Optochemical control of RNA interference in mammalian cells

Short interfering RNAs (siRNAs) and microRNAs (nniRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules

Upregulation of the Nrf2 antioxidant pathway characterizes the transition from productive to latent infection in CD4+ T cells

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Short-duration splice promoting compound enables a tunable mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. SMA results from insufficient survival motor neuron (SMN) protein due to alternative splicing. Antisense oligonucleotides, gene therapy and splicing modifiers recently received FDA approval. Although severe SMA transgenic mouse models have been beneficial for testing therapeutic efficacy, models mimicking milder cases that manifest post-infancy have proven challenging to develop. We established a titratable model of mild and moderate SMA using the splicing compound NVS-SM2. Administration for 30 d prevented development of the SMA phenotype in severe SMA mice, which typically show rapid weakness and succumb by postnatal day 11. Furthermore, administration at day eight resulted in phenotypic recovery. Remarkably, acute dosing limited to the first 3 d of life significantly enhanced survival in two severe SMA mice models, easing the burden on neonates and demonstrating the compound as suitable for evaluation of follow-on therapies without potential drug-drug interactions. This pharmacologically tunable SMA model represents a useful tool to investigate cellular and molecular pathogenesis at different stages of disease

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic, and metabolomic analyses, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a “shock and kill effect” decreasing proviral DNA in cells from people living with HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies

Discovery of a small molecule probe that post-translationally stabilizes the survival motor neuron protein for the treatment of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is the leading genetic cause of infant death. We previously developed a high-throughput assay that employs an SMN2-luciferase reporter allowing identification of compounds that act transcriptionally, enhance exon recognition, or stabilize the SMN protein. We describe optimization and characterization of an analog suitable for in vivo testing. Initially, we identified analog 4m that had good in vitro properties but low plasma and brain exposure in a mouse PK experiment due to short plasma stability; this was overcome by reversing the amide bond and changing the heterocycle. Thiazole 27 showed excellent in vitro properties and a promising mouse PK profile, making it suitable for in vivo testing. This series post-translationally stabilizes the SMN protein, unrelated to global proteasome or autophagy inhibition, revealing a novel therapeutic mechanism that should complement other modalities for treatment of SMA

The Human Mitochondrial tRNAMet: Structure/Function Relationship of a Unique Modification in the Decoding of Unconventional Codons

Human mitochondrial mRNAs utilize the universal AUG and the unconventional isoleucine AUA codons for methionine. In contrast to translation in the cytoplasm, human mitochondria use one tRNA, hmtRNAMetCAU, to read AUG and AUA codons at both the peptidyl- (P-), and aminoacyl-(A-) sites of the ribosome. The hmtRNAMetCAU has a unique post-transcriptional modification, 5-formylcytidine, at the wobble position 34 (f5C34), and a cytidine substituting for the invariant uridine at position 33 of the canonical “U-turn” in tRNAs. The structure of the tRNA's anticodon stem and loop domain (hmtASLMetCAU), determined by NMR restrained molecular modeling, revealed how the f5C34 modification facilitates the decoding of AUA at the P- and A-sites. The f5C34 defined a reduced conformational space for the nucleoside, in what appears to have restricted the conformational dynamics of the anticodon bases of the modified hmtASLMetCAU. The hmtASLMetCAU exhibited a “C-turn” conformation that has some characteristics of the U-turn motif. Codon binding studies with both E. coli and bovine mitochondrial ribosomes revealed that the f5C34 facilitates AUA binding in the A-site and suggested that the modification favorably alters the ASL's binding kinetics. Mitochondrial translation by many organisms including humans sometimes initiates with the universal isoleucine codons AUU and AUC. The f5C34 enabled P-site codon binding to these normally isoleucine codons. Thus, the physicochemical properties of this one modification, f5C34, expand codon recognition from the traditional AUG to the non-traditional, synonymous codons AUU and AUC as well as AUA, in the reassignment of universal codons in the mitochondria

The FDA-Approved Drug Cobicistat Synergizes with Remdesivir To Inhibit SARS-CoV-2 Replication In Vitro and Decreases Viral Titers and Disease Progression in Syrian Hamsters

