Serine-arginine protein kinases and their targets in viral infection and their inhibition.

Accumulating evidence has consolidated the interaction between viral infection and host alternative splicing. Serine-arginine (SR) proteins are a class of highly conserved splicing factors critical for the spliceosome maturation, alternative splicing and RNA metabolism. Serine-arginine protein kinases (SRPKs) are important kinases that specifically phosphorylate SR proteins to regulate their distribution and activities in the central pre-mRNA splicing and other cellular processes. In addition to the predominant SR proteins, other cytoplasmic proteins containing a serine-arginine repeat domain, including viral proteins, have been identified as substrates of SRPKs. Viral infection triggers a myriad of cellular events in the host and it is therefore not surprising that viruses explore SRPKs-mediated phosphorylation as an important regulatory node in virus-host interactions. In this review, we briefly summarize the regulation and biological function of SRPKs, highlighting their involvement in the infection process of several viruses, such as viral replication, transcription and capsid assembly. In addition, we review the structure-function relationships of currently available inhibitors of SRPKs and discuss their putative use as antivirals against well-characterized viruses or newly emerging viruses. We also highlight the viral proteins and cellular substrates targeted by SRPKs as potential antiviral therapeutic candidates.

Alternative structures of RNA control gene expression and offer therapeutic strategies

RNA viruses are diverse and abundant pathogens responsible for numerous human ailments, from common colds to AIDS, SARS, Ebola, and other dangerous diseases. RNA viruses possess relatively compact genomes and have therefore evolved multiple mechanisms to maximize their coding capacities, often using overlapping reading frames. In this way, one RNA sequence can encode multiple proteins via mechanisms including alternative splicing and ribosomal frameshifting. Many such processes in gene expression involve the RNA folding into three-dimensional structures that can recruit ribosomes without initiation factors, hijack host proteins, cause ribosomes to frameshift, and expose or occlude regulatory protein binding motifs to ultimately control each key process in the viral life cycle. I will discuss the RNA structure of HIV-1 and SARS-CoV-2 and the importance of alternative conformations assumed by the same RNA sequence in controlling gene expression of viruses and bacteria.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Optimized nanoparticle-mediated base editing of cystic fibrosis-causing variants

Background: Next-generation SARS-CoV-2 vaccines demonstrated that nanoparticle messenger ribonucleic acid (mRNA) delivery is effective and safe for in vivo delivery in humans. Current treatments for cystic fibrosis (CF) primarily focus on modulator drug therapies designed to correct malfunctioning CF transmembrane conductance regulator (CFTR) protein, but these modulators are ineffective for the 10% of people with CF with variants that do not allow protein production. Among these is the splice variant 3120 + 1G >A, the most common CF-causing mutation in native Africans. Gene editing would allow production of CFTR protein and enhancement of function using available CFTR modulators. We have demonstrated that electroporation of a modified CRISPR-Cas9 base editor to primary human bronchial epithelial cells carrying 3120 + 1G >A and F508del mutant alleles achieved 75% genome editing of the splice variant, resulting in approximately 40% wild-type (WT) CFTR function [1]. Here,we evaluate the effectiveness of several new nanoparticle formulations at delivering green fluorescent protein (GFP) mRNA to CF bronchial epithelial (CFBE41o-) cells. Using the optimal formulation,we then tested the efficacy correction of the 3120 + 1G >Avariant in a CFTR expression minigene (EMG) integrated into the genome of isogenic CFBE cells using mRNA and plasmid deoxyribonucleic acid (DNA) encoding adenine base editor (ABE) and guide (g)RNA. Method(s): GFP served as a reporter to evaluate transfection efficiency, cell viability, and mean fluorescence intensity (MFI) for three dosages (150, 75, 32.5 ng of mRNA), four polymer-to-mRNA to weight (w/w) ratios (60, 40, 30, 20), and four polymers (R, Y, G, B). 7-AAD served as a live/dead stain to quantify viability, with flow cytometry results analyzed using FlowJo software. CFBE cells stably expressing the 3120 + 1G >A EMG were transfected with the optimized nanoparticle formulation to deliver ABE and gRNA at two dosages (150, 75 ng) of mRNA and DNA. CFTR function in CFBE cellswas measured by short circuit current, forskolin stimulation, and inh-172 inhibition as a measure of editing efficiency. Result(s): Flow cytometry showed that polymer R achieved more than 85% GFP transfection, compared with a maximum of approximately 35% for the other three polymers at the maximum 150-ng dose, with approximately 80% viability normalized to untreated cells. In addition, polymer R achieved GFP MFI more than one order of magnitude as high as other formulations (~30 000 vs 2700 MFI) for the other three polymers at 150-ng dose and 40 w/w ratio. CFBE cells transfected with polymer R nanoparticles containing ABE and guide RNA at 75 ng and 150 ng showed mean CFTR function increase to 10 muA 6 (standard error of the mean [SEM] 1.1 muA) (~10% of WT) and 6.3 muA (SEM 0.9 muA) (~6% of WT), respectively. Greater toxicity at the higher dose could explain the larger increase in CFTR current at the lower dose. DNA-encoded ABE plasmid and gRNA showed a less robust increase in CFTR function (2.9 muA [SEM 0.4 muA] for 75-ng dose;3.0 muA [SEM 0.4 muA] for 150-ng dose), which was probably a result of the nanoparticle formulation being optimized for RNA instead of DNA cargo or the additional intracellular barriers that must be overcome for successful DNA delivery. Conclusion(s): We demonstrated that an optimized nanoparticle formulation containing ABE and gRNA can correct splicing of isogenic cells bearing the 3120 + 1G >A CFTR variant, resulting in recovery of CFTR function. In ongoing work, we are adapting these nanoparticles for RNA- and DNAencoded ABE and gRNA delivery to primary human bronchial epithelial cells.Copyright © 2022, European Cystic Fibrosis Society. All rights reserved

