic cells. Because melanoma cells metastasize primarily via lymphatic vessels, our data suggest that blocking lymphangiogenesis by repressing either melanosome release or type I IFN signaling will prevent melanoma progression to the deadly metastatic stage.

Effects of cell size and bicarbonate on single photon response variability in retinal rods.

Accurate photon counting requires that rods generate highly amplified, reproducible single photon responses (SPRs). The SPR is generated within the rod outer segment (ROS), a multilayered structure built from membranous disks that house rhodopsin. Photoisomerization of rhodopsin at the disk rim causes a local depletion of cGMP that closes ion channels in the plasmalemma located nearby with relative rapidity. In contrast, a photoisomerization at the disk center, distant from the plasmalemma, has a delayed impact on the ion channels due to the time required for cGMP redistribution. Radial differences should be greatest in large diameter rods. By affecting membrane guanylate cyclase activity, bicarbonate could impact spatial inhomogeneity in cGMP content. It was previously known that in the absence of bicarbonate, SPRs are larger and faster at the base of a toad ROS (where the ROS attaches to the rest of the cell) than at the distal tip. Given that bicarbonate enters the ROS at the base and diffuses to the tip and that it expedites flash response recovery, there should be an axial concentration gradient for bicarbonate that would accentuate the base-to-tip SPR differences. Seeking to understand how ROS geometry and bicarbonate affect SPR variability, we used mathematical modeling and made electrophysiological recordings of single rods. Modeling predicted and our experiments confirmed minor radial SPR variability in large diameter, salamander rods that was essentially unchanged by bicarbonate. SPRs elicited at the base and tip of salamander rods were similar in the absence of bicarbonate, but when treated with 30 mM bicarbonate, SPRs at the base became slightly faster than those at the tip, verifying the existence of an axial gradient for bicarbonate. The differences were small and unlikely to undermine visual signaling. However, in toad rods with longer ROSs, bicarbonate somehow suppressed the substantial, axial SPR variability that is naturally present in the absence of bicarbonate. Modeling suggested that the axial gradient of bicarbonate might dampen the primary phototransduction cascade at the base of the ROS. This novel effect of bicarbonate solves a mystery as to how toad vision is able to function effectively in extremely dim light.

Two- and Three-Dimensional Tracking of MFA2 mRNA Molecules in Mating Yeast.

Intracellular mRNA transport contributes to the spatio-temporal regulation of mRNA function and localized translation. In the budding yeast, Saccharomyces cerevisiae, asymmetric mRNA transport localizes ~30 specific mRNAs including those encoding polarity and secretion factors, to the bud tip. The underlying process involves RNA-binding proteins (RBPs), molecular motors, processing bodies (PBs), and the actin cytoskeleton. Recently, pheromone a-factor expression in mating yeast was discovered to depend on proper localization of its mRNA, MFA2 mRNAs in conjunction with PBs cluster at the shmoo tip to form "mating bodies", from which a-factor is locally expressed. The mechanism ensuring the correct targeting of mRNA to the shmoo tip is poorly understood. Here we analyzed the kinetics and trajectories of MFA2 mRNA transport in living, alpha-factor treated yeast. Two- (2D) and three-dimensional (3D) analyses allowed us to reconstruct the granule tracks and estimate granule velocities. Tracking analysis of single MFA2 mRNA granules, labeled using a fluorescent aptamer system, demonstrated three types movement: vibrational, oscillatory and translocational. The mRNA granule transport was complex; a granule could change its movement behavior and composition during its journey to the shmoo. Processing body assembly and the actin-based motor, Myo4p, were involved in movement of MFA2 mRNA to the shmoo, but neither was required, indicating that multiple mechanisms for translocation were at play. Our visualization studies present a dynamic view of the localization mechanism in shmoo-bearing cells.

Modes of Accessing Bicarbonate for the Regulation of Membrane Guanylate Cyclase (ROS-GC) in Retinal Rods and Cones.

