RNA Targeting Research Articles

CRISPR RNA binding drives structural ordering that primes Cas7-11 for target cleavage.

Type III-E CRISPR-Cas effectors, of which Cas7-11 is the first, are single proteins that cleave target RNAs without nonspecific collateral cleavage, opening new possibilities for RNA editing. Biochemical experiments combined with amide hydrogen-deuterium exchange (HDX-MS) experiments provide a first glimpse of the conformational dynamics of apo Cas7-11. HDX-MS revealed the backbone comprised of the four Cas7 zinc-binding RRM folds are well-folded but insertion sequences are highly dynamic and fold upon binding crRNA. The crRNA causes folding of disordered catalytic loops and β-hairpins, stronger interactions at domain-domain interfaces, and folding of the Cas7.1 processing site. Target RNA binding causes only minor ordering around the catalytic loops of Cas7.2 and Cas7.3. We show that Cas7-11 cannot fully process the CRISPR array and that binding of partially processed crRNA induces multiple states in Cas7-11 and reduces target RNA cleavage. The insertion domain shows the most ordering upon binding of mature crRNA. Finally, we show a crRNA-induced conformational change in one of the TPR-CHAT binding sites providing an explanation for why crRNA binding facilitates TPR-CHAT binding. The results provide the first glimpse of the apo state of Cas7-11 and reveal how its structure and function are regulated by crRNA binding.

Machine learning-augmented molecular dynamics simulations (MD) reveal insights into the disconnect between affinity and activation of ZTP riboswitch ligands.

The challenge of targeting RNA with small molecules necessitates a better understanding of RNA-ligand interaction mechanisms. However, the dynamic nature of nucleic acids, their ligand-induced stabilization, and how conformational changes influence gene expression pose significant difficulties for experimental investigation. This work employs a combination of computational and experimental methods to address these challenges. By integrating structure-informed design, crystallography, and machine learning-augmented all-atom molecular dynamics simulations (MD) we synthesized, biophysically and biochemically characterized, and studied the dissociation of a library of small molecule activators of the ZTP riboswitch, a ligand-binding RNA motif that regulates bacterial gene expression. We uncovered key interaction mechanisms, revealing valuable insights into the role of ligand binding kinetics on riboswitch activation. Further, we established that ligand on-rates determine activation potency as opposed to binding affinity and elucidated RNA structural differences, which provide mechanistic insights into the interplay of RNA structure on riboswitch activation.

Microfluidic-free droplet digital RAA/RT-RAA assisted CRISPR/Cas12a system for the detection of DNA/RNA viruses

Digital PCR (dPCR) is the most sensitive method for virus detection by accurate quantifying the nucleic acids. However, the requirement of sophisticated and expensive instrumentations greatly limits its application in clinical diagnosis particularly in poorly resourced settings. Herein, we developed a simple digital assay based on polydisperse droplet digital recombinase-aided amplification/reverse transcription recombinase-aided amplification dependent CRISPR/Cas12a system (PddRaaCas/PddRRaaCas) for viral DNA/RNA detection with minimal instruments. This assay does not require any microfluidic chip for droplet generation and can be completed about 1 hour. We found that the PddRaaCas and PddRRaaCas enabled to detect 0.015 fM target DNA and 10 fM target RNA respectively. In addition, we applied this method to detect human papillomavirus type 18 (HPV18) and severe acute respiratory syndrome coronavirus (SARS-CoV-2) in clinical samples, which represented the detection of DNA virus and RNA virus respectively. The sensitivity and specificity were 100 % and 100 % for both DNA and RNA virus detection respectively. Taken together, these results highlight that PddRaaCas/PddRRaaCas is a sensitive, rapid, and easy-to-use strategy for the detection of DNA/RNA viruses, which has great potential application for the control of infectious diseases.

