Formation Of Double-stranded RNA Research Articles

Engineered mRNAs With Stable Structures Minimize Double-stranded RNA Formation and Increase Protein Expression

The therapeutic use of synthetic message RNA (mRNA) has been validated in COVID-19 vaccines and shows enormous potential in developing infectious and oncological vaccines. However, double-stranded RNA (dsRNA) byproducts generated during the in vitro transcription (IVT) process can diminish the efficacy of mRNA-based therapeutics and provoke innate immune responses. Existing methods to eliminate dsRNA byproducts are often cumbersome and labor-intensive. In this study, we revealed that a loose mRNA secondary structure and more unpaired U bases in the sequence generally lead to the formation of more dsRNA byproducts during the IVT process. We further developed a predictive model for dsRNA byproducts formation based on sequence characteristics to guide the optimization of mRNA sequences, helping to minimize unwanted immune response and improve the protein expression of mRNA products. Collectively, our study provides novel clues and methodologies for developing effective mRNA therapeutics with minimized dsRNA byproducts and increased protein expression.

GNAS knockout potentiates HDAC3 inhibition through viral mimicry-related interferon responses in lymphoma

Despite selective HDAC3 inhibition showing promise in a subset of lymphomas with CREBBP mutations, wild-type tumors generally exhibit resistance. Here, using unbiased genome-wide CRISPR screening, we identify GNAS knockout (KO) as a sensitizer of resistant lymphoma cells to HDAC3 inhibition. Mechanistically, GNAS KO-induced sensitization is independent of the canonical G-protein activities but unexpectedly mediated by viral mimicry-related interferon (IFN) responses, characterized by TBK1 and IRF3 activation, double-stranded RNA formation, and transposable element (TE) expression. GNAS KO additionally synergizes with HDAC3 inhibition to enhance CD8+ T cell-induced cytotoxicity. Moreover, we observe in human lymphoma patients that low GNAS expression is associated with high baseline TE expression and upregulated IFN signaling and shares common disrupted biological activities with GNAS KO in histone modification, mRNA processing, and transcriptional regulation. Collectively, our findings establish an unprecedented link between HDAC3 inhibition and viral mimicry in lymphoma. We suggest low GNAS expression as a potential biomarker that reflects viral mimicry priming for enhanced response to HDAC3 inhibition in the clinical treatment of lymphoma, especially the CREBBP wild-type cases.

Phase 1 dose escalation and cohort expansion study evaluating safety, PK, PD and clinical activity of STC-15, a METTL-3 inhibitor, in patients with advanced malignancies.

2586 Background: RNA modifications are involved in cancer initiation and progression. The most abundant modification of mRNA is m6A generated by METTL3. Inhibition of METTL3 causes double-stranded RNA formation and activation of cellular T1 IFN response. IFN signaling induces IFN-stimulated gene families i.e. IFIT and OAS, characteristic of an anti-viral response, and secretion of cytokines and chemokines. STC-15, a first in class small molecule inhibitor of METTL3 developed by Storm Therapeutics, demonstrated in pre-clinical models, activation of IFN signaling and remodeling of the TME towards pro-inflammatory state. We report the dose escalation results from a FIH trial in patients with advanced malignancies. Methods: This is a multi-center, open-label, dose escalation study. STC-15 oral capsules are administered daily or t.i.w. in 21-d treatment cycles. Dose escalation follows 3+3 modified Fibonacci regimen. Primary objectives are safety and PK. Secondary objectives are preliminary evidence of anticancer activity and RP2D. Exploratory objectives are PD (i.e. target engagement and immune activation biomarkers) and correlation of primary pharmacology with observed clinical efficacy. Results: As of 02/01/2024, 31 patients enrolled across 4 dose levels and 5 cohorts: 60mg QD (6 pats), 60mg t.i.w. (3 pats), 100mg t.i.w. (14 pats), 160 mg t.i.w. (5 pats) and 200mg t.i.w. (3 pats). PK profile supports t.i.w. dosing. A total of 169 AEs were observed with 45 AEs attributed to STC-15 treatment. AEs were manageable, mostly hematology (thrombocytopenia 31%; 4% Grade 3), skin (pruritis, rash 14% G1/2) and GI (N/V and diarrhea 14% G1/2). 12 SAEs occurred; 1 pt (60mg QD) had DLT with G3 pneumonitis. Of 14 patients with at least 1 on-treatment scan, DCR is 78% with 2 confirmed PR ongoing in angiosarcoma (60mg, 32 weeks) and IO-refractory NSCLC (100mg, 33 weeks) and 9 SD. An average of 63% reduction in m6A on mRNA in peripheral blood within the first 24h post dosing was observed in 60mg cohorts, confirming target engagement. Whole blood Nanostring and pathway analysis of gene expression confirms upregulation of innate immune pathways (i.e. T1,2 IFN activation and anti-viral responses) as early as 8h after first dose and throughout the treatment cycle. Updated PK/PD and clinical data will be presented. Conclusions: Treatment with STC-15 is well tolerated across pharmacologically active dose range with encouraging signs of clinical activity. Early biomarker data provide proof of mechanism in target engagement, strong activation of innate immune responses and correlation of pharmacological activity with clinical response. The study is ongoing and expansion cohorts are underway to further evaluate PK/PD, safety and clinical efficacy at optimized pharmacologically active doses. Clinical trial information: NCT05584111 .

