RNA 5-methylcytosine marks mitochondrial double-stranded RNAs for degradation and cytosolic release.

Mitochondria are essential regulators of innate immunity. They generate long mitochondrial double-stranded RNAs (mt-dsRNAs) and release them into the cytosol to trigger an immune response under pathological stress conditions. Yet the regulation of these self-immunogenic RNAs remains largely unknown. Here, we employ CRISPR screening on mitochondrial RNA (mtRNA)-binding proteins and identify NOP2/Sun RNA methyltransferase 4 (NSUN4) as a key regulator of mt-dsRNA expression in human cells. We find that NSUN4 induces 5-methylcytosine (m5C) modification on mtRNAs, especially on the termini of light-strand long noncoding RNAs. These m5C-modified RNAs are recognized by complement C1q-binding protein (C1QBP), which recruits polyribonucleotide nucleotidyltransferase to facilitate RNA turnover. Suppression of NSUN4 or C1QBP results in increased mt-dsRNA expression, while C1QBP deficiency also leads to increased cytosolic mt-dsRNAs and subsequent immune activation. Collectively, our study unveils the mechanism underlying the selective degradation of light-strand mtRNAs and establishes a molecular mark for mtRNA decay and cytosolic release.

Inverted Alu repeats: friends or foes in the human transcriptome.

Alu elements are highly abundant primate-specific short interspersed nuclear elements that account for ~10% of the human genome. Due to their preferential location in gene-rich regions, especially in introns and 3' UTRs, Alu elements can exert regulatory effects on the expression of both host and neighboring genes. When two Alu elements with inverse orientations are positioned in close proximity, their transcription results in the generation of distinct double-stranded RNAs (dsRNAs), known as inverted Alu repeats (IRAlus). IRAlus are key immunogenic self-dsRNAs and post-transcriptional cis-regulatory elements that play a role in circular RNA biogenesis, as well as RNA transport and stability. Recently, IRAlus dsRNAs have emerged as regulators of transcription and activators of Z-DNA-binding proteins. The formation and activity of IRAlus can be modulated through RNA editing and interactions with RNA-binding proteins, and misregulation of IRAlus has been implicated in several immune-associated disorders. In this review, we summarize the emerging functions of IRAlus dsRNAs, the regulatory mechanisms governing IRAlus activity, and their relevance in the pathogenesis of human diseases.

Meet the authors: Jayoung Ku, Keonyong Lee, and Yoosik Kim.

We spoke with authors co-first authors Jayoung Ku, Keonyong Lee, and lead author Yoosik Kim about their paper "Alternative polyadenylation determines the functional landscape of inverted Alu repeats" (this issue of Molecular Cell), finding renewed energy from attending scientific meetings, and the open questions they are most interested in investigating next.

Alternative polyadenylation determines the functional landscape of inverted Alu repeats.

Inverted Alu repeats (IRAlus) are abundantly found in the transcriptome, especially in introns and 3' untranslated regions (UTRs). Yet, the biological significance of IRAlus embedded in 3' UTRs remains largely unknown. Here, we find that 3' UTR IRAlus silences genes involved in essential signaling pathways. We utilize J2 antibody to directly capture and map the double-stranded RNA structure of 3' UTR IRAlus in the transcriptome. Bioinformatic analysis reveals alternative polyadenylation as a major axis of IRAlus-mediated gene regulation. Notably, the expression of mouse double minute 2 (MDM2), an inhibitor of p53, is upregulated by the exclusion of IRAlus during UTR shortening, which is exploited to silence p53 during tumorigenesis. Moreover, the transcriptome-wide UTR lengthening in neural progenitor cells results in the global downregulation of genes associated with neurodegenerative diseases, including amyotrophic lateral sclerosis, via IRAlus inclusion. Our study establishes the functional landscape of 3' UTR IRAlus and its role in human pathophysiology.

Multi-targeted therapy resistance via drug-induced secretome fucosylation.