Inhibition of SARS-CoV-2 growth in the lungs of mice by a peptide-conjugated morpholino oligomer targeting viral RNA

Further development of direct-acting antiviral agents against human SARS-CoV-2 infections remains a public health priority. Here, we report that an antisense peptide-conjugated morpholino oligomer (PPMO), named 5’END-2, targeting a highly conserved sequence in the 5’UTR of SARS-CoV-2 genomic RNA, potently suppressed SARS-CoV-2 growth in vitro and in vivo. In Hela-ACE 2 cells, 5’END-2 produced IC50 values of between 40 nM and 1.15 μM in challenges using six genetically disparate strains of SARS-CoV-2, including JN.1. In vivo, using K18-hACE2 mice and the WA-1/2020 virus isolate, two doses of 5’END-2 at 10 mg/kg, administered intranasally on the day before and the day after infection, produced approximately 1.4 log10 virus titer reduction in lung tissue at 3 days post-infection. Under a similar dosing schedule, intratracheal administration of 1.0 to 2.0 mg/kg 5’END-2 produced over 3.5 log10 virus growth suppression in mouse lungs. Electrophoretic mobility shift assays characterized specific binding of 5’END-2 to its complementary target RNA. Further, using reporter constructs containing SARS-CoV-2 5’UTR leader sequence, in an in-cell system, we observed that 5’END-2 could interfere with translation in a sequence-specific manner. The results demonstrate that direct pulmonary delivery of 5’END-2 PPMO is a promising antiviral strategy against SARS-CoV-2 infections and warrants further development.

Making target sites in large structured RNAs accessible to RNA-cleaving DNAzymes through hybridization with synthetic DNA oligonucleotides.

The 10-23 DNAzyme is one of the most active DNA-based enzymes, and in theory, can be designed to target any purine-pyrimidine junction within an RNA sequence for cleavage. However, purine-pyrimidine junctions within a large, structured RNA (lsRNA) molecule of biological origin are not always accessible to 10-23, negating its general utility as an RNA-cutting molecular scissor. Herein, we report a generalizable strategy that allows 10-23 to access any purine-pyrimidine junction within an lsRNA. Using three large SARS-CoV-2 mRNA sequences of 566, 584 and 831 nucleotides in length as model systems, we show that the use of antisense DNA oligonucleotides (ASOs) that target the upstream and downstream regions flanking the cleavage site can restore the activity (kobs) of previously poorly active 10-23 DNAzyme systems by up to 2000-fold. We corroborated these findings mechanistically using in-line probing to demonstrate that ASOs reduced 10-23 DNAzyme target site structure within the lsRNA substrates. This approach represents a simple, efficient, cost-effective, and generalizable way to improve the accessibility of 10-23 to a chosen target site within an lsRNA molecule, especially where direct access to the genomic RNA target is necessary.

AMPK-mediated regulation of cardiac energy metabolism: Implications for thermotolerance in Argopecten irradians irradians

AMPK is a key regulator of metabolism in eukaryotes across various pathways related to energy regulation. Although extensive investigations of AMPK have been conducted in mammals and some model organisms, research on AMPK in scallops is comparatively limited. In this study, three AMPK family genes (AiAMPKα, AiAMPKβ and AiAMPKγ) in scallop Argopecten irradians irradians were identified through genome scanning. Structure prediction and phylogenetic analyses of AiAMPKs were performed to determine their structural features and evolutionary relationships. Spatiotemporal expression patterns of AiAMPKs at different developmental stages and in healthy adult tissues were analyzed to elucidate the function of AiAMPKs in bay scallops’ growth and development. The spatiotemporally specific expression of AiAMPKs implied their important roles in growth and development of bay scallops. Heat stress experiment was performed to determine the regulations of AiAMPKs in four kinds of thermosensitive tissues. Expression profiles revealed distinct molecular mechanisms of AiAMPKs in different tissues in response to heat stress: significant down-regulations in mobile hemocytes, but dominant up-regulations occurring in stationary gills, mantles and hearts. Functional verification including knock-down of AiAMPKα and inhibition of AiAMPK was separately conducted in the thermotolerant tissue heart at the post-transcription and translation levels. The thermotolerant index Arrhenius break temperature (ABT) showed a significant decrease and the rate-amplitude product (RAP) peaked earlier in the individuals after RNAi targeting AiAMPKα, displaying an earlier transition to anaerobic metabolism under heat stress, indicating an impairing ability of aerobic metabolism. After AiAMPK inhibition, widespread down-regulations of genes in key energy metabolism pathways, RNA polymerase II-mediated transcription, and aminoacyl-tRNA synthesis pathways were obviously observed, revealing the post-translational inhibition of AiAMPK hindered cardiac energy metabolism, basal transcription and translation. Overall, our findings provide evidences for exploring the molecular mechanisms of energy regulation in thermotolerant traits in bay scallops under ongoing global warming.

