Antisense Strand Research Articles

Reduction of antisense transcription affects bovine leukemia virus replication and oncogenesis.

In sheep infected with bovine leukemia virus (BLV), transcription of structural, enzymatic, and accessory genes is silenced. However, the BLV provirus transcribes a series of non-coding RNAs that remain undetected by the host immune response. Specifically, three RNAs (AS1-L, AS1-S, and AS2) are consistently expressed from the antisense strand, originating from transcriptional initiation at the 3'-Long Terminal Repeat (LTR). To investigate the role of these non-coding RNAs in viral replication and pathogenesis, a reverse genetics approach was devised, capitalizing on a mechanistic disparity in transcription initiation between the 5' and 3' promoters. A two-nucleotide mutation (GG>TA) in the TFIIB-recognition element (BRE) impaired antisense transcription originating from the 3'-LTR. In the context of the provirus, this 2bp mutation significantly diminished the expression of antisense RNAs, while not notably affecting sense transcription. When inoculated to sheep, the mutated provirus was infectious but exhibited reduced replication levels, shedding light on the role of antisense transcription in vivo. In comparison to lymphoid organs in sheep infected with a wild-type (WT) provirus, the mutant demonstrated alterations in both the spatial distribution and rates of cell proliferation in the lymph nodes and the spleen. Analysis through RNA sequencing and RT-qPCR unveiled an upregulation of the Hmcn1/hemicentin-1 gene in B-lymphocytes from sheep infected with the mutated provirus. Further examination via confocal microscopy and immunohistochemistry revealed an increase in the amount of hemicentin-1 protein encoded by Hmcn1 in peripheral blood mononuclear cells (PBMCs) and lymphoid organs of sheep infected with the mutant. RNA interference targeting Hmcn1 expression impacted the migration of ovine kidney (OVK) cells in vitro. In contrast to the WT, the mutated provirus showed reduced oncogenicity when inoculated into sheep. Collectively, this study underscores the essential role of antisense transcription in BLV replication and pathogenicity. These findings may offer valuable insights into understanding the relevance of antisense transcription in the context of human T-cell leukemia virus (HTLV-1).

Enhancing Target Detection: A Fluorescence-Based Streptavidin-Bead Displacement Assay.

Fluorescence-based aptasensors have been regarded as innovative analytical tools for the detection and quantification of analytes in many fields, including medicine and therapeutics. Using DNA aptamers as the biosensor recognition component, conventional molecular beacon aptasensor designs utilise target-induced structural switches of the DNA aptamers to generate a measurable fluorescent signal. However, not all DNA aptamers undergo sufficient target-specific conformational changes for significant fluorescence measurements. Here, the use of complementary 'antisense' strands is proposed to enable fluorescence measurement through strand displacement upon target binding. Using a published target-specific DNA aptamer against the receptor binding domain of SARS-CoV-2, we designed a streptavidin-aptamer bead complex as a fluorescence displacement assay for target detection. The developed assay demonstrates a linear range from 50 to 800 nanomolar (nM) with a limit of detection calculated at 67.5 nM and a limit of quantification calculated at 204.5 nM. This provides a 'fit-for-purpose' model assay for the detection and quantification of any target of interest by adapting and functionalising a suitable target-specific DNA aptamer and its complementary antisense strand.

SARS-CoV-2 replicates in the placenta after maternal infection during pregnancy.

Pregnant women are at increased risk for severe SARS-CoV-2 infection and adverse neonatal outcome, primarily preterm birth and stillbirth. Our study aimed to investigate to which extent SARS-CoV-2 affects placental tissue and if viral replication within the placenta is evident, thus if there is a correlation between placental damage and adverse pregnancy outcome such as stillbirth. We prospectively collected placentas from 61 SARS-CoV-2 infected pregnant women and 10 controls. Histopathological, immunohistochemical, and in situ hybridization studies were performed on all placentas with antibodies for SARS-CoV-2 proteins, ACE2, various immune cells, and inflammatory markers or probes for SARS-CoV-2 genes and an antisense strand. The measured scores of SARS-CoV-2 glycoprotein, nucleocapsid, and antisense strand indicating replication correlated with both the severity of maternal symptoms and presence of stillbirth. Specifically, 15/61 placentas exhibited replication, while the three cases with stillbirth had high or maximal replication scores. ACE2-H-score was significantly higher in COVID-19 patients, while the expression of various immune cells did not differ statistically. In multivariate analysis, presence of maternal comorbidities correlated with presence of severe COVID-19 infection. We report evidence of active in vivo SARS-CoV-2 replication in the placenta after maternal infection in pregnancy in a case-control setting in a large population. Intensity of placental viral replication as well as viral levels were higher in women with severe or critical COVID-19 disease, supporting the rationale that severity of maternal SARS-CoV-2 infection could correlate with the severity of placentitis. Replication was maximal in cases of stillbirth, which suggests direct placental involvement in the pathophysiology of this dramatic outcome. Continuing to advocate for preventive measures against COVID-19 during pregnancy, including (re)vaccination, as well as appropriately counseling women with diagnosed infection, are of utter importance.

Nonclinical Pharmacokinetics Study of OLX702A-075-16, N-Acetylgalactosamine Conjugated Asymmetric Small Interfering RNA (GalNAc-asiRNA).