Combinations of direct-acting antivirals are needed to minimize drug resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and testing of a number of drug regimens has led to conflicting results. Here, we show that cobicistat, which is an FDA-approved drug booster that blocks the activity of the drug-metabolizing proteins cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Two independent cell-to-cell membrane fusion assays showed that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low-micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. This was consistent with the effects of cobicistat on two of its known targets, CYP3A4 and P-gp, the silencing of which boosted the in vitro antiviral activity of remdesivir in a cobicistat-like manner. When administered in vivo to Syrian hamsters at a high dose, cobicistat decreased viral load and mitigated clinical progression. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19

Cell line-dependent variability in HIV activation employing DNMT inhibitors

Long-lived reservoirs of Human Immunodeficiency Virus (HIV) latently infected cells present the main barrier to a cure for HIV infection. Much interest has focused on identifying strategies to activate HIV, which would be used together with antiretrovirals to attack reservoirs. Several HIV activating agents, including Tumor Necrosis Factor alpha (TNFα) and other agents that activate via NF-kB are not fully effective in all latent infection models due to epigenetic restrictions, such as DNA methylation and the state of histone acetylation. DNA methyltransferases (DNMT) inhibitors like 5-aza-2'deoxycytidine (Aza-CdR) and histone deacetylase (HDAC) inhibitors like Trichostatin A (TSA) have been proposed as agents to enhance reactivation and have shown activity in model systems. However, it is not clear how the activities of DNMT and HDAC inhibitors range across different latently infected cell lines, potential models for the many different latently infected cells within an HIV patient. We determined HIV activation following treatment with TNFα, TSA and Aza-CdR across a range of well known latently infected cell lines. We assessed the activity of these compounds in four different Jurkat T cell-derived J-Lat cell lines (6.3, 8.4, 9.2 and 10.6), which have a latent HIV provirus in which GFP replaces Nef coding sequence, and ACH-2 and J1.1 (T cell-derived), and U1 (promonocyte-derived) cell lines with full-length provirus. We found that Aza-CdR plus TNFα activated HIV at least twice as well as TNFα alone for almost all J-Lat cells, as previously described, but not for J-Lat 10.6, in which TNFα plus Aza-CdR moderately decreased activation compared to TNFα alone. Surprisingly, a much greater reduction of TNFα-stimulated activation with Aza-CdR was detected for ACH-2, J1.1 and U1 cells. Reaching the highest reduction in U1 cells with a 75% reduction. Interestingly, Aza-CdR not only decreased TNFα induction of HIV expression in certain cell lines, but also decreased activation by TSA. Since DNMT inhibitors reduce the activity of provirus activators in some HIV latently infected cell lines the use of epigenetic modifying agents may need to be carefully optimized if they are to find clinical utility in therapies aimed at attacking latent HIV reservoirs

Non-destructive determination of floral staging in cereals using X-ray micro computed tomography (µCT)

Background Accurate floral staging is required to aid research into pollen and flower development, in particular male development. Pollen development is highly sensitive to stress and is critical for crop yields. Research into male development under environmental change is important to help target increased yields. This is hindered in monocots as the flower develops internally in the pseudostem. Floral staging studies therefore typically rely on destructive analysis, such as removal from the plant, fixation, staining and sectioning. This time-consuming analysis therefore prevents follow up studies and analysis past the point of the floral staging. Results This study focuses on using X-ray µCT scanning to allow quick and detailed non-destructive internal 3D phenotypic information to allow accurate staging of Arabidopsis thaliana L. and Barley (Hordeum vulgare L.) flowers. X-ray µCT has previously relied on fixation methods for above ground tissue, therefore two contrast agents (Lugol’s iodine and Bismuth) were observed in Arabidopsis and Barley in planta to circumvent this step. 3D models and 2D slices were generated from the X-ray µCT images providing insightful information normally only available through destructive time-consuming processes such as sectioning and microscopy. Barley growth and development was also monitored over three weeks by X-ray µCT to observe flower development in situ. By measuring spike size in the developing tillers accurate non-destructive staging at the flower and anther stages could be performed; this staging was confirmed using traditional destructive microscopic analysis. Conclusion The use of X-ray micro computed tomography (µCT) scanning of living plant tissue offers immense benefits for plant phenotyping, for successive developmental measurements and for accurate developmental timing for scientific measurements. Nevertheless, X-ray µCT remains underused in plant sciences, especially in above-ground organs, despite its unique potential in delivering detailed non-destructive internal 3D phenotypic information. This work represents a novel application of X-ray µCT that could enhance research undertaken in monocot species to enable effective non-destructive staging and developmental analysis for molecular genetic studies and to determine effects of stresses at particular growth stages