Insights on the molecular mechanisms of SINEUP activity

Translation initiation during protein synthesis is one of the rate limiting steps in regulating gene expression in eukaryotes. It may occur through a cap- dependent or independent mechanism. Cap-independent translation initiation usually takes place when the canonical cap-dependent process involving the cap-eIF4F complex and/or the ternary complex (TC) are inhibited, mostly during cellular stress. It can be activated by modular RNA elements like internal ribosome entering sites (IRESs) that can act alone or in conjunction with other RNA cis-acting elements such as upstream open reading frames (uORF), terminal oligopyrimidines (TOPs) and N6-methyladenosine (m6A). Recently, antisense (AS) long non-coding RNAs (lncRNAs) to mouse and human gene targets were reported to up-regulate translation of those genes, representing a new functional class of lncRNAs termed SINEUPs, for SINE sequences UP-regulating translation. SINEUP modular organization consists of a binding domain (BD) which specifically targets the mRNA of interest by an antisense sequence to its 5\u2019UTR, and an effector domain (ED) constituted by an embedded SINE sequence which confers the biological function of translation activation. However, the underlying molecular mechanism mediated by SINEUPs is unknown. Here, I show using both SINEUP- and IRES-activity assays that the ED of AS Uchl1, the lncRNA representative member of SINEUPs, promotes cap-independent translation through an internal ribosome entry site that is mediated by the inverted SINEB2 stem loop 1 (SL1) hairpin structure, which probably recruits the ribosome using an IRES-like mechanism similar to the one in Hepatitis Virus C. Since synthetic SINEUPs can be targeted to potentially any gene of interest by swapping the BDs, they can be used as molecular tools, in protein manufacturing and RNA therapeutics to increase protein synthesis. Therefore, in this work I optimized a miniaturized version, the synthetic microSINEUP, and showed for the first time that SINEUPs are functional in Drosophila cells. In addition, taking advantage of SINEUP modular organization, I synthesized IRES-containing SINEUPs, called SINEUP-IRESs, that present cellular and viral IRESs acting as ED in SINEUP RNA molecules and therefore proving that IRES sequences can activate translation in trans through a BD. By the study of IRES-containing c-myc mRNA, I discovered the ability of natural IRES elements to increase endogenously expressed, targeted proteins synthesis in trans. These data are the first evidence for a potential new molecular mechanism of gene expression control with long-range consequences in health and disease

SINEUPs: a novel toolbox for RNA therapeutics

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic

Connexin Hemichannel Activation by S-Nitrosoglutathione Synergizes Strongly with Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca2+) waves propagated from irradiated to bystander cells promoting intracellular Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of apoptotic pathways. Combination treatment with S-nitrosoglutathione (GSNO), an endogenous nitric oxide (NO) donor that biases HCs towards the open state, greatly potentiated anti-tumor bystander killing via enhanced Ca2+ signaling, leading to a significant reduction of post-irradiation tumor mass. Our results demonstrate that HCs can be exploited to dramatically increase cytotoxic bystander effects and reveal a previously unappreciated role for HCs in tumor eradication promoted by PDT

Biophysical Aspects of Neurodegenerative and Neurodevelopmental Disorders Involving Endo-/Lysosomal CLC Cl⁻/H⁺ Antiporters

Endosomes and lysosomes are intracellular vesicular organelles with important roles in cell functions such as protein homeostasis, clearance of extracellular material, and autophagy. Endolysosomes are characterized by an acidic luminal pH that is critical for proper function. Five members of the gene family of voltage-gated ChLoride Channels (CLC proteins) are localized to endolysosomal membranes, carrying out anion/proton exchange activity and thereby regulating pH and chloride concentration. Mutations in these vesicular CLCs cause global developmental delay, intellectual disability, various psychiatric conditions, lysosomal storage diseases, and neurodegeneration, resulting in severe pathologies or even death. Currently, there is no cure for any of these diseases. Here, we review the various diseases in which these proteins are involved and discuss the peculiar biophysical properties of the WT transporter and how these properties are altered in specific neurodegenerative and neurodevelopmental disorders