Targeting RNA with small molecules, from RNA structures to precision medicines: IUPHAR review: 40.

RNA plays important roles in regulating both health and disease biology in all kingdoms of life. Notably, RNA can form intricate three-dimensional structures, and their biological functions are dependent on these structures. Targeting the structured regions of RNA with small molecules has gained increasing attention over the past decade, because it provides both chemical probes to study fundamental biology processes and lead medicines for diseases with unmet medical needs. Recent advances in RNA structure prediction and determination and RNA biology have accelerated the rational design and development of RNA-targeted small molecules to modulate disease pathology. However, challenges remain in advancing RNA-targeted small molecules towards clinical applications. This review summarizes strategies to study RNA structures, to identify small molecules recognizing these structures, and to augment the functionality of RNA-binding small molecules. We focus on recent advances in developing RNA-targeted small molecules as potential therapeutics in a variety of diseases, encompassing different modes of actions and targeting strategies. Furthermore, we present the current gaps between early-stage discovery of RNA-binding small molecules and their clinical applications, as well as a roadmap to overcome these challenges in the near future.

Programming Fast DNA Amplifier Circuits with Versatile Toehold Exchange Pathway.

DNA amplifier circuits establish powerful tools to dynamically control molecular assembly for computation, sensing, and biological applications. However, the slow reaction speed remains a major barrier to their practical utility. Here, diverse fast DNA amplifier circuits termed toehold exchange polymerization (TEP) and toehold exchange catalysis (TEC) using toehold exchange-mediated assembly as a fundamental mechanism are built. Both TEP and TEC with a duplex and a hairpin can respond within minutes to diverse nucleic acid inputs with high fidelity. In addition, the circuits can amplify live-cell signals for fluorescence imaging target RNA dynamics and discriminating different cell lines. Compared with existing DNA circuits that involve time scales of hours for transducing small signals, TEP and TEC exhibit much faster dynamics, simpler design, and comparable sensitivity. These features make TEP and TEC promising platforms to develop programmable nucleic acid tools and devices and to create fast sensing and processing systems, amenable to wide practical applications.

The RNA binding protein Arid5a drives IL-17-dependent autoantibody-induced glomerulonephritis.

Autoantibody-mediated glomerulonephritis (AGN) arises from dysregulated renal inflammation, with urgent need for improved treatments. IL-17 is implicated in AGN and drives pathology in a kidney-intrinsic manner via renal tubular epithelial cells (RTECs). Nonetheless, downstream signaling mechanisms provoking kidney pathology are poorly understood. A noncanonical RNA binding protein (RBP), Arid5a, was upregulated in human and mouse AGN. Arid5a-/- mice were refractory to AGN, with attenuated myeloid infiltration and impaired expression of IL-17-dependent cytokines and transcription factors (C/EBPβ, C/EBPδ). Transcriptome-wide RIP-Seq revealed that Arid5a inducibly interacts with conventional IL-17 target mRNAs, including CEBPB and CEBPD. Unexpectedly, many Arid5a RNA targets corresponded to translational regulation and RNA processing pathways, including rRNAs. Indeed, global protein synthesis was repressed in Arid5a-deficient cells, and C/EBPs were controlled at the level of protein rather than RNA accumulation. IL-17 prompted Arid5a nuclear export and association with 18S rRNA, a 40S ribosome constituent. Accordingly, IL-17-dependent renal autoimmunity is driven by Arid5a at the level of ribosome interactions and translation.