Possible Biological Mechanisms Underlying the Association between COVID-19 Severity and HLA-C*04:01

A strong link between COVID-19 severity and HLAC* 04:01 allele has been replicated in several Caucasian populations including Armenians. The results have led to an idea that HLA-C*04:01 may affect the immune response via three biological mechanisms: (i) disruption of the HLA-C mediated protection harnessing natural killer cells (NK);(ii) causing NK hypo-responsiveness through KIR2DL1;or (iii) over-activation and exhaustion of CTL and NK cells by stimulating functional KIR2DS4. To test those hypotheses, we re-analyzed HLA-genotypes and RNA-sequencing data of Overmyer et al. [Cell Systems 2021;12:23-40]. An ordinal regression of patients' status (i.e., non-COVID vs. COVIDnon- ICU vs. COVID-ICU) against HLA-C has corroborated the increase in the disease severity with increasing HLA-C*04:01 dosage (p< 0.003). DESeq2 analyses of the transcriptome (16444 loci) within COVID subset mapped 3586 down-regulated and 4031 up-regulated loci to the disease severity at FDR p<0.05. The results of enrichment analyses of those 7617 genes indicated aberrations in processes, such as T cell activation, inflammatory response, positive regulation of both NK-mediated cytotoxicity and interferon-gamma production. However, only 563 down- and 341 up-regulated loci had nominally associated with the HLA-C*04:01 carriage, reflecting its genetic association with severe symptoms. Using GTEx data and rs5010528 as proxy for HLAC* 04:01 (R2 = 0.97, 1kG EUR cohort), we found that HLA-C*04:01 was associated with multiple tissue (e.g., lung, heart and blood) RNA expressional and splicing changes in >10 protein-coding loci situated close to HLA-C. The ontology analysis of the loci implicated HLA-C*04:01 in altering antigen processing and presentation of endogenous peptide antigen via HLA class I via ER pathway (FDR p<0.0001), protection from NKmediated cytotoxicity (p<0.004), and innate immune response to other organisms (p<0.009). The work was supported by the Science Committee of RA (grant E17).

Post-transcriptional control of haemostatic genes: mechanisms and emerging therapeutic concepts in thrombo-inflammatory disorders.