In this study, the nonclinical pharmacokinetics of OLX702A-075-16, an RNA interference therapeutic currently in development, were investigated. OLX702A-075-16 is a novel N-acetylgalactosamine conjugated asymmetric small-interfering RNA (GalNAc-asiRNA) used for the treatment of an undisclosed liver disease. Its unique 16/21-mer asymmetric structure reduces nonspecific off-target effects without compromising efficacy. We investigated the plasma concentration, tissue distribution, metabolism, and renal excretion of OLX702A-075-16 following a subcutaneous administration in mice and rats. For bioanalysis, high-performance liquid chromatography with fluorescence detection was used. The results showed rapid clearance from plasma (0.5 to 1.5 hours of half-life) and predominant distribution to the liver and/or kidney. Less than 1% of the liver concentration of OLX702A-075-16 was detected in the other tissues. Metabolite profiling using liquid chromatography coupled with high-resolution mass spectrometry revealed that the intact duplex OLX702A-075-16 was the major compound in plasma. The GalNAc moiety was predominantly metabolized from the sense strand in the liver, with the unconjugated sense strand of OLX702A-075-16 accounting for more than 95% of the total exposure in the rat liver. Meanwhile, the antisense strand was metabolized by the sequential loss of nucleotides from the 3'-terminus by exonuclease, with the rat liver samples yielding the most diverse truncated forms of metabolites. Urinary excretion over 96 hours was less than 1% of the administered dose in rats. High plasma protein binding of OLX702A-075-16 likely inhibited its clearance through renal filtration. SIGNIFICANCE STATEMENT: This study presents the first comprehensive characterization of the in vivo pharmacokinetics of GalNAc-asiRNA. The pharmacokinetic insights gained from this research will aid in understanding toxicology and efficacy, optimizing delivery platforms, and improving the predictive power of preclinical species data for human applications.

The primate-specific presence of interferon regulatory factor-5 pseudogene 1.

Interferon regulatory factor 5 (IRF5) is a key transcription factor in inflammatory and immune responses, with its dysregulation linked to autoimmune diseases. Using bioinformatic approaches, including Basic Local Alignment Search Tool (BLAST) for sequence similarity searches, BLAST-Like Alignment Tool (BLAT) for genome-wide alignments, and several phylogenetics software, such as Multiple Alignment using Fast Fourier Transform (MAFFT), for phylogenetic analyses, we characterized the structure, origin, and evolutionary history of the human IRF5 pseudogene 1 (IRF5P1). Our analyses reveal that IRF5P1 is a chimeric processed pseudogene containing sequences derived from multiple sources, including IRF5-like sequences from disparate organisms. We find that IRF5P1 is specific to higher primates, likely originating through an ancient retroviral integration event approximately 60 million years ago. Interestingly, IRF5P1 resides within the triple QxxK/R motif-containing (TRIQK) gene, and its antisense strand is predominantly expressed as part of the TRIQK pre-messenger RNA (mRNA). Analysis of publicly available RNA-seq data suggests potential expression of antisense IRF5P1 RNA. We hypothesize that this antisense RNA may regulate IRF5 expression through complementary binding to IRF5 mRNA, with human genetic variants potentially modulating this interaction. The conservation of IRF5P1 in the primate lineage suggests its positive effects on primate evolution and innate immunity. This study highlights the importance of investigating pseudogenes and their potential regulatory roles in shaping lineage-specific immune adaptations.

Therapeutic siRNA Loaded to RISC as Single and Double Strands Requires an Appropriate Quantitative Assay for RISC PK Assessment.

In recent years, therapeutic siRNA projects are booming in the biotech and pharmaceutical industries. As these drugs act by silencing the target gene expression, a critical step is the binding of antisense strands of siRNA to RNA-induced silencing complex (RISC) and then degrading their target mRNA. However, data that we recently obtained suggest that double-stranded siRNA can also load to RISC. This brings a new understanding of the mechanism of RISC loading which may have a potential impact on how quantification of RISC loaded siRNA should be performed. By combining RNA immune precipitation and probe-based hybridization LC-fluorescence approach, we have developed a novel assay that can accurately quantify the RISC-bound antisense strand, irrespective of which form (double-stranded or single-stranded) is loaded on RISC. In addition, this novel assay can discriminate between the 5'-phosphorylated antisense (5'p-AS) and the nonphosphorylated forms, therefore specifically quantifying the RISC bound 5'p-AS. In comparison, stem-loop qPCR assay does not provide discrimination and accurate quantification when the oligonucleotide analyte exists as a mixture of double and single-stranded forms. Taking together, RISC loading assay with probe-hybridization LC-fluorescence technique would be a more accurate and specific quantitative approach for RISC-associated pharmacokinetic assessment.

Separation and Characterization of Therapeutic Oligonucleotide Isomer Impurities by Cyclic Ion Mobility Mass Spectrometry.

Therapeutic oligonucleotides such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA) are among the most remarkable modalities in modern medicine. ASOs and siRNA are composed of single- or double-stranded 15-25 mer synthesized oligonucleotides, which can be used to modulate gene expression. Liquid chromatography-mass spectrometry (LC/MS) is a necessary technique for the quality control of therapeutic oligonucleotides; it is used to evaluate the quantities of target oligonucleotides and their impurities. The widely applied oligonucleotide therapeutic quantitation method uses both ultraviolet (UV) absorbance and the MS signal intensity. Peaks separated from the main peak, which contains full-length product, are generally quantitated by UV. However, coeluting impurities, such as n - 1 shortmers, abasic oligonucleotides, and PS → PO (phosphorothiate to phosphodiester) oligonucleotides, are quantitated by MS. These coeluting impurities can also be comprised of various isomers with the same modification, thