RNA Silencing Research Articles

High throughput screen identifies lysosomal acid phosphatase 2 (ACP2) to regulate IFN-1 responses to potentiate oncolytic VSV∆51 activity.

Strategies in genetic and pharmacological modulation of innate immunity to enhance oncolytic virotherapy (OV) efficacy are being explored. We have recently characterized the ability for vanadium-based compounds, a class of pan-phosphatase (PP) inhibitors, to potentiate OVs. We next sought to identify PPs that could be targeted to enhance OVs, akin to vanadium. By conducting a high-throughput screen of a library of silencing RNA (siRNA) targeting human PPs, we uncovered several PPs that robustly enhanced infectivity and oncolysis of the oncolytic vesicular stomatitis virus (VSV∆51). Knockdown of our top validated hit, lysosomal acid phosphatase 2 (ACP2), increased VSV∆51 viral titers by over 20-fold. In silico analysis by RNA sequencing revealed ACP2 to regulate antiviral type I interferon (IFN-1) signaling pathways, similar to vanadium. To further exploit this mechanism for therapeutic gain, we encoded a short-hairpin RNA (shRNA) against ACP2 into oncolytic vesicular stomatitis virus (VSV∆51) under a miR-30 promoter. This bioengineered OV demonstrated expression of the miR-30 promoter, knockdown of ACP2, repression and ultimately, showed markedly enhanced viral VSV∆51 particle production compared to its non-targeting control counterpart. Altogether, this study identifies IFN-1 regulating PP targets, namely ACP2, that may prove instrumental in increasing the therapeutic efficacy of OVs.

Plant Viral Synergism: Co-Expression of P1 and NIa-Pro Cistrons of Wheat Streak Mosaic Virus and Triticum Mosaic Virus Is Required for Synergistic Interactions in Wheat.

Synergistic interactions among unrelated viruses in mixed infections can cause significant yield losses, and viral determinants of these interactions are poorly understood. Wheat (Triticum aestivum L.) co-infection with wheat curl mite-transmitted wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) results in disease synergism with a drastically increased symptom phenotype of stunted growth, leaf bleaching, and enhanced titers of both viruses. In this study, we examined the viral determinants responsible for WSMV-TriMV disease synergism through transient expression of select cistrons of WSMV in wheat through TriMV and vice-versa. We found that expression of WSMV P1, NIa, or NIaPro cistrons in wheat through TriMV or vice-versa elicited moderate to severe symptoms with a moderate or no increase in virus titer. However, co-expression of P1 and NIaPro cistrons of WSMV in wheat through TriMV or vice-versa exhibited a WSMV-TriMV disease synergism-like phenotype. Additionally, we found that the P3 cistron of both viruses is dispensable for synergism, whereas HCPro and NIaVPg cistrons of WSMV and TriMV are not the primary determinants but might have a minor role in efficient synergism. In co-infected wheat, accumulation of vsiRNAs was increased, similar to viral genomic RNA copies, despite the presence of dual viral RNA silencing suppressors (VRSS), which function through sequestration of vsiRNAs. Our findings revealed that WSMV-TriMV disease synergism is not caused by the suppression of host post-transcriptional gene silencing by dual VRSS in co-infected wheat and the P1 and NIaPro cistrons of both viruses collectively drive synergistic interactions between WSMV and TriMV in wheat.

IMMU-17. ADENOSINE DEAMINASE ACTING ON RNA (ADAR) SILENCES TUMOR-INTRINSIC INTERFERON RESPONSE IN DIFFUSE MIDLINE GLIOMA

Abstract INTRODUCTION Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children and is currently incurable. Clinical trials using immune checkpoint blockade (ICB) therapy have failed to show efficacy due to multifactorial mechanisms including limited lymphocyte infiltration, low immune checkpoint (IC) expression, and a low mutational burden. Adenosine deaminase acting on RNA (ADAR) is an RNA editor, which limits anti-tumor immunity by editing/preventing the sensing of endogenous double-stranded RNA (dsRNA). We have found that ADAR protein levels are significantly elevated in DMG patients, suggesting that ADAR’s immune-silencing function is relevant to DMG immune suppression. The onco-histone driver H3K27M promotes epigenetic dysregulation in DMGs, de-suppressing retroelement transcription, a major source of endogenous dsRNA. By inhibiting ADAR, we expect that the increased levels of dsRNA will stimulate intracellular type-1 interferon (IFN) responses, induce chemokine secretion, and promote antitumor CD8+ cytotoxicity. METHODS Human DMG cells and patient-derived glioma spheres were treated with silencing RNA to transiently knockdown (KD) ADAR levels for assays involving in vitro viability and proliferation. Affected pathways were identified using immunoblotting and quantitative PCR. RESULTS Our data shows that ADAR protein levels are elevated in DMG patient proteomic datasets and cell lines. ADAR-depleted patient-derived DMG cell lines exhibit significantly less proliferation compared to control siRNA and ADAR-depleted hTERT-astrocytes (Mann-Whitney test, P-value <0.0001, ****). CONCLUSION Our results demonstrate that DMG tumor cells have an ADAR selective dependency in vitro. In ongoing studies, we aim to determine if ADAR-depletion improves in-vivo survival in both immunodeficient and immunocompetent models in combination with immunotherapies against DMG.

Identification of an RNA silencing suppressor encoded by an Indian citrus ringspot virus

Identification of an RNA silencing suppressor encoded by an Indian citrus ringspot virus

Involvement of a Microplusin-like Gene (HlonML-1) in the Olfactory Chemosensation of Haemophysalis longicornis: Expression, RNA Silencing, and Behavioral Implications

The study of tick olfaction is relatively new compared to that of insects, and the molecular mechanisms involved remain poorly understood. Despite several potential chemosensory genes identified in multiple tick species, these are yet to be validated through independent functional experiments. In this research, we cloned and analyzed a microplusin-like gene, HlonML-1, and investigated its role in the chemosensory activities of H. longicornis. The results showed that this gene’s amino acid sequences lack histidine residues essential for antimicrobial activity, and it is evolutionarily linked to putative chemosensory microplusins in ticks. Gene expression analyses indicated that HlonML-1 was significantly more abundant in ticks exposed to potential attractants and in the forelegs of H. longicornis than in non-exposed ticks and the hindlegs, respectively. Tick forelegs support the Haller’s organ, which is a sensory structure mostly specialized for chemosensation. Furthermore, Y-tube olfactometer assays indicated that silencing HlonML-1 significantly impaired adult ticks’ ability to detect selected odors, while their gustatory-related behavior remained unaffected compared to the control groups. Given its unique sequences, relative abundance in chemosensory tissues, and impact on odor detection, HlonML-1 is likely involved in the olfactory chemosensation of H. longicornis. Future research validating putative chemosensory microplusins in the genomes of various tick species may enhance our understanding of their olfactory functions in tick and lead to the identification of new molecular targets for developing tick repellents.

Capturing Hydrophilic Chemotherapeutics Agents Into siRNA-Encapsulated Vesicle-Like Nanoparticles for Convenient ICB-Chemo Combination Therapy.

Clinical evidence has demonstrated that combining immune checkpoint blockade (ICB) therapy with chemotherapy significantly improves response rates to ICB therapy and therapeutic efficacy in various tumor types. However, a convenient method for achieving synergistic ICB therapy and chemotherapy with precise co-delivery of both agents is still highly desirable. In this study, a strategy for co-delivering small interfering RNA (siRNA) encapsulated in vesicle-like nanoparticles (VNPsiRNA) and chemotherapeutic drugs is aimed to develop. It is discovered that the hydrophilic chemotherapeutic drug mitoxantrone hydrochloride (MTO·2HCl) can be captured into VNPsiRNA. The resulting VNPsiRNACpMTO can simultaneously block immune checkpoints via RNA silencing and induce chemotherapeutic effects on tumor cells. The mechanism of MTO·2HCl is elucidates, captures, and demonstrates the superior therapeutic effect of VNPsiRNACpMTO through chemo-immunotherapy. This strategy can also be extended to deliver other hydrochloride anticancer drugs, such as doxorubicin hydrochloride (DOX·HCl), for achieving synergistic combination therapy. This study provides a facile strategy for enhancing combined ICB and chemotherapy via co-delivering siRNA and chemotherapeutic drugs, offering a promising approach to cancer treatment.

Quest for HBV functional cure: what have we learnt from silencing RNAs?

Quest for HBV functional cure: what have we learnt from silencing RNAs?

Epigenetic control of T-DNA during transgenesis and pathogenesis.

Mobile elements known as T-DNAs are transferred from pathogenic Agrobacterium to plants and reprogram the host cell to form hairy roots or tumors. Disarmed non-oncogenic T-DNAs are extensively used to deliver transgenes in plant genetic engineering. Such T-DNAs were the first known targets of RNA silencing mechanisms, which detect foreign RNA in plant cells and produce small RNAs that induce transcript degradation. These T-DNAs can also be transcriptionally silenced by the deposition of epigenetic marks such as DNA methylation and the dimethylation of lysine 9 (H3K9me2) in plants. Here, we review the targeting and the roles of RNA silencing and DNA methylation on T-DNAs in transgenic plants as well as during pathogenesis. In addition, we discuss the crosstalk between T-DNAs and genome-wide changes in DNA methylation during pathogenesis. We also cover recently discovered regulatory phenomena, such as T-DNA suppression and RNA silencing-independent and epigenetic-independent mechanisms that can silence T-DNAs. Finally, we discuss the implications of findings on T-DNA silencing for the improvement of plant genetic engineering.

ALTERED MERISTEM PROGRAM1 impairs RNA silencing by repressing the biogenesis of a subset of inverted repeat-derived siRNAs.

RNA silencing negatively regulates gene expression at the transcriptional and post-transcriptional levels through DNA methylation, histone modification, mRNA cleavage, and translational inhibition. Small interfering RNAs (siRNAs) of 21 to 24 nucleotides are processed from double-stranded RNAs by Dicer-like enzymes and play essential roles in RNA silencing in plants. Here, we demonstrated that ALTERED MERISTEM PROGRAM1 (AMP1) and its putative paralog LIKE AMP1 (LAMP1) impair RNA silencing by repressing the biogenesis of a subset of inverted repeat (IR)-derived siRNAs in Arabidopsis (Arabidopsis thaliana). AMP1 and LAMP1 inhibit Pol II-dependent IR gene transcription by suppressing ARGONAUTE 1 (AGO1) protein levels. Genetic analysis indicates that AMP1 acts upstream of RNA polymerase IV subunit 1 (NRPD1), RNA-dependent RNA polymerase 2 (RDR2), and Dicer-Like 4 (DCL4), which are required for IR-induced RNA silencing. We also show that AMP1 and LAMP1 inhibit siRNA-mediated silencing in a different mechanism from AGO4 and DCL3. Together, these results reveal two previously unknown players in siRNA biogenesis from IRs - AGO1, which promotes IR transcription, and AMP1, which inhibits IR transcription indirectly through the repression of AGO1 expression.

Betasatellite: A Hijacker of Plant Innate Immunity

Betasatellites associated with begomoviruses significantly exacerbate viral infections in economically important crops, prompting the evolution of various plant defense mechanisms, including RNA interference, phytohormone signalling and cellular modifications using autophagy and ubiquitination. Approximately 13.5-kDa βC1 protein has two key functions: It boosts viral replication and weakens plant defenses. By interfering with signal transduction pathways and RNA silencing, it makes the plant more vulnerable to infection. Additionally, βC1 impairs chloroplast function, complicating the plant's defense strategies. In contrast, the nuclear shuttle protein (βV1) protein, though less understood, appears to facilitate viral replication and interacts with the helper virus replication enhancer protein (REn), leading to altered localization within the cell. As only 40% of betasatellites encode the βV1 protein, further investigation into its molecular interactions and functions could provide crucial insights for developing effective antiviral strategies against geminivirus infections. This review highlights the complex interplay between betasatellites and host defenses, offering avenues for future research in crop protection.

The SlbHLH92 transcription factor enhances salt stress resilience by fine-tuning hydrogen sulfide biosynthesis in tomato

Ongoing soil salinization severely hampers plant growth and the sustainability of global crops production. Hydrogen sulfide (H2S), acting as a critical gaseous signaling molecule, plays a vital role in plant response to various environmental cues such as salt stress. Nonetheless, it is not well understood how the transcriptional network regulates H2S production in response to salt stress in tomato. Herein, we determine that the bHLH transcription factor SlbHLH92 functions as a transcriptional activator in tomato (Solanum lycopersicum L.), upregulating the expression of the L-CYSTEINE DESULFHYDRASE 1 (SlLCD1) gene involved in H2S biosynthesis, thereby enhancing the plants' tolerance to salt stress. When exposed to salt stress, overexpression of SlbHLH92 in tomato leads to enhanced salt tolerance compared to wild-type plants. In contrast, suppression of SlbHLH92 expression with RNAi silencing results in increased sensitivity to salt stress. Subsequent molecular and biochemical investigations confirm that the salt-induced SlbHLH92 upregulates the expression of SlLCD1, leading to an increase in H₂S levels, as well as other salt-responsive genes (SlCBL10 and SlVQ16), by directly binding to specific cis-elements in their promoter regions. Furthermore, the VQ-motif containing protein SlVQ16 physically interacts with SlbHLH92, thereby promoting an increase in its transcriptional activity. Taken together, our study reveals an emerging mechanism in which the SlbHLH92-SlVQ16-H2S signaling cascade contributes to enhancing salt tolerance in tomato, presenting potential genetic targets for breeding salt-tolerant tomato cultivars.

RNA silencing is activated by N gene-mediated hypersensitive response and plays a key role in local and systemic virus resistance

AbstractIn plants, recognition between resistance gene (R) and virus induces a local hypersensitive response (HR), which is accompanied by systemic acquired resistance (SAR). The dominant resistance gene N in tobacco confers resistance to tobacco mosaic virus (TMV) at both locally inoculated tissues and systemically infected tissues. However, the mechanisms underlying HR- and SAR-mediated viral inhibition are not fully revealed. In this study, we find that Nicotiana glutinosa RNA-binding protein (NgRBP) is an RNA silencing suppressor which enhances TMV-triggered HR. Stronger HR could result in stronger local and systemic RNA silencing as well as SAR. Enhanced RNA silencing in the systemically infected leaves induced by the NgRBP gene is compromised by transient expression of NahG. These results indicate that RNA silencing is activated by HR and plays a crucial role in local and systemic virus resistance. Our results reveal a crosstalk between N gene-mediated virus resistance and RNA silencing which would deepen our understanding of the established HR and SAR models.

Resistance gene Ty-1 restricts TYLCV infection in tomato by increasing RNA silencing

A major antiviral mechanism in plants is mediated by RNA silencing through the action of DICER-like (DCL) proteins, which cleave dsRNA into discrete small RNA fragments, and ARGONAUTE (AGO) proteins, which use the small RNAs to target single-stranded RNA. RNA silencing can also be amplified through the action of RNA-dependent RNA polymerases (RDRs), which use single stranded RNA to generate dsRNA that in turn is targeted by DCL proteins. As a counter-defense, plant viruses encode viral suppressors of RNA silencing (VSRs) that target different components in the RNA silencing pathway. The tomato Ty-1 gene confers resistance to the DNA virus tomato yellow leaf curl virus (TYLCV) and has been reported to encode an RDRγ protein. However, the molecular mechanisms by which Ty-1 controls TYLCV infection, including whether Ty-1 is involved in RNA silencing, are unknown. Here, by using a transient expression assay, we have confirmed that Ty-1 shows antiviral activity against TYLCV in Nicotiana benthamiana. Also, in transient expression-based silencing assays, Ty-1 augmented systemic transgene silencing in GFP transgenic N. benthamiana plants. Furthermore, co-expression of Ty-1 or other RDRγ proteins from N. benthamiana or Arabidopsis with various proteins resulted in lower protein expression. These results are consistent with a model wherein Ty-1-mediated resistance to TYLCV is due, at least in part, to an increase in RNA silencing activity.