The haemostatic system is pivotal to maintaining vascular integrity. Multiple components involved in blood coagulation have central functions in inflammation and immunity. A derailed haemostasis is common in prevalent pathologies such as sepsis, cardiovascular disorders, and lately, COVID-19. Physiological mechanisms limit the deleterious consequences of a hyperactivated haemostatic system through adaptive changes in gene expression. While this is mainly regulated at the level of transcription, co- and posttranscriptional mechanisms are increasingly perceived as central hubs governing multiple facets of the haemostatic system. This layer of regulation modulates the biogenesis of haemostatic components, for example in situations of increased turnover and demand. However, they can also be 'hijacked' in disease processes, thereby perpetuating and even causally entertaining associated pathologies. This review summarizes examples and emerging concepts that illustrate the importance of posttranscriptional mechanisms in haemostatic control and crosstalk with the immune system. It also discusses how such regulatory principles can be used to usher in new therapeutic concepts to combat global medical threats such as sepsis or cardiovascular disorders.

Genome-wide detection of human variants that disrupt intronic branchpoints.

Pre-messenger RNA splicing is initiated with the recognition of a single-nucleotide intronic branchpoint (BP) within a BP motif by spliceosome elements. Forty-eight rare variants in 43 human genes have been reported to alter splicing and cause disease by disrupting BP. However, until now, no computational approach was available to efficiently detect such variants in massively parallel sequencing data. We established a comprehensive human genome-wide BP database by integrating existing BP data and generating new BP data from RNA sequencing of lariat debranching enzyme DBR1-mutated patients and from machine-learning predictions. We characterized multiple features of BP in major and minor introns and found that BP and BP-2 (two nucleotides upstream of BP) positions exhibit a lower rate of variation in human populations and higher evolutionary conservation than the intronic background, while being comparable to the exonic background. We developed BPHunter as a genome-wide computational approach to systematically and efficiently detect intronic variants that may disrupt BP recognition. BPHunter retrospectively identified 40 of the 48 known pathogenic BP variants, in which we summarized a strategy for prioritizing BP variant candidates. The remaining eight variants all create AG-dinucleotides between the BP and acceptor site, which is the likely reason for missplicing. We demonstrated the practical utility of BPHunter prospectively by using it to identify a novel germline heterozygous BP variant of STAT2 in a patient with critical COVID-19 pneumonia and a novel somatic intronic 59-nucleotide deletion of ITPKB in a lymphoma patient, both of which were validated experimentally. BPHunter is publicly available from https://hgidsoft.rockefeller.edu/BPHunter and https://github.com/casanova-lab/BPHunter.

Time-course RNA-Seq profiling reveals isoform-level gene expression dynamics of the cGAS-STING pathway.

The cGAS-STING pathway, orchestrating complicated transcriptome-wide immune responses, is essential for host antiviral defense but can also drive immunopathology in severe COVID-19. Here, we performed time-course RNA-Seq experiments to dissect the transcriptome expression dynamics at the gene-isoform level after cGAS-STING pathway activation. The in-depth time-course transcriptome after cGAS-STING pathway activation within 12 h enabled quantification of 48,685 gene isoforms. By employing regression models, we obtained 13,232 gene isoforms with expression patterns significantly associated with the process of cGAS-STING pathway activation, which were named activation-associated isoforms. The combination of hierarchical and k-means clustering algorithms revealed four major expression patterns of activation-associated isoforms, including two clusters with increased expression patterns enriched in cell cycle, autophagy, antiviral innate-immune functions, and COVID-19 coronavirus disease pathway, and two clusters showing decreased expression pattern that mainly involved in ncRNA metabolism, translation process, and mRNA processing. Importantly, by merging four clusters of activation-associated isoforms, we identified three types of genes that underwent isoform usage alteration during the cGAS-STING pathway activation. We further found that genes exhibiting protein-coding and non-protein-coding gene isoform usage alteration were strongly enriched for the factors involved in innate immunity and RNA splicing. Notably, overexpression of an enriched splicing factor, EFTUD2, shifted transcriptome towards the cGAS-STING pathway activated status and promoted protein-coding isoform abundance of several key regulators of the cGAS-STING pathway. Taken together, our results revealed the isoform-level gene expression dynamics of the cGAS-STING pathway and uncovered novel roles of splicing factors in regulating cGAS-STING pathway mediated immune responses.

Circular RNAs: Non-Canonical Observations on Non-Canonical RNAs.