Cancer secretome is a reservoir for aberrant glycosylation. How therapies alter this post- translational cancer hallmark and the consequences thereof remain elusive. Here, we show that an elevated secretome fucosylation is a pan-cancer signature of both response and resistance to multiple targeted therapies. Large-scale pharmacogenomics revealed that fucosylation genes display widespread association with resistance to these therapies. In cancer cell cultures, xenograft mouse models, and patients, targeted kinase inhibitors distinctively induced core fucosylation of secreted proteins less than 60 kDa. Label-free proteomics of N-glycoproteomes identified fucosylation of the antioxidant PON1 as a critical component of the therapy-induced secretome (TIS). N-glycosylation of TIS and target core fucosylation of PON1 are mediated by the fucose salvage-FUT8-SLC35C1 axis with PON3 directly modulating GDP-Fuc transfer on PON1 scaffolds. Core fucosylation in the Golgi impacts PON1 stability and folding prior to secretion, promoting a more degradation-resistant PON1. Global and PON1-specific secretome de-N-glycosylation both limited the expansion of resistant clones in a tumor regression model. We defined the resistance-associated transcription factors (TFs) and genes modulated by the N-glycosylated TIS via a focused and transcriptome-wide analyses. These genes characterize the oxidative stress, inflammatory niche, and unfolded protein response as important factors for this modulation. Our findings demonstrate that core fucosylation is a common modification indirectly induced by targeted therapies that paradoxically promotes resistance.

Mitochondrial double-stranded RNAs govern the stress response in chondrocytes to promote osteoarthritis development.

Protein kinase R (PKR) is an immune response protein that becomes activated by double-stranded RNAs (dsRNAs). PKR overactivation is associated with degenerative diseases with inflammation, including osteoarthritis (OA), but the dsRNA activator remains largely unknown. Here, we find that mitochondrial dsRNA (mt-dsRNA) expression and its cytosolic efflux are facilitated in chondrocytes under OA-eliciting conditions, leading to innate immune activation. Moreover, mt-dsRNAs are released to the extracellular space and activate Toll-like receptor 3 at the plasma membrane. Elevated levels of mt-dsRNAs in the synovial fluids and damaged cartilage of OA patients and in the cartilage of surgery-induced OA mice further support our data. Importantly, autophagy prevents PKR activation and protects chondrocytes from mitochondrial stress partly by removing cytosolic mtRNAs. Our study provides a comprehensive understanding of innate immune activation by mt-dsRNAs during stress responses that underlie the development of OA and suggests mt-dsRNAs as a potential target for chondroprotective intervention.

Potential toxicity of inorganic ions in particulate matter: Ion permeation in lung and disruption of cell metabolism.

Exposure to ambient particulate matter (PM) is associated with adverse health effects. Yet, due to the complexity of its chemical composition, the molecular effects of PM exposure and the mechanism of PM-mediated toxicity remain largely unknown. Here, we show that water-soluble inorganics such as nitrate and sulfate ions, rather than PM itself, rapidly penetrate the lung surfactant barrier to the alveolar region and perturb gene expression in the lungs. Through high-throughput sequencing of lung adenocarcinoma cells, we find that exposure to nitrate and sulfate ions activates the cholesterol biosynthetic metabolism and induces the expression of genes related to tumorigenesis. Transcriptome analysis of mouse lungs exposed to nitrate/sulfate aerosols reveals interferon gamma-associated immune response. Interestingly, we find that exposure to a nitrate/sulfate mixture leads to a unique gene expression pattern that is not observed when nitrate or sulfate is treated alone. Our work suggests that the water-soluble ions are a potential source of PM-mediated toxicity and provides a roadmap to unveil the molecular mechanism of health hazards from PM exposure.

Chemotherapy confers a conserved secondary tolerance to EGFR inhibition via AXL-mediated signaling bypass.