Paper‐Based RNase Digestion toward Viral Nucleic Acid Self‐Tests

AbstractHome‐based Nucleic Acid Amplification Tests (NAATs) for viral infections would be an important step to improve public health, but sample preparation remains an important obstacle, particularly for the protection of target RNA from RNases in samples. Here, a new method for RNase deactivation in saliva samples is presented. This method uses Proteinase K (PK) immobilized on nitrocellulose membrane to store and deliver the enzyme, capable of digesting nucleases present in the sample. The immobilized PK is also separated from the amplification reagents, so that it does not disrupt DNA amplification, thus omitting the need for heat‐deactivation or dilution steps. Treatment by PK nitrocellulose at 50 °C dramatically decreases the RNase activity of RNase A, in diluted saliva samples, and even shows promising results at 42 °C. The potential of this method to protect RNA from digestion is further demonstrated, by pretreating diluted saliva samples spiked with Influenza Virus A (IVA) genomic RNA, which allows its amplification and colorimetric detection by lateral flow strips. In the absence of PK pretreatment, the RNA is digested by RNase, which leads to false negative results. These findings show that immobilized PK enables the integration of sample preparation of viral samples toward home‐based NAATs.

Abstract 73: Extracellular vesicles-derived miR-125b-5p/miR-125a-5p promotes hypertension by regulating angiotensin-converting enzyme 2

Therapeutic targeting of non-coding RNAs, particularly microRNA (miRNA), presents an appealing approach in the treatment of chronic diseases such as resistant hypertension. Recently, miRNA have emerged as promising targets for therapeutic intervention, capable of regulating critical genes to provide a suitable solution to the lack of efficacy of current treatments. Our comprehensive miRNA profiling of plasma extracellular vesicles (EV) from DOCA-salt hypertensive mice of both sexes, compared to sham controls, identified two miRNA implicated in salt-sensitive hypertension, namely miR-125b-5p and miR-125a-5p. Our bioinformatic analysis suggests that these miRNA could be involved in angiotensin-converting enzyme 2 (ACE2) downregulation in hypertension. The present study intended to validate ACE2 as a target of miR-125. Quantitative RT-PCR and capillary western analysis of hypothalami from C57BL/6J DOCA+Salt hypertensive male and female mice following 3-weeks of DOCA+Salt treatment (n=5-6 per group, 3-month old), detected a significant decrease in ACE2 mRNA (males: 0.34±0.03 vs. 1.02±0.02; females: 0.65±0.08 vs. 1.01±0.01; P<0.05) and protein (males: 0.17±0.02 vs. 0.31±0.05; females: 0.16±0.03 vs. 0.37±0.07; P<0.05) expression for both sexes. Further investigation in bEND.3 cells treated with different concentrations of Ang-II (0, 10, 100 nM) for 12, 24, and 48 hours, demonstrated a dose-dependent negative correlation between miR-125b-5p (r≈-0.9978) or miR-125a-5p (r≈-1.002) and ACE2 gene expression, independently of time. When treated with miR-125 mimics the cells demonstrated a consistent downregulation of ACE2 protein levels (miR-125b-5p: 0.6±0.1 and miR-125a-5p: 2.0±0.2 vs. control: 2.8±0.3). Conversely, no significant change in ACE2 protein levels was observed with the respective miRNA inhibitors. Using a firefly/Renilla luciferase assay, we further identified ACE2 as a validated primary target for these miRNA. While miR-125b-5p indirectly modulates the expression of the Ace2 gene by targeting important transcription factors like Sp1 and STAT3, our data further elucidate that miR-125b-5p and/or miR-125a-5p directly reduce ACE2 levels by targeting ACE2 3’UTR mRNA. Additional work is warranted to investigate these specific regulatory mechanisms based on cell specificity, physiological conditions, and sex differences.

Abstract P104: Kidney arteriolar responses to liver-targeted small interference RNA (siRNA) targeting angiotensinogen in diabetic rats: comparison with other renin-angiotensin system blockers