The existence of circular RNA (circRNA) research in mainstream science can be attributed to the contemporary synergism of big data and keen attention to detail by several research groups worldwide. Since the re-emergence of these non-canonical RNA transcripts, seminal advances have been made in understanding their biogenesis, interactome, and functions in diverse fields and a myriad of human diseases. However, most research outputs to date have focused on the ability of highly stable circRNAs to interact with, and impact signalling through, microRNAs. This is likely to be the result of seminal papers in the field ascribing a few remarkable circRNAs as "miRNA sponges". However, the stoichiometric ratio between the (often-lowly-expressed) circRNA and their (commonly-more-abundant) target is rarely in favour of a biologically relevant and functional consequence of these interactions. It is time for yet another revolution in circRNA research to uncover functions beyond their documented ability to bind miRNAs. This Special Issue aims to highlight non-canonical functions for this non-canonical family of RNA molecules.

Blood RNA alternative splicing events as diagnostic biomarkers for infectious disease.

Assays detecting blood transcriptome changes are studied for infectious disease diagnosis. Blood-based RNA alternative splicing (AS) events, which have not been well characterized in pathogen infection, have potential normalization and assay platform stability advantages over gene expression for diagnosis. Here, we present a computational framework for developing AS diagnostic biomarkers. Leveraging a large prospective cohort of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and whole-blood RNA sequencing (RNA-seq) data, we identify a major functional AS program switch upon viral infection. Using an independent cohort, we demonstrate the improved accuracy of AS biomarkers for SARS-CoV-2 diagnosis compared with six reported transcriptome signatures. We then optimize a subset of AS-based biomarkers to develop microfluidic PCR diagnostic assays. This assay achieves nearly perfect test accuracy (61/62 = 98.4%) using a naive principal component classifier, significantly more accurate than a gene expression PCR assay in the same cohort. Therefore, our RNA splicing computational framework enables a promising avenue for host-response diagnosis of infection.

The Landscape of Expressed Chimeric Transcripts in the Blood of Severe COVID-19 Infected Patients.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 infections has quickly developed into a global public health threat. COVID-19 patients show distinct clinical features, and in some cases, during the severe stage of the condition, the disease severity leads to an acute respiratory disorder. In spite of several pieces of research in this area, the molecular mechanisms behind the development of disease severity are still not clearly understood. Recent studies demonstrated that SARS-CoV-2 alters the host cell splicing and transcriptional response to overcome the host immune response that provides the virus with favorable conditions to replicate efficiently within the host cells. In several disease conditions, aberrant splicing could lead to the development of novel chimeric transcripts that could promote the functional alternations of the cell. As severe SARS-CoV-2 infection was reported to cause abnormal splicing in the infected cells, we could expect the generation and expression of novel chimeric transcripts. However, no study so far has attempted to check whether novel chimeric transcripts are expressed in severe SARS-CoV-2 infections. In this study, we analyzed several publicly available blood transcriptome datasets of severe COVID-19, mild COVID-19, other severe respiratory viral infected patients, and healthy individuals. We identified 424 severe COVID-19 -specific chimeric transcripts, 42 of which were recurrent. Further, we detected 189 chimeric transcripts common to severe COVID-19 and multiple severe respiratory viral infections. Pathway and gene enrichment analysis of the parental genes of these two subsets of chimeric transcripts reveals that these are potentially involved in immune-related processes, interferon signaling, and inflammatory responses, which signify their potential association with immune dysfunction leading to the development of disease severity. Our study provides the first detailed expression landscape of chimeric transcripts in severe COVID-19 and other severe respiratory viral infections.

What Can RNA-Based Therapy Do for Monogenic Diseases?

The use of RNA-based approaches to treat monogenic diseases (i.e., hereditary disorders caused by mutations in single genes) has been developed on different fronts. One approach uses small antisense oligonucleotides (ASOs) to modulate RNA processing at various stages; namely, to enhance correct splicing, to stimulate exon skipping (to exclude premature termination codon variants), to avoid undesired messenger RNA (mRNA) transcript degradation via the nonsense-mediated decay (NMD) pathway, or to induce mRNA degradation where they encode toxic proteins (e.g., in dominant diseases). Another approach consists in administering mRNA, which, like gene therapy, is a mutation-agnostic approach with potential application to any recessive monogenic disease. This is simpler than gene therapy because instead of requiring targeting of the nucleus, the mRNA only needs to be delivered to the cytoplasm. Although very promising (as demonstrated by COVID-19 vaccines), these approaches still have potential for optimisation, namely regarding delivery efficiency, adverse drug reactions and toxicity.