Drug resistance remains the major culprit of therapy failure in disseminated cancers. Simultaneous resistance to multiple, chemically different drugs feeds this failure resulting in cancer relapse. Here, we investigate co-resistance signatures shared between antimitotic drugs (AMDs) and inhibitors of receptor tyrosine kinases (RTKs) to probe mechanisms of secondary resistance. We map co-resistance ranks in multiple drug pairs and identified a more widespread occurrence of co-resistance to the EGFR-tyrosine kinase inhibitor (TKI) gefitinib in hundreds of cancer cell lines resistant to at least 11 AMDs. By surveying different parameters of genomic alterations, we find that the two RTKs EGFR and AXL displayed similar alteration and expression signatures. Using acquired paclitaxel and epothilone B resistance as first-line AMD failure models, we show that a stable collateral resistance to gefitinib can be relayed by entering a dynamic, drug-tolerant persister state where AXL acts as bypass signal. Delayed AXL degradation rendered this persistence to become stably resistant. We probed this degradation process using a new EGFR-TKI candidate YD and demonstrated that AXL bypass-driven collateral resistance can be suppressed pharmacologically. The findings emphasize that AXL bypass track is employed by chemoresistant cancer cells upon EGFR inhibition to enter a persister state and evolve resistance to EGFR-TKIs.

Single-cell analysis of AIMP2 splice variants informs on drug sensitivity and prognosis in hematologic cancer.

Aminoacyl-tRNA synthetase-interacting multifunctional protein 2 (AIMP2) is a non-enzymatic component required for the multi-tRNA synthetase complex. While exon 2 skipping alternatively spliced variant of AIMP2 (AIMP2-DX2) compromises AIMP2 activity and is associated with carcinogenesis, its clinical potential awaits further validation. Here, we found that AIMP2-DX2/AIMP2 expression ratio is strongly correlated with major cancer signaling pathways and poor prognosis, particularly in acute myeloid leukemia (AML). Analysis of a clinical patient cohort revealed that AIMP2-DX2 positive AML patients show decreased overall survival and progression-free survival. We also developed targeted RNA-sequencing and single-molecule RNA-FISH tools to quantitatively analyze AIMP2-DX2/AIMP2 ratios at the single-cell level. By subclassifying hematologic cancer cells based on their AIMP2-DX2/AIMP2 ratios, we found that downregulating AIMP2-DX2 sensitizes cells to anticancer drugs only for a subgroup of cells while it has adverse effects on others. Collectively, our study establishes AIMP2-DX2 as a potential biomarker and a therapeutic target for hematologic cancer.

Reactive Polymer Targeting dsRNA as Universal Virus Detection Platform with Enhanced Sensitivity.

Reactive poly(pentafluorophenyl acrylate) (PPFPA)-grafted surfaces offer a versatile platform to immobilize biomolecules. Here, we utilize PPFPA-grafted surface and double-stranded RNA (dsRNA) recognizing J2 antibody to construct a universal virus detection platform with enhanced sensitivity. PPFPA on silicon substrates is prepared, and surface hydrophilicity is modulated by partial substitution of the pentafluorophenyl units with poly(ethylene glycol). Following dsRNA antibody immobilization, the prepared surfaces can distinguish long dsRNAs from single-stranded RNAs of the same length and short dsRNAs. As long dsRNAs are common byproducts of viral transcription/replication, these surfaces can detect the presence of different kinds of viruses without prior knowledge of their genomic sequences. To increase dsRNA detection sensitivity, a two-step method is devised where the captured dsRNAs are visualized with multiple fluorophore-tagged J2 antibodies. We show that the developed platform can differentiate foreign long dsRNAs from cellular dsRNAs and other biomolecules present in the cell lysate. Moreover, when tested against cells infected with hepatitis A or C viruses, both viruses are successfully detected using a single platform. Our study shows that the developed PPFPA platform immobilized with J2 antibody can serve as a primary diagnostic tool to determine the infection status for a wide range of viruses.

Prior acquired resistance to paclitaxel relays diverse EGFR-targeted therapy persistence mechanisms.

Secondary drug resistance stems from dynamic clonal evolution during the development of a prior primary resistance. This collateral type of resistance is often a characteristic of cancer recurrence. Yet, mechanisms that drive this collateral resistance and their drug-specific trajectories are still poorly understood. Using resistance selection and small-scale pharmacological screens, we find that cancer cells with primary acquired resistance to the microtubule-stabilizing drug paclitaxel often develop tolerance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), leading to formation of more stable resistant cell populations. We show that paclitaxel-resistant cancer cells follow distinct selection paths under EGFR-TKIs by enriching the stemness program, developing a highly glycolytic adaptive stress response, and rewiring an apoptosis control pathway. Collectively, our work demonstrates the alterations in cellular state stemming from paclitaxel failure that result in collateral resistance to EGFR-TKIs and points to new exploitable vulnerabilities during resistance evolution in the second-line treatment setting.

Evidence of Aberrant Immune Response by Endogenous Double-Stranded RNAs: Attack from Within.

Many innate immune response proteins recognize foreign nucleic acids from invading pathogens to initiate antiviral signaling. These proteins mostly rely on structural characteristics of the nucleic acids rather than their specific sequences to distinguish self and nonself. One feature utilized by RNA sensors is the extended stretch of double-stranded RNA (dsRNA) base pairs. However, the criteria for recognizing nonself dsRNAs are rather lenient, and hairpin structure of self-RNAs can also trigger an immune response. Consequently, aberrant activation of RNA sensors has been reported in numerous human diseases. Yet, in most cases, the activating antigens remain unknown. Recent studies have developed sequencing techniques tailored to specifically capture dsRNAs and identified that various noncoding elements in the nuclear and the mitochondrial genome can generate dsRNAs. Here, the identity of endogenous dsRNAs, their recognition by dsRNA sensors, and their implications in the pathogenesis of human diseases ranging from inflammatory to degenerative are presented.

Preparation of Poly(pentafluorophenyl acrylate) Functionalized SiO2 Beads for Protein Purification.

We demonstrate a simple method to prepare poly(pentafluorophenyl acrylate) (poly(PFPA)) grafted silica beads for antibody immobilization and subsequent immunoprecipitation (IP) application. The poly(PFPA) grafted surface is prepared via a simple two-step process. In the first step, 3-aminopropyltriethoxysilane (APTES) is deposited as a linker molecule onto the silica surface. In the second step, poly(PFPA) homopolymer, synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization, is grafted to the linker molecule through the exchange reaction between the pentafluorophenyl (PFP) units on the polymer and the amine groups on APTES. The deposition of APTES and poly(PFPA) on the silica particles are confirmed by X-ray photoelectron spectroscopy (XPS), as well as monitored by the particle size change measured via dynamic light scattering (DLS). To improve the surface hydrophilicity of the beads, partial substitution of poly(PFPA) with amine-functionalized poly(ethylene glycol) (amino-PEG) is also performed. The PEG-substituted poly(PFPA) grafted silica beads are then immobilized with antibodies for IP application. For demonstration, an antibody against protein kinase RNA-activated (PKR) is employed, and IP efficiency is determined by Western blotting. The analysis results show that the antibody immobilized beads can indeed be used to enrich PKR while non-specific protein interactions are minimal.

Amine-Reactive Poly(pentafluorophenyl acrylate) Brush Platforms for Cleaner Protein Purification.

Reactive pentafluorophenyl acrylate (PFPA) polymer brushes grafted on silica particles were prepared using surface-initiated reversible addition and fragmentation chain transfer polymerization. The polymer brush was successfully immobilized with antibody, then used for protein separation. The immunoprecipitated proteins showed successful enrichment of target protein, with reduced nonspecific background and less contamination from eluted antibodies. To further improve protein recovery, the hydrophobic poly(PFPA) brush was modified with hydrophilic poly(ethylene glycol) (PEG). The partially PEG-substituted poly(PFPA) brush showed better dispersion in aqueous solution, leading to improved antibody immobilization efficiency. By optimizing both the brush molecular weight and the degree of PEG substitution, an optimal balance between surface hydrophilicity and number of available PFP units was found, leading to efficient target protein purification. This study shows that poly(PFPA) platform offers a versatile approach to prepare biomolecule-activated surfaces with tunable surface property, which has potential applications in protein separation and other areas.