Objective: Inhibition of the renin-angiotensin system (RAS) with ACE inhibitors (ACEi) or angiotensin receptor blockers (ARB) is associated with concentric thickening of renal arteries and arterioles and parenchymal disease. Here we evaluated to what degree this also occurs during RAS inhibition with siRNA targeting hepatic angiotensinogen (AGT). Methods: Male hypertensive heterozygous transgenic rats overexpressing mouse renin (TGR(mRen2)27 rats), made diabetic with streptozotocin, were treated with the ARB valsartan (4 mg/kg/day), the ACEi captopril (6 mg/kg/day), AGT siRNA (30 mg/kg every 2 weeks), AGT siRNA + valsartan, or captopril + valsartan for 3 weeks, starting at 15 weeks after induction of diabetes. After 3 weeks of treatment, kidneys were removed, and immunocytochemistry was performed on Bouin’s fixed kidney sections using antibodies against renin or α-smooth muscle actin. Juxtaglomerular index and wall thickness were assessed using tiling pictures of both renin and α-smooth muscle actin sections from the slides. Tubular degeneration was scored by summing up scores for tubular atrophy, interstitial fibrosis, and tubulointerstitial inflammation. Concentric vascular hypertrophy was assessed in a blinded manner. Results: All treatments increased juxtaglomerular index, and the largest increases were observed in dual treatment groups, consistent with the highest degree of RAS blockade in these groups. Tubular scores increased after induction of diabetes, and the highest scores were seen in dual treatment groups, significance versus vehicle being reached in the valsartan + captopril group only (P<0.05). Afferent arteriole wall thickness increased in all treatment groups, and the greatest increases were observed during dual treatment. Finally, concentric vascular hypertrophy was observed in 1 of 7 (14%) diabetic Ren2 rats; this percentage increased during RAS blocker treatment of these rats, numerically reaching the highest values during dual treatment. Conclusions: Our data suggest that, in the hypertensive diabetic rat model, high levels of RAS blockade, irrespective of the type of blockade, may result in an increase in afferent arteriole thickness and concentric vascular hypertrophy. AGT siRNA in combination with RAS inhibitors should be further explored in clinical studies.

The role of brevican regulation in the antidepressant effects of electroacupuncture in a chronic stress rat model

ObjectiveTo investigate the mechanism of electroacupuncture (EA) for treating depression and to explore the role of brevican in the medial prefrontal cortex (mPFC) in modulating stress susceptibility and the antidepressant effects of EA in rats. MethodsTwenty-four Sprague–Dawley (SD) rats were equally divided into three groups: green fluorescent protein (GFP) + control, GFP + chronic unpredicted mild stress (CUMS), and short-hairpin RNA targeting on brevican (shBcan) + CUMS. Another 24 SD rats were equally divided into CUMS + GFP, CUMS + GFP + EA, and CUMS + shBcan + EA groups. Behavioral tests were conducted to assess depression-like behavior. Western blot analysis was used to evaluate the expression of brevican, aggrecan, GLuA1, and PSD95 in mPFC subregions. ResultsBehavioral parameter evaluation show that rats in the shBcan + CUMS group exhibited a significantly reduced sucrose preference (P = .0002) and increased immobility time (P = .0011) compared to those in rats in the GFP + CUMS group. Western blotting showed that brevican expression was significantly downregulated in the PrL of the shBcan + CUMS group compared with that in the GFP + CUMS group (P = .0192). Furthermore, compared to the CUMS + GFP + EA group, the CUMS + shBcan + EA group exhibited a significantly decreased sucrose preference (P = .0334), increased immobility time (P = .0465), and increased latency to food (P = .0261). In the CUMS + shBcan + EA group, the EA-induced brevican and PSD95 overexpression was reversed, compared with that in the CUMS + GFP + EA group (P = .0454 and P = .0198, respectively). ConclusionEA exerts its antidepressant effects through the modulation of brevican expression in rats. Our findings highlight the important role for brevican in stress susceptibility, which could be a potential target for treating depression.

RNA m6A methylation at the juxtaposition of apoptosis and RNA therapeutics

Targeting RNA m6A marks in apoptosis-related transcripts holds promise for RNA therapeutics. However, pathway-specific RNA m6A sites on pro- or antiapoptotic transcripts have not been fully unveiled, let alone characterized. This article summarizes the current knowledge and gaps in the cellular response modulated by apoptotic stimulus-specific RNA m6A marks.