SRSF5-Mediated Alternative Splicing of M Gene is Essential for Influenza A Virus Replication: A Host-Directed Target Against Influenza Virus.

Splicing of influenza A virus (IAV) RNA is an essential process in the viral life cycle that involves the co-opting of host factors. Here, it is demonstrated that induction of host serine and arginine-rich splicing factor 5 (SRSF5) by IAV facilitated viral replication by enhancing viral M mRNA splicing. Mechanistically, SRSF5 with its RRM2 domain directly bounds M mRNA at conserved sites (M mRNA position 163, 709, and 712), and interacts with U1 small nuclear ribonucleoprotein (snRNP) to promote M mRNA splicing and M2 production. Mutations introduced to the three binding sites, without changing amino acid code, significantly attenuates virus replication and pathogenesis in vivo. Likewise, SRSF5 conditional knockout in the lung protects mice against lethal IAV challenge. Furthermore, anidulafungin, an approved antifungal drug, is identified as an inhibitor of SRSF5 that effectively blocks IAV replication in vitro and in vivo. In conclusion, SRSF5 as an activator of M mRNA splicing promotes IAV replication and is a host-derived antiviral target.

Descriptions of the Process of New COVID-19 Virus Infection and Transmission Using the Idea of Splicing LEGO Bricks

The Idea of splicing LEGO bricks is proposed for describing the structure of a composite viral infection, which makes it possible to explain the possibility of a recurrence of the disease and the pattern of peaks in the spread of the disease. Comparing the RNA sequences/fragments of a virus to LEGO bricks, the number of LEGO bricks an individual acquires can be used to estimate the probability of disease and death for that individual. The aim is to advise and help public health security develop appropriate measures to reduce infection, recurrence and death from disease during a pandemic. The purpose is to provide the basis for recommendations and measures developed for public health safety to reduce disease infection, recurrence and death during a pandemic. © 2022 IEEE.

Circular RNA: An emerging frontier in RNA therapeutic targets, RNA therapeutics, and mRNA vaccines.

Circular RNAs (circRNA) is a class of natural (biogenic) or synthetic closed RNA without 5' or 3' ends. Meanwhile, their unique covalently-closed structures of circRNA prevent RNA degradation by exonucleases, thereby empowering them with high pharmaceutical stability and biostability relative to current standard-of-care linear mRNA. Natural circRNA can be non-coding RNAs as well as protein-coding RNA, the latter of which was recently discovered. The physiological functions of biogenic circRNAs, which largely remain elusive, include protein and gene sponges, cell activity modulators, and protein translation. The discovery that the circRNA levels can be correlated with some human diseases empowers circRNA with the potential as a novel type of disease biomarkers and a noncanonical class of therapeutic targets. Recently, synthetic circRNA have been engineered to explore their applications as a novel class of mRNA therapeutics and vaccines. In this review, we will discuss the current understanding of the biogenesis and physiological functions of natural circRNAs, the approaches to circRNA synthesis, and current research in the exploration of endogenous circRNAs as novel therapeutic targets and testing circRNAs as an emerging class of RNA therapeutics and vaccines.

Pre-infection antiviral innate immunity contributes to sex differences in SARS-CoV-2 infection.

Male sex is a major risk factor for SARS-CoV-2 infection severity. To understand the basis for this sex difference, we studied SARS-CoV-2 infection in a young adult cohort of United States Marine recruits. Among 2,641 male and 244 female unvaccinated and seronegative recruits studied longitudinally, SARS-CoV-2 infections occurred in 1,033 males and 137 females. We identified sex differences in symptoms, viral load, blood transcriptome, RNA splicing, and proteomic signatures. Females had higher pre-infection expression of antiviral interferon-stimulated gene (ISG) programs. Causal mediation analysis implicated ISG differences in number of symptoms, levels of ISGs, and differential splicing of CD45 lymphocyte phosphatase during infection. Our results indicate that the antiviral innate immunity set point causally contributes to sex differences in response to SARS-CoV-2 infection. A record of this paper's transparent peer review process is included in the supplemental information.

The Medicinal Chemistry of Artificial Nucleic Acids and Therapeutic Oligonucleotides.