Abstract 3254: Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines

Abstract mRNA-based therapeutics have shown clinical efficacy, evidenced by the success of the mRNA vaccines for SARS-CoV-2. Personalized mRNA vaccines containing patient-derived tumor mutations are now entering the clinic and have shown promise. Delivery of mRNA-encoded therapeutic proteins (e.g., antibodies) is also under development. mRNA drug substance is typically generated by in vitro transcription (IVT). One byproduct and contaminant of IVT is the formation of double-stranded RNA (dsRNA). dsRNA is highly immunogenic, can induce inflammation and cell death, and inhibit protein translation, including the protein encoded by the therapeutic mRNA. As such, dsRNA must be measured for all IVT products to ensure safety and efficacy. However, existing dot bot and ELISA technology to detect dsRNA is neither sensitive nor quantitative. Herein, we describe the development of novel assays for sensitive and specific quantitation of dsRNA in IVT products and following encapsulation of IVT products in lipid nanoparticles (LNPs). First, NanoBiT® split luciferase technology was applied to detect dsRNA in IVT products. dsRNA binding domains were genetically attached to either SmBiT or LgBiT. In the presence of dsRNA, the SmBiT and LgBiT constructs bind via the dsRNA binding domains, resulting in dimerization and the complementation of functional NanoBiT® luciferase. NanoBiT® then generates light which is proportional to the amount of dsRNA in the sample. The assay detects dsRNA greater than or equal to 30 base pairs and is independent of the size and sequence of the dsRNA contaminant. The assay does not cross-react with single-stranded RNA or DNA and is highly sensitive, with a limit of detection less than 100 pg/ml. As a complementary tool to the biochemical assay, we developed a cell-based bioassay to detect dsRNA encapsulated in LNPs. dsRNA/LNPs are taken up by endocytosis where Toll-like receptor 3 (TLR3) detects dsRNA. Activation of TLR3 initiates a signaling pathway that is detected by a TLR3 pathway-specific promoter driving luciferase expression. This pathway likely represents the most physiologically-relevant readout of dsRNA bioactivity. Both assays are developed in “add-mix-read” format with no wash steps or media transfer, and exhibit robust, sensitive, and repeatable performance for specific detection of dsRNA in mixed solutions. Citation Format: Rich Moravec, Jun Wang, Jim Hartnett, Mei Cong, Jamison Grailer. Development of novel luminescent assays for sensitive and specific quantitation of double-stranded RNA in mRNA vaccines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3254.