KHDRBS3 facilitates self-renewal and temozolomide resistance of glioblastoma cell lines

Glioblastoma is a deadly tumor which possesses glioblastoma stem cell populations involved in temozolomide (TMZ) resistance. To gain insight into the mechanisms of self-renewing and therapy-resistant cancer stem cells, subcellular proteomics was utilized to identify proteins whose expression is enriched in U251-derived glioblastoma stem-like cells. The KH RNA Binding Domain Containing, Signal Transduction Associated 3, KHDRBS3, was successfully identified as a gene up-regulated in the cancer stem cell population compared with its differentiated derivatives. Depletion of KHDRBS3 by RNA silencing led to a decrease in cell proliferation, neurosphere formation, migration, and expression of genes involved in glioblastoma stemness. Importantly, TMZ sensitivity can be induced by the gene knockdown. Collectively, our results highlight KHDRBS3 as a novel factor associated with self-renewal of glioblastoma stem-like cells and TMZ resistance. As a consequence, targeting KHDRBS3 may help eradicate glioblastoma stem-like cells.

Information entropy-assisted intuitionistic fuzzy rough feature subset selection

Fuzzy-rough set (FRS) has been effectively implemented as a powerful pre-processing tool to cope with the issues such as vagueness, imprecision, noise and uncertainty in high-dimensional data analysis. However, FRS fails to handle the uncertainty due to identification, which is very much common in heterogeneous data. Moreover, it is always challenging to handle different issues such as vagueness, uncertainty, and noise during elimination of irrelevant and redundant features. This paper investigates an intuitionistic fuzzy rough set (IFRS)-aided feature selection method by using information entropy notion to tackle these issues simultaneously. We establish an intuitionistic fuzzy (IF) similarity relation. Then IFRS model is discussed based on this relation. Next, we present a novel IF granular structure. Based on the granular structure, we present lambda-conditional entropy for IF framework. Further, relevant mathematical theorems are proved to justify the theoretical aspect of the models. Moreover, a positive region preserved attribute selection method is proposed. A comprehensive experimental study is discussed to demonstrate the effectiveness and practical validation of the proposed method. Finally, a methodology is developed based on proposed models, which increases accuracy for the minority RNA silencing suppressors against majority class non-suppressors as evident from better sensitivity and G-means metrics.

Silencing of maternally expressed RNAs in Dlk1-Dio3 domain causes fatal vascular injury in the fetal liver

The mammalian imprinted Dlk1-Dio3 domain contains multiple lncRNAs, mRNAs, the largest miRNA cluster in the genome and four differentially methylated regions (DMRs), and deletion of maternally expressed RNA within this locus results in embryonic lethality, but the mechanism by which this occurs is not clear. Here, we optimized the model of maternally expressed RNAs transcription termination in the domain and found that the cause of embryonic death was apoptosis in the embryo, particularly in the liver. We generated a mouse model of maternally expressed RNAs silencing in the Dlk1-Dio3 domain by inserting a 3 × polyA termination sequence into the Gtl2 locus. By analyzing RNA-seq data of mouse embryos combined with histological analysis, we found that silencing of maternally expressed RNAs in the domain activated apoptosis, causing vascular rupture of the fetal liver, resulting in hemorrhage and injury. Mechanistically, termination of Gtl2 transcription results in the silencing of maternally expressed RNAs and activation of paternally expressed genes in the interval, and it is the gene itself rather than the IG-DMR and Gtl2-DMR that causes the aforementioned phenotypes. In conclusion, these findings illuminate a novel mechanism by which the silencing of maternally expressed RNAs within Dlk1-Dio3 domain leads to hepatic hemorrhage and embryonic death through the activation of apoptosis.

Tetramerization-dependent activation of the Sir2-associated short prokaryotic Argonaute immune system

Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids or bacteriophages. The Sir2-domain associated short pAgo (SPARSA) immune system defends against invaders by depleting NAD+ and triggering cell death. However, the molecular mechanism underlying SPARSA activation remains unknown. Here, we present cryo-EM structures of inactive monomeric, active tetrameric and active NAD+-bound tetrameric SPARSA complexes, elucidating mechanisms underlying SPARSA assembly, guide RNA preference, target ssDNA-triggered SPARSA tetramerization, and tetrameric-dependent NADase activation. Short pAgos form heterodimers with Sir2-APAZ, favoring short guide RNA with a 5′-AU from ColE-like plasmids. RNA-guided recognition of the target ssDNA triggers SPARSA tetramerization via pAgo- and Sir2-mediated interactions. The resulting tetrameric Sir2 rearrangement aligns catalytic residue H186 for NAD+ hydrolysis. These insights advance our understanding of Sir2-domain associated pAgos immune systems and should facilitate the development of a short pAgo-associated biotechnological toolbox.