Nucleic acids play a central role in human biology, making them suitable and attractive tools for therapeutic applications. While conventional drugs generally target proteins and induce transient therapeutic effects, nucleic acid medicines can achieve long-lasting or curative effects by targeting the genetic bases of diseases. However, native oligonucleotides are characterized by low in vivo stability due to nuclease sensitivity and unfavourable physicochemical properties due to their polyanionic nature, which are obstacles to their therapeutic use. A myriad of synthetic oligonucleotides have been prepared in the last few decades and it has been shown that proper chemical modifications to either the nucleobase, the ribofuranose unit or the phosphate backbone can protect the nucleic acids from degradation, enable efficient cellular uptake and target localization ensuring the efficiency of the oligonucleotide-based therapy. In this review, we present a summary of structure and properties of artificial nucleic acids containing nucleobase, sugar or backbone modifications, and provide an overview of the structure and mechanism of action of approved oligonucleotide drugs including gene silencing agents, aptamers and mRNA vaccines.

Transcript-Targeted Therapy Based on RNA Interference and Antisense Oligonucleotides: Current Applications and Novel Molecular Targets.

The development of novel target therapies based on the use of RNA interference (RNAi) and antisense oligonucleotides (ASOs) is growing in an exponential way, challenging the chance for the treatment of the genetic diseases and cancer by hitting selectively targeted RNA in a sequence-dependent manner. Multiple opportunities are taking shape, able to remove defective protein by silencing RNA (e.g., Inclisiran targets mRNA of protein PCSK9, permitting a longer half-life of LDL receptors in heterozygous familial hypercholesteremia), by arresting mRNA translation (i.e., Fomivirsen that binds to UL123-RNA and blocks the translation into IE2 protein in CMV-retinitis), or by reactivating modified functional protein (e.g., Eteplirsen able to restore a functional shorter dystrophin by skipping the exon 51 in Duchenne muscular dystrophy) or a not very functional protein. In this last case, the use of ASOs permits modifying the expression of specific proteins by modulating splicing of specific pre-RNAs (e.g., Nusinersen acts on the splicing of exon 7 in SMN2 mRNA normally not expressed; it is used for spinal muscular atrophy) or by downregulation of transcript levels (e.g., Inotersen acts on the transthryretin mRNA to reduce its expression; it is prescribed for the treatment of hereditary transthyretin amyloidosis) in order to restore the biochemical/physiological condition and ameliorate quality of life. In the era of precision medicine, recently, an experimental splice-modulating antisense oligonucleotide, Milasen, was designed and used to treat an 8-year-old girl affected by a rare, fatal, progressive form of neurodegenerative disease leading to death during adolescence. In this review, we summarize the main transcriptional therapeutic drugs approved to date for the treatment of genetic diseases by principal regulatory government agencies and recent clinical trials aimed at the treatment of cancer. Their mechanism of action, chemical structure, administration, and biomedical performance are predominantly discussed.

RNA solutions to treat inborn errors of metabolism.

RNA-based therapies are a new, rapidly growing class of drugs that until a few years ago were being used mainly in research in rare diseases. However, the clinical efficacy of recently approved oligonucleotide drugs and the massive success of COVID-19 RNA vaccines has boosted the interest in this type of molecules of both scientists and industry, as wells as of the lay public. RNA drugs are easy to design and cost effective, with greatly improved pharmacokinetic properties thanks to progress in oligonucleotide chemistry over the years. Depending on the type of strategy employed, RNA therapies offer the versatility to replace, supplement, correct, suppress, or eliminate the expression of a targeted gene. Currently, there are more than a dozen RNA-based drugs approved for clinical use, including some for specific inborn errors of metabolism (IEM), and many other in different stages of development. New initiatives in n-of-1 RNA drug development offer new hope for patients with rare diseases and/or ultra-rare mutations. RNA-based therapeutics include antisense oligonucleotides, aptamers, small interfering RNAs, small activating RNAs, microRNAs, lncRNAs and messenger RNAs. Further research and collaborations in the fields of chemistry, biology and medicine will help to overcome major challenges in their delivery to target tissues. Herein, we review the mechanism of action of the different therapeutic approaches using RNA drugs, focusing on those approved or in clinical trials to treat IEM.

ZAP isoforms regulate unfolded protein response and epithelial- mesenchymal transition.