Abstract B069: Double-stranded RNA derived from tandem repeat sequences induces mesenchymal transition in pancreatic cancer cells by regulating alternative splicing

Abstract Human satellite II (HSATII) composed of tandem repeats in pericentromeric regions in the human genome is aberrantly transcribed in epithelial cancers, particularly pancreatic cancer. Dysregulation of the repetitive elements in cancer tissues can facilitate incidental double-stranded RNA formation. However, it remains controversial whether double-stranded RNAs play tumor-promoting or tumor-suppressing roles during cancer progression. Therefore, we focused on the formation of double-stranded HSATII (dsHSATII) RNA and explored its molecular functions. The overexpression of dsHSATII RNA promoted mesenchymal-like morphological changes and enhanced the invasiveness of pancreatic cancer cells. We identified an RNA-binding protein, STRBP, which preferentially binds to dsHSATII RNA rather than single-stranded HSATII RNA. Depletion of STRBP induced the mesenchymal transition and enhanced the invasiveness. Conversely, the epithelial–mesenchymal transition (EMT) of dsHSATII-expressing cells was rescued by STRBP overexpression. Mechanistically, STRBP is involved in the alternative splicing of genes associated with the EMT events, which was interfered by the overexpression of dsHSATII RNA. Finally, we confirmed that isoform switching of CLSTN1, one of the target genes of STRBP-associated alternative splicing, induced EMT-like morphological changes. These findings reveal a novel cell-autonomous function of dsHSATII RNA in enhancing malignant potential, providing a better understanding of the biological significance of aberrant expression of satellite arrays in malignant tumors. Citation Format: Takahiro Kishikawa, Takuma Iwata, Motoyuki Otsuka, Mitsuhiro Fujishiro. Double-stranded RNA derived from tandem repeat sequences induces mesenchymal transition in pancreatic cancer cells by regulating alternative splicing [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B069.

PRMT1 Inhibition Activates the Interferon Pathway to Potentiate Antitumor Immunity and Enhance Checkpoint Blockade Efficacy in Melanoma.

Targeting PRMT1 stimulates interferon signaling by increasing expression of endogenous retroviral elements and double-stranded RNA through repression of DNMT1, which induces antitumor immunity and synergizes with immunotherapy to suppress tumor progression.

Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation.

Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, ELAVL3, and ELAVL4) can increase (i) 3'UTR length, (ii) dsRNA load, and (iii) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR-mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense "self" dsRNAs to preemptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.

Purification of linearized template plasmid DNA decreases double-stranded RNA formation during IVT reaction.

After the COVID-19 pandemic, messenger RNA (mRNA) has revolutionized traditional vaccine manufacturing. With the increasing number of RNA-based therapeutics, valuable new scientific insights into these molecules have emerged. One fascinating area of study is the formation of double-stranded RNA (dsRNA) during in vitro transcription (IVT) which is considered a significant impurity, as it has been identified as a major trigger in the cellular immune response pathway. Therefore, there is a growing importance placed to develop and optimize purification processes for the removal of this by-product. Traditionally, efforts have primarily focused on mRNA purification after IVT through chromatographic separations, with anion exchange and reverse phase chromatography emerging as effective tools for this purpose. However, to the best of our knowledge, the influence and significance of the quality of the linearized plasmid have not been thoroughly investigated. Plasmids production involves the growth of bacterial cultures, bacterial harvesting and lysis, and multiple filtration steps for plasmid DNA purification. The inherent complexity of these molecules, along with the multitude of purification steps involved in their processing, including the subsequent linearization and the less-developed purification techniques for linearized plasmids, often result in inconsistent batches with limited control over by-products such as dsRNA. This study aims to demonstrate how the purification process employed for linearized plasmids can impact the formation of dsRNA. Several techniques for the purification of linearized plasmids based on both, resin filtration and chromatographic separations, have been studied. As a result of that, we have optimized a chromatographic method for purifying linearized plasmids using monolithic columns with C4 chemistry (butyl chains located in the surface of the particles), which has proven successful for mRNAs of various sizes. This chromatographic separation facilitates the generation of homogeneous linearized plasmids, leading to mRNA batches with lower levels of dsRNA during subsequent IVT processes. This finding reveals that dsRNA formation is influenced not only by RNA polymerase and IVT conditions but also by the quality of the linearized template. The results suggest that plasmid impurities may contribute to the production of dsRNA by providing additional templates that can be transcribed into sequences that anneal with the mRNA molecules. This highlights the importance of considering the quality of plasmid purification in relation to dsRNA generation during transcription. Further investigation is needed to fully understand the mechanisms and implications of plasmid-derived dsRNA. This discovery could shift the focus in mRNA vaccine production, placing more emphasis on the purification of linearized plasmids and potentially saving, in some instances, a purification step for mRNA following IVT.