Computational Analysis Suggests That AsnGTT 3′-tRNA-Derived Fragments Are Potential Biomarkers in Papillary Thyroid Carcinoma

Transfer-RNA-derived fragments (tRFs) are a novel class of small non-coding RNAs that have been implicated in oncogenesis. tRFs may act as post-transcriptional regulators by recruiting AGO proteins and binding to highly complementary regions of mRNA at seed regions, resulting in the knockdown of the transcript. Therefore, tRFs may be critical to tumorigenesis and warrant investigation as potential biomarkers. Meanwhile, the incidence of papillary thyroid carcinoma (PTC) has increased in recent decades and current diagnostic technology stands to benefit from new detection methods. Although small non-coding RNAs have been studied for their role in oncogenesis, there is currently no standard for their use as PTC biomarkers, and tRFs are especially underexplored. Accordingly, we aim to identify dysregulated tRFs in PTC that may serve as biomarker candidates. We identified dysregulated tRFs and driver genes between PTC primary tumor samples (n = 511) and adjacent normal tissue samples (n = 59). Expression data were obtained from MINTbase v2.0 and The Cancer Genome Atlas. Dysregulated tRFs and genes were analyzed in tandem to find pairs with anticorrelated expression. Significantly anticorrelated tRF-gene pairs were then tested for potential binding affinity using RNA22—if a heteroduplex can form via complementary binding, this would support the hypothesized RNA silencing mechanism. Four tRFs were significantly dysregulated in PTC tissue (p < 0.05), with only AsnGTT 3′-tRF being upregulated. Binding affinity analysis revealed that tRF-30-RY73W0K5KKOV (AsnGTT 3′-tRF) exhibits sufficient complementarity to potentially bind to and regulate transcripts of SLC26A4, SLC5A8, DIO2, and TPO, which were all found to be downregulated in PTC tissue. In the present study, we identified dysregulated tRFs in PTC and found that AsnGTT 3′-tRF is a potential post-transcriptional regulator and biomarker.

Protein P5 of pear chlorotic leaf spot-associated virus is a pathogenic factor that suppresses RNA silencing and enhances virus movement.

Pear chlorotic leaf spot-associated virus (PCLSaV) is a newly described emaravirus that infects pear trees. The virus genome consists of at least five single-stranded, negative-sense RNAs. The P5 encoded by RNA5 is unique to PCLSaV. In this study, the RNA silencing suppression (RSS) activity of P5 and its subcellular localization were determined in Nicotiana benthamiana plants by Agrobacterium tumefaciens-mediated expression assays and green fluorescent protein RNA silencing induction. Protein P5 partially suppressed local RNA silencing, strongly suppressed systemic RNA silencing and triggered reactive oxygen species accumulation. The P5 self-interacted and showed subcellular locations in plasmodesmata, endoplasmic reticulum and nucleus. Furthermore, P5 rescued the cell-to-cell movement of a movement defective mutant PVXΔP25 of potato virus X (PVX) and enhanced the pathogenicity of PVX. The N-terminal 1-89 amino acids of the P5 were responsible for the self-interaction ability and RSS activity, for which the signal peptide at positions 1-19 was indispensable. This study demonstrated the function of an emaravirus protein as a pathogenic factor suppressing plant RNA silencing to enhance virus infection and as an enhancer of virus movement.

RNAi, a sword of plant seeds to combat viral infections

Vertical transmission of plant viruses through seeds has been known for a century, yet the mechanism for seeds to combat viral infection remains unclear. In this issue of Cell Host & Microbe, Liu and Ding demonstrate the genetic requirement of RNA silencing (RNAi) pathway for plants to suppress seed transmission.