Human ZAP inhibits many viruses, including HIV and coronaviruses, by binding to viral RNAs to promote their degradation and/or translation suppression. However, the regulatory role of ZAP in host mRNAs is largely unknown. Two major alternatively spliced ZAP isoforms, the constitutively expressed ZAPL and the infection-inducible ZAPS, play overlapping yet different antiviral and other roles that need further characterization. We found that the splicing factors hnRNPA1/A2, PTBP1/2, and U1-snRNP inhibit ZAPS production and demonstrated the feasibility to modulate the ZAPL/S balance by splice-switching antisense oligonucleotides in human cells. Transcriptomic analysis of ZAP-isoform-specific knockout cells revealed uncharacterized host mRNAs targeted by ZAPL/S with broad cellular functions such as unfolded protein response (UPR), epithelial-mesenchymal transition (EMT), and innate immunity. We established that endogenous ZAPL and ZAPS localize to membrane compartments and cytosol, respectively, and that the differential localization correlates with their target-RNA specificity. We showed that the ZAP isoforms regulated different UPR branches under resting and stress conditions and affected cell viability during ER stress. We also provided evidence for a different function of the ZAP isoforms in EMT-related cell migration, with effects that are cell-type dependent. Overall, this study demonstrates that the competition between splicing and IPA is a potential target for the modulation of the ZAPL/S balance, and reports new cellular transcripts and processes regulated by the ZAP isoforms.

IRE1A/ XBP1s Axis of ER Stress Promotes DRP1 Phosphorylation at Specific Serine Residue (S616) and Induces Mitochondrial Fragmentation During Airway Inflammation

RATIONALE: Airway inflammation plays a role in airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), chronic bronchitis, and COVID-19 that affect millions of people worldwide. Previously, we showed that acute (24-h) exposure to the pro-inflammatory cytokine tumor necrosis factor α (TNFα) triggers an endoplasmic reticulum (ER) stress response in human airway smooth muscle (hASM) cells. In hASM cells, TNFα selectively activates the inositol requiring enzyme 1α (IRE1α) ER stress pathway with downstream splicing of X-box binding protein 1 (XBP1s), which transcriptionally activates expression of target genes that include proteins mediating phosphorylation of dynamin-related protein 1 (pDRP1) at the Ser616 (S616) residue. Increased pDRP1 at S616 is associated with mitochondrial fission (fragmentation);however, DRP1 is also phosphorylated at Ser637 (S637) residue, and the balance between phosphorylation at S616 and S637 regulates the translocation of DRP1 from cytosol to mitochondria and subsequent fragmentation of mitochondria. In the present study, we hypothesized that TNFα induces ER stress leading to XBP1s mediated increase in the expression of specific kinases that phosphorylate DRP1 at S616 and promote mitochondrial fragmentation. METHODS: hASM cells, dissociated from bronchial tissue obtained from patients with no history of respiratory diseases, were exposed to TNFα (20 ng/ ml for 6-h). As an inhibitor of fragmentation, cells were treated with Mdivi1 (50 μM for 6-h), GTPase inhibitor of DRP1. The expression and phosphorylation status of IRE1α, DRP1, XBP1, cyclin dependent kinases (CDK1, CDK5) and cyclin B1 were quantified by Western blot and immunohistochemistry. Mitochondrial morphology was assessed by 3D confocal microscopy using MitoTracker. XBP1-targets were confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. RESULTS: Bioinformatics analysis predicted putative binding sites of XBP1 in the promoter region of CDK1, CDK5 and cyclin B1 genes that are reported to phosphorylate DRP1 at S616. Consistent with our previous findings, we found that TNFα increases IRE1α phosphorylation and XBP1 splicing. The TNFα induced increase in XBP1s transcriptionally activated expression of CDK1, CDK5 and cyclin B1 and leads to subsequent phosphorylation of DRP1 at S616 with no change in S637 phosphorylation. As a result, TNFα mediated increase in the ratio of S616/ S637 phosphorylation, which promoted translocation of DRP1 from cytosol to mitochondria and mitochondrial fragmentation. We also showed that Mdivi1 mediated inhibition of DRP1-GTPase activity ameliorated phosphorylation at S616 residue and significantly reduced mitochondrial fragmentation. CONCLUSIONS: The present study elucidates the mechanism underlying TNFα induced ER stress and mitochondrial fragmentation.