Templated 3ʹ terminal fluorescent labeling of RNA using Klenow DNA polymerase

A long-standing challenge in the study of RNA structure-function dynamics using fluorescence-based methods has been the precise attachment of fluorophores to structured RNA molecules. Despite significant advancements in the field, existing techniques have limitations, especially for 3ʹ end labeling of long, structured RNAs. In response to this challenge, we developed a chemo-enzymatic method that uses Klenow DNA polymerase to label RNAs. In this method:•Klenow DNA polymerase adds an amino-modified nucleotide to the 3ʹ end of the RNA, guided by the DNA oligonucleotide template.•An NHS-ester dye is then conjugated to the amino-modified RNA, forming a covalent amide bond.•For highly structured RNAs, DNA oligonucleotides complementary to the RNA disrupt pre-existing intramolecular RNA structures.This methodological advancement enables site-specific incorporation of a single modified nucleotide at the 3′ terminus of various RNA substrates, irrespective of their length or secondary structure. The user-friendly nature of the technique, with minimal modifications required for different RNA targets, makes it readily adaptable by a broad range of researchers. This approach has the potential to significantly improve the development of functionalized RNA for various applications.

PUF Proteins as Critical RNA-Binding Proteins in TriTryp Parasites: A Review Article.

In eukaryotes, translation is a fundamental step in the long pathway of protein synthesis within the cell. In this process, several proteins and factors have involved directly or indirectly, individually or in association with other elements to contact mRNA. For perfect translation, many essential modifications should be done, such as cis-splicing to remove introns and two main events for capping and poly A polymerization in 5' and 3' end of mRNA, respectively. Gene expression is then regulated at both translation and stability of the target mRNA molecule levels. Pumilio/FBFs (PUFs) are the main group of RNA-binding proteins which bind to the 3'-UTR of target RNA and thereby regulate the fate, stability and subcellular localization of mRNAs and adjust the translated protein level. PUF proteins have been found both in nucleus where that bind to precursor mRNA, for processing and maturation of rRNA, and in cytoplasm where that bind to mRNA, stall the ribosomes, suppress the translation and localization of the mRNA. They can regulate the expression of mRNAs through activation or suppression of translation. Therefore, these proteins have recently garnered much attention as new generation of therapeutic targets against diseases such as cancer and neurological disorders. In comparison to other eukaryotes, trypanosomatids have a high number of PUF proteins, which function not only as gene expression regulatory factors but also in several biological processes such as differentiation and life-cycle progression of the cells. Here, we review the molecular and biological roles of known PUF proteins in TriTryp parasites (Trypanosome brucei, T. cruzi and Leishmania) beside some other parasites.

Differential microRNA expression in adolescent anxiety proneness.

Biological mechanisms underlying anxiety proneness (AP), the tendency to react fearfully to stressors due to the belief that experiencing anxiety has harmful consequences, remain unclear. Epigenetic mechanisms, such as microRNAs (small, non-coding RNAs 19-20 nucleotides long), may be contributory. This study investigated AP-associated differences in microRNA expression among South African adolescents with variable exposure to childhood trauma (CT). AP was assessed using a composite score reflecting trait anxiety and anxiety sensitivity, while CT exposure was assessed with the Childhood Trauma Questionnaire. High-quality total RNA (n = 88) extracted from whole blood underwent microRNA-sequencing. Differential microRNA expression analysis was conducted with DESeq2 in R, messenger RNA target prediction analysis was performed using TargetScan and DIANA-microT, and the DIANA mirPATH tool was used for KEGG pathway analysis. The majority of participants were female (75.86%) with an average age of 15 (±1.19) years. MicroRNA expression analysis identified upregulation of hsa-miR-28-5p and downregulation of hsa-miR-502-3p and hsa-miR-500a-3p in high-AP individuals, irrespective of CT. Four KEGG pathways, each with ≥10% of their constituent genes predicted to be targets of the differentially expressed microRNAs, were identified and were enriched for genes involved in calcineurin and glutamate signalling. These findings suggest that epigenetically mediated effects on neuronal function contribute to the molecular aetiology of AP.

FMRP cooperates with miRISC components to repress translation and regulate neurite morphogenesis in Drosophila

ABSTRACT Fragile X Syndrome (FXS) is the most common inherited form of intellectual disability and is caused by mutations in the gene encoding the Fragile X messenger ribonucleoprotein (FMRP). FMRP is an evolutionarily conserved and neuronally enriched RNA-binding protein (RBP) with functions in RNA editing, RNA transport, and protein translation. Specific target RNAs play critical roles in neurodevelopment, including the regulation of neurite morphogenesis, synaptic plasticity, and cognitive function. The different biological functions of FMRP are modulated by its cooperative interaction with distinct sets of neuronal RNA and protein-binding partners. Here, we focus on interactions between FMRP and components of the microRNA (miRNA) pathway. Using the Drosophila S2 cell model system, we show that the Drosophila ortholog of FMRP (dFMRP) can repress translation when directly tethered to a reporter mRNA. This repression requires the activity of AGO1, GW182, and MOV10/Armitage, conserved proteins associated with the miRNA-containing RNA-induced silencing complex (miRISC). Additionally, we find that untagged dFMRP can interact with a short stem-loop sequence in the translational reporter, a prerequisite for repression by exogenous miR-958. Finally, we demonstrate that dFmr1 interacts genetically with GW182 to control neurite morphogenesis. These data suggest that dFMRP may recruit the miRISC to nearby miRNA binding sites and repress translation via its cooperative interactions with evolutionarily conserved components of the miRNA pathway.

Strategic deactivation of mRNA COVID-19 vaccines: New applications for siRNA therapy and RIBOTACs.

The rapid development and authorization of messenger ribonucleic acid (mRNA) vaccines by Pfizer-BioNTech (BNT162b2) and Moderna (mRNA-1273) in 2020 marked a significant milestone in human mRNA product application, overcoming previous obstacles such as mRNA instability and immunogenicity. This paper reviews the strategic modifications incorporated into these vaccines to enhance mRNA stability and translation efficiency, such as the inclusion of nucleoside modifications and optimized mRNA design elements including the 5' cap and poly(A) tail. We highlight emerging concerns regarding the wide systemic biodistribution of these mRNA vaccines leading to prolonged inflammatory responses and other safety concerns. The regulatory framework guiding the biodistribution studies is pivotal in assessing the safety profiles of new mRNA formulations in use today. The stability of mRNA vaccines, their pervasive distribution, and the longevity of the encapsulated mRNA along with unlimited production of the damaging and potentially lethal spike (S) protein call for strategies to mitigate potential adverse effects. Here, we explore the potential of small interfering RNA (siRNA) and ribonuclease targeting chimeras (RIBOTACs) as promising solutions to target, inactivate, and degrade residual and persistent vaccine mRNA, thereby potentially preventing uncontrolled S protein production and reducing toxicity. The targeted nature of siRNA and RIBOTACs allows for precise intervention, offering a path to prevent and mitigate adverse events of mRNA-based therapies. This review calls for further research into siRNA and RIBOTAC applications as antidotes and detoxication products for mRNA vaccine technology.

The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites.

RNA molecules are localized to subcellular regions through interactions between localization-regulatory cis-elements and trans-acting RNA binding proteins (RBPs). However, the identities of RNAs whose localization is regulated by a specific RBP as well as the impacts of that RNA localization on cell function have generally remained unknown. Here, we demonstrate that the RBP HNRNPA2B1 acts to keep specific RNAs out of neuronal projections. Using subcellular fractionation, high-throughput sequencing, and single molecule RNA FISH, we find that hundreds of RNAs demonstrate markedly increased abundance in neurites in HNRNPA2B1 knockout cells. These RNAs often encode motor proteins and are enriched for known HNRNPA2B1 binding sites and motifs in their 3' UTRs. The speed and processivity of microtubule-based transport is impaired in these cells, specifically in their neurites. HNRNPA2B1 point mutations that increase its cytoplasmic abundance relative to wildtype lead to stronger suppression of RNA mislocalization defects than seen with wildtype HNRNPA2B1. We further find that the subcellular localizations of HNRNPA2B1 target RNAs are sensitive to perturbations of RNA decay machinery, suggesting that it is HNRNPA2B1's known role in regulating RNA stability that may explain these observations. These findings establish HNRNPA2B1 as a negative regulator of neurite RNA abundance and link the subcellular activities of motor proteins with the subcellular abundance of the RNAs that encode them.