The membrane guanylate cyclase, ROS-GC, that synthesizes cyclic GMP for use as a second messenger for visual transduction in retinal rods and cones, is stimulated by bicarbonate. Bicarbonate acts directly on ROS-GC1, because it enhanced the enzymatic activity of a purified, recombinant fragment of bovine ROS-GC1 consisting solely of the core catalytic domain. Moreover, recombinant ROS-GC1 proved to be a true sensor of bicarbonate, rather than a sensor for CO2. Access to bicarbonate differed in rods and cones of larval salamander, Ambystoma tigrinum, of unknown sex. In rods, bicarbonate entered at the synapse and diffused to the outer segment, where it was removed by Cl--dependent exchange. In contrast, cones generated bicarbonate internally from endogenous CO2 or from exogenous CO2 that was present in extracellular solutions of bicarbonate. Bicarbonate production from both sources of CO2 was blocked by the carbonic anhydrase inhibitor, acetazolamide. Carbonic anhydrase II expression was verified immunohistochemically in cones but not in rods. In addition, cones acquired bicarbonate at their outer segments as well as at their inner segments. The multiple pathways for access in cones may support greater uptake of bicarbonate than in rods and buffer changes in its intracellular concentration.

Network analysis of microRNAs, genes and their regulation in diffuse and follicular B-cell lymphomas.

MicroRNAs (miRs) are short non-coding regulatory RNAs that control gene expression at the post-transcriptional level and play an important role in cancer development and progression, acting either as oncogenes or as tumor suppressors. Identification of aberrantly expressed miRs in patients with hematological malignancies as compared to healthy individuals has suggested that these molecules may serve as novel clinical diagnostic and prognostic biomarkers. We conducted a systematic literature review of articles published between 2007 and 2017 and re-analyzed experimentally-validated human miR expression signatures in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) from various biological sources (tumor tissue, peripheral blood, bone marrow and cell lines). A unique miR expression pattern was observed for each disease. Compared to healthy individuals, 61 miRs were aberrantly expressed in DLBCL and 85 in FL; 20-30% of aberrantly expressed miRs overlapped between the two lymphoma subtypes. Analysis of integrative positive and negative miRNA-mRNA relationships using the Ingenuity Pathway Analysis (IPA) system revealed 970 miR-mRNA pairs for DLBCL and 90 for FL. Through gene ontology analysis, we found potential regulatory pathways that are deregulated in DLBCL and FL due to improper expression of miR target genes. By comparing the expression level of the aberrantly expressed miRs in DLBCL to their expression levels in other malignancies, we identified seven miRs that are aberrantly expressed in DLBCL tumor tissues (miR-15a, miR-16, miR-17, miR-106, miR-21, miR-155 and miR-34a-5p). This specific expression pattern may be a potential diagnostic tool for DLBCL.

Increased copper bioremediation ability of new transgenic and adapted Saccharomyces cerevisiae strains.

Environmental pollution with heavy metals is a very serious ecological problem, which can be solved by bioremediation of metal ions by microorganisms. Yeast cells, especially Saccharomyces cerevisiae, are known to exhibit a good natural ability to remove heavy metal ions from an aqueous phase. In the present work, an attempt was made to increase the copper-binding properties of S. cerevisiae. For this purpose, new strains of S. cerevisiae were produced by construction and integration of recombinant human MT2 and GFP-hMT2 genes into yeast cells. The ySA4001 strain expressed GFP-hMT2p under the constitutive pADH1 promoter and the ySA4002 and ySA4003 strains expressed hMT2 and GFP-hMT2 under the inducible pCUP1 promoter. An additional yMNWTA01 strain was obtained by adaptation of the BY4743 wild type S. cerevisiae strain to high copper concentrations. The yMNWTA01, ySA4002, and ySA4003 strains exhibited an enhanced ability for copper ion bioremediation.