A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+](cyt)) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues

The Age-Related Changes in Vertebrate Retina: Insights from Studies in Inbred and Outbred Mouse Strains

The vertebrate retina is a complex and highly organized tissue that plays a critical role in vision. As individuals age, the retina undergoes a variety of changes, including a decline in photoreceptor function, thinning of the layers, and alterations in the structure and function of glial retinal cells, that can impact visual function [1]. Such changes have been associated with a variety of visual disorders, including age-related macular degeneration and diabetic retinopathy [2,3]. Understanding the molecular mechanisms underlying these alterations is crucial for developing effective therapies to treat these disorders. Animal models provide valuable tools to study the aging of the retina and, based on their different genetic background, the combinatory use of both inbred and outbred mouse strains may lead to a more comprehensive understanding of the aging process. In this context, we propose a multimodal approach to identify and characterize stages of age-related retinal degeneration with high temporal resolution. To achieve this goal, we conducted in vivo, and ex vivo analyses using optical computed tomography (OCT), electroretinogram (ERG) and immunofluorescence (IF) experiments, respectively, to estimate the age-dependent decline of retina function. Mouse retinas from two different strains, an inbred (C57BL/6) and an outbred (CD-1), both male and female, were analyzed at four different time points (2, 6, 12, and 18 months). The research was approved by the Ministry of Health with protocol 1177/2020-PR. OCT provided structural information on the thickness of each retinal layer, documenting a decrease in retinal thickness associated with aging. ERG measures the light-induced electrical activity of the retina, and results indicate a progressive reduction of amplitudes of both a- and b- waves. Additionally, we conducted IF using retinal-specific markers to label key retinal structures, such as GNAT-2 for photoreceptor cells (PRCs), PKCα for rod bipolar cells and Calbindin for horizontal cells (HCs). We also investigated synaptophysin as a marker of cell synapses between photoreceptors and horizontal cells. Consistent with the in vivo data, IF analysis demonstrated a significant age-dependent reduction of PRCs, HCs, RBSs, as well as the area of synapses between PRCs and HCs. Taken together these findings demonstrate a decline in retinal layers associated with aging and suggest potential implications for age-related visual impairment in both mouse strains

Organ-on-chip model shows that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge in the developing cochlea

Prior work supports the hypothesis that ATP release through connexin hemichannels drives spontaneous Ca2+ signaling in non-sensory cells of the greater epithelial ridge (GER) in the developing cochlea; however, direct proof is lacking. To address this issue, we plated cochlear organotypic cultures (COCs) and whole cell-based biosensors with nM ATP sensitivity (ATP-WCBs) at the bottom and top of an ad hoc designed transparent microfluidic chamber, respectively. By performing dual multiphoton Ca2+ imaging, we monitored the propagation of intercellular Ca2+ waves in the GER of COCs and ATP-dependent Ca2+ responses in overlying ATP-WCBs. Ca2+ signals in both COCs and ATP-WCBs were inhibited by supplementing the extracellular medium with ATP diphosphohydrolase (apyrase). Spontaneous Ca2+ signals were strongly depressed in the presence of Gjb6-/- COCs, in which connexin 30 (Cx30) is absent and connexin 26 (Cx26) is strongly downregulated. In contrast, spontaneous Ca2+ signals were not affected by replacement of Panx1-/- with Panx1+/+ COCs in the microfluidic chamber. Similar results were obtained by estimating ATP release from COCs using a classical luciferin-luciferase bioluminescence assay. Therefore, connexin hemichannels and not pannexin 1 channels mediate the release of ATP that is responsible for Ca2+ wave propagation in the developing mouse cochlea. The technological advances presented here have the potential to shed light on a plethora of unrelated open issues that involve paracrine signaling in physiology and pathology and cannot be addressed with standard methods