The non-coding RNA SVALKA locus produces a cis-natural antisense transcript that negatively regulates the expression of CBF1 and biomass production at normal temperatures

Non-coding transcription is present in all eukaryotic genomes, but we lack fundamental knowledge about its importance for an organism’s ability to develop properly. In plants, emerging evidence highlights the essential biological role of non-coding transcription in the regulation of coding transcription. However, we have few molecular insights into this regulation. Here, we show that a long isoform of the long non-coding RNA SVALKA-L (SVK-L) forms a natural antisense transcript to the host gene CBF1 and negatively regulates CBF1 mRNA levels at normal temperatures in the model plant Arabidopsis thaliana. Furthermore, we show detailed evidence for the specific mode of action of SVK-L. This pathway includes the formation of double-stranded RNA that is recognized by the DICER proteins and subsequent downregulation of CBF1 mRNA levels. Thus, the CBF1-SVK regulatory circuit is not only important for its previously known role in cold temperature acclimation but also for biomass production at normal temperatures. Our study characterizes the developmental role of SVK-L and offers mechanistic insight into how biologically important overlapping natural antisense transcripts can act on and fine-tune the steady-state levels of their host gene’s mRNA.

The BRCA1 BRCT promotes antisense RNA production and double-stranded RNA formation to suppress ribosomal R-loops

R-loops, or RNA:DNA hybrids, can induce DNA damage, which requires DNA repair factors including breast cancer type 1 susceptibility protein (BRCA1) to restore genomic integrity. To date, several pathogenic mutations have been found within the tandem BRCA1 carboxyl-terminal (BRCT) domains that mediate BRCA1 interactions with proteins and DNA in response to DNA damage. Here, we describe a nonrepair role of BRCA1 BRCT in suppressing ribosomal R-loops via two mechanisms. Through its RNA binding and annealing activities, BRCA1 BRCT facilitates the formation of double-stranded RNA between ribosomal RNA (rRNA) and antisense-rRNA (as-rRNA), hereby minimizing rRNA hybridization to ribosomal DNA to form R-loops. BRCA1 BRCT also promotes RNA polymerase I-dependent transcription of as-rRNA to enhance double-stranded rRNA (ds-rRNA) formation. In addition, BRCA1 BRCT-mediated as-rRNA production restricts rRNA maturation in unperturbed cells. Hence, impairing as-rRNA transcription and ds-rRNA formation due to BRCA1 BRCT deficiency deregulates rRNA processing and increases ribosomal R-loops and DNA breaks. Our results link ribosomal biogenesis dysfunction to BRCA1-associated genomic instability.

Abstract B019: Inflammatory response to dietary carbohydrates contributes to the formation of hepatic metastatic niche

Abstract Mortality from colon and pancreatic cancers arises from cancer metastatic dissemination to the liver. The glycemic load from carbohydrates (carbs) has been specifically linked to the risk of metastatic recurrence of stage 3 colorectal cancer, but the mechanism of how dietary carbs contribute to the formation of liver metastases remains poorly understood. Our analyses now demonstrate that feeding mice a high carb diet (HCD) promotes liver colonization by tumor cells, and supports the survival of these cells, once in the liver. HCD also induces expression of a Type I interferon (IFN) inflammatory gene signatures which may contribute to its ability to promote metastatic seeding of the liver. Notably, this inflammatory phenotype in hepatocytes was completely abolished if animals are fed a diet enriched in fats (75% of calories), indicating a direct link between liver inflammation and consumption of carbohydrates. Specifically, we have discovered that: 1) dietary carbohydrates, but not a high-fat ketogenic diet, activate multiple independent inflammatory cascades in hepatocytes, including Type-I IFN/STAT1, NFkB, and IL6-STAT3 pathways; 2) the inflammatory signaling in hepatocytes is retained in Scid and NSG mice, indicating that it occurs in the absence of adaptive immunity; 3) HCD, but not fasting or ketogenic diet, markedly increases abundance of retained intronic sequences and formation of double-stranded RNA (dsRNAs). We nominate these yet uncharacterized dsRNAs as potential viral mimics which may trigger the Type I IFN signature observed in HCD-fed livers; and (4) a ketogenic diet showed a trend to reduce liver metastatic colonization, but was associated with mild hepatic steatosis. Overall, this study establishes a new paradigm in nutrient sensing by normal hepatocytes and nominates alternative RNA splicing as the potential trigger of inflammatory responses to HCDs via accumulation of dsRNAs. We suggest that a reduction of dietary carbohydrates, by suppressing the diet-induced hepatic inflammation, will improve the survival of cancer patients, and reduce the risk of liver metastases. Citation Format: Aizhan Surumbayeva, Riley Williams, Michael Kotliar, Maryclare Taylor, Débora B. Vendramini Costa, Charline Ogier, Diana Restifo, Kathy Q. Cai, Xueyang He, Paul Boutz, Siddharth Balachandran, Igor Astsaturov. Inflammatory response to dietary carbohydrates contributes to the formation of hepatic metastatic niche [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B019.

Interdependent Transcription of a Natural Sense/Antisense Transcripts Pair (SLC34A1/PFN3).

Natural antisense transcripts (NATs) constitute a significant group of regulatory, long noncoding RNAs. They are prominently expressed in testis but are also detectable in other organs. NATs are transcribed at low levels and co-expressed with related protein coding sense transcripts. Nowadays NATs are generally considered as regulatory, long noncoding RNAs without closer focus on the inevitable interference between sense and antisense expression. This work describes a cellular system where sense and antisense transcription of a specific locus (SLC34A1/PFN3) is induced using epigenetic modifiers and CRISPR-Cas9. The renal cell lines HEK293 and HKC-8 do not express SLC34A1/PFN3 under normal culture conditions. Five-day exposure to dexamethasone significantly stimulates sense transcript (SLC34A1) levels and antisense (PFN3) minimally; the effect is only seen in HEK293 cells. Enhanced expression is paralleled by reduced sense promoter methylation and an increase in activating histone marks. Expression is further modulated by cassettes that stimulate the expression of sense or antisense transcript but disrupt protein coding potential. Constitutive expression of a 5′-truncated SLC34A1 transcript increases sense expression independent of dexamethasone induction but also stimulates antisense expression. Concordant expression is confirmed with the antisense knock-in that also enhances sense expression. The antisense effect acts on transcription in cis since transient transfection with sense or antisense constructs fails to stimulate the expression of the opposite transcript. These results suggest that bi-directional transcription of the SLC34A1/PFN3 locus has a stimulatory influence on the expression of the opposite transcript involving epigenetic changes of the promoters. In perspective of extensive, previous research into bi-directionally transcribed SLC34A loci, the findings underpin a hypothesis where NATs display different biological roles in soma and germ cells. Accordingly, we propose that in somatic cells, NATs act like lncRNAs–with the benefit of close proximity to a potential target gene. In germ cells, however, recent evidence suggests different biological roles for NATs that require RNA complementarity and double-stranded RNA formation.

Model-Based Design of Synthetic Antisense RNA for Predictable Gene Repression.

Our enhanced understanding of RNA folding and function has increased the use of small RNA regulators. Among these RNA regulators, synthetic antisense RNA (asRNA) is designed to contain an RNA sequence complementary to the target mRNA sequence, and the formation of double-stranded RNA (dsRNA) facilitates gene repression due to dsRNA degradation or prevention of ribosome access to the mRNA. Despite the simple complementarity rule, however, predictably tunable repression has been challenging when synthetic asRNAs are used. Here, the protocol for model-based asRNA design is described. This model can predict synthetic asRNA-mediated repression efficiency using two parameters: the change in free energy of complex formation (ΔGCF) and percent mismatch of the target binding region (TBR). The model has been experimentally validated in both Gram-positive and Gram-negative bacteria as well as for target genes in both plasmids and chromosomes. These asRNAs can be created by simply replacing the TBR sequence with one that is complementary to the target mRNA sequence of interest. In principle, this protocol can be applied to design and build asRNAs for predictable gene repression in various contexts, including multiple target genes and organisms, making asRNAs predictably tunable regulators for broad applications.

Structural transition of replicable RNAs during in vitro evolution with Qβ replicase.

Single-stranded RNAs (ssRNAs) are utilized as genomes in some viruses and also in experimental models of ancient life-forms, owing to their simplicity. One of the largest problems for ssRNA replication is the formation of double-stranded RNA (dsRNA), a dead-end product for ssRNA replication. A possible strategy to avoid dsRNA formation is to create strong intramolecular secondary structures of ssRNA. To design ssRNAs that efficiently replicate by Qβ replicase with minimum dsRNA formation, we previously proposed the “fewer unpaired GC rule.” According to this rule, ssRNAs that have fewer unpaired G and C bases in the secondary structure should efficiently replicate with less dsRNA formation. However, the validity of this rule still needs to be examined, especially for longer ssRNAs. Here, we analyze nine long ssRNAs that successively appeared during an in vitro evolution of replicable ssRNA by Qβ replicase and examine whether this rule can explain the structural transitions of the RNAs. We found that these ssRNAs improved their template abilities step-by-step with decreasing dsRNA formation as mutations accumulated. We then examine the secondary structures of all the RNAs by a chemical modification method. The analysis of the structures revealed that the probabilities of unpaired G and C bases tended to decrease gradually in the course of evolution. The decreases were caused by the local structural changes around the mutation sites in most of the cases. These results support the validity of the “fewer unpaired GC rule” to efficiently design replicable ssRNAs by Qβ replicase, useful for more complex ssRNA replication systems.

Tet2 promotes pathogen infection-induced myelopoiesis through mRNA oxidation.

Varieties of RNA modification form the epitranscriptome for post-transcriptional regulation. 5-Methylcytosine (5-mC) is a sparse RNA modification in messenger RNA (mRNA) under physiological conditions. The function of RNA 5-hydroxymethylcytosine (5-hmC) oxidized by ten-eleven translocation (Tet) proteins in Drosophila has been revealed more recently. However, the turnover and function of 5-mC in mammalian mRNA have been largely unknown. Tet2 suppresses myeloid malignancies mostly in an enzymatic activity-dependent manner, and is important in resolving inflammatory response in an enzymatic activity-independent way. Myelopoiesis is a common host immune response in acute and chronic infections; however, its epigenetic mechanism needs to be identified. Here we demonstrate that Tet2 promotes infection-induced myelopoiesis in an mRNA oxidation-dependent manner through Adar1-mediated repression of Socs3 expression at the post-transcription level. Tet2 promotes both abdominal sepsis-induced emergency myelopoiesis and parasite-induced mast cell expansion through decreasing mRNA levels of Socs3, a key negative regulator of the JAK-STAT pathway that is critical for cytokine-induced myelopoiesis. Tet2 represses Socs3 expression through Adar1, which binds and destabilizes Socs3 mRNA in a RNA editing-independent manner. For the underlying mechanism of Tet2 regulation at the mRNA level, Tet2 mediates oxidation of 5-mC in mRNA. Tet2 deficiency leads to the transcriptome-wide appearance of methylated cytosines, including ones in the 3' untranslated region of Socs3, which influences double-stranded RNA formation for Adar1 binding, probably through cytosine methylation-specific readers, such as RNA helicases. Our study reveals a previously unknown regulatory role of Tet2 at the epitranscriptomic level, promoting myelopoiesis during infection in the mammalian system by decreasing 5-mCs in mRNAs. Moreover, the inhibitory function of cytosine methylation on double-stranded RNA formation and Adar1 binding in mRNA reveals its new physiological role in the mammalian system.

Biological functions of natural antisense transcripts.

Natural antisense transcripts (NATs) are RNA molecules that originate from opposite DNA strands of the same genomic locus (cis-NAT) or unlinked genomic loci (trans-NAT). NATs may play various regulatory functions at the transcriptional level via transcriptional interference. NATs may also regulate gene expression levels post-transcriptionally via induction of epigenetic changes or double-stranded RNA formation, which may lead to endogenous RNA interference, RNA editing or RNA masking. The true biological significance of the natural antisense transcripts remains controversial despite many years of research. Here, we summarize the current state of knowledge and discuss the sense-antisense overlap regulatory mechanisms and their potential.

GM plants with RNAi - golden mosaic resistant bean

GM plants with RNAi - golden mosaic resistant bean

Template Role of Double-Stranded RNA in Tombusvirus Replication

Replication of plus-strand RNA [(+)RNA] viruses of plants is a relatively simple process that involves complementary minus-strand RNA [(-)RNA] synthesis and subsequent (+)RNA synthesis. However, the actual replicative form of the (-)RNA template in the case of plant (+)RNA viruses is not yet established unambiguously. In this paper, using a cell-free replication assay supporting a full cycle of viral replication, we show that replication of Tomato bushy stunt virus (TBSV) leads to the formation of double-stranded RNA (dsRNA). Using RNase digestion, DNAzyme, and RNA mobility shift assays, we demonstrate the absence of naked (-)RNA templates during replication. Time course experiments showed the rapid appearance of dsRNA earlier than the bulk production of new (+)RNAs, suggesting an active role for dsRNA in replication. Radioactive nucleotide chase experiments showed that the mechanism of TBSV replication involves the use of dsRNA templates in strand displacement reactions, where the newly synthesized plus strand replaces the original (+)RNA in the dsRNA. We propose that the use of dsRNA as a template for (+)RNA synthesis by the viral replicase is facilitated by recruited host DEAD box helicases and the viral p33 RNA chaperone protein. Altogether, this replication strategy allows TBSV to separate minus- and plus-strand syntheses in time and regulate asymmetrical RNA replication that leads to abundant (+)RNA progeny. Positive-stranded RNA viruses of plants use their RNAs as the templates for replication. First, the minus strand is synthesized by the viral replicase complex (VRC), which then serves as a template for new plus-strand synthesis. To characterize the nature of the (-)RNA in the membrane-bound viral replicase, we performed complete RNA replication of Tomato bushy stunt virus (TBSV) in yeast cell-free extracts and in plant extracts. The experiments demonstrated that the TBSV (-)RNA is present as a double-stranded RNA that serves as the template for TBSV replication. During the production of new plus strands, the viral replicase displaces the old plus strand in the dsRNA template, leading to asymmetrical RNA synthesis. The presented data are in agreement with the model that the dsRNA is present in nuclease-resistant membranous VRCs. This strategy likely allows TBSV to protect the replicating viral RNA from degradation as well as to evade the early detection of viral dsRNAs by the host surveillance system.

Kinetic model of double-stranded RNA formation during long RNA replication by Qβ replicase

Qβ replicase is an RNA-dependent RNA polymerase, which synthesizes the complementary RNA using a single-stranded RNA as a template. The formation of non-replicable double-stranded RNA (dsRNA) by hybridization between newly synthesized RNA and the template RNA hinders the broader application of Qβ replicase. Here, we developed a kinetic model of Qβ RNA replication consisting of two reaction pathways of dsRNA formation, which quantitatively explains the dynamics of dsRNA formation of three template RNAs. We also found that part of the Qβ phage genomic RNA sequence including the central hairpin loop significantly decreases the rate of dsRNA formation.

Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis

Owing to their sessile nature, plants have evolved sophisticated genetic and epigenetic regulatory systems to respond quickly and reversibly to daily and seasonal temperature changes. However, our knowledge of how plants sense and respond to warming ambient temperatures is rather limited. Here we show that an increase in growth temperature from 22 °C to 30 °C effectively inhibited transgene-induced posttranscriptional gene silencing (PTGS) in Arabidopsis. Interestingly, warmth-induced PTGS release exhibited transgenerational epigenetic inheritance. We discovered that the warmth-induced PTGS release occurred during a critical step that leads to the formation of double-stranded RNA (dsRNA) for producing small interfering RNAs (siRNAs). Deep sequencing of small RNAs and RNA blot analysis indicated that the 22-30 °C increase resulted in a significant reduction in the abundance of many trans-acting siRNAs that require dsRNA for biogenesis. We discovered that the temperature increase reduced the protein abundance of SUPPRESSOR OF GENE SILENCING 3, as a consequence, attenuating the formation of stable dsRNAs required for siRNA biogenesis. Importantly, SUPPRESSOR OF GENE SILENCING 3 overexpression released the warmth-triggered inhibition of siRNA biogenesis and reduced the transgenerational epigenetic memory. Thus, our study reveals a previously undescribed association between warming temperatures, an epigenetic system, and siRNA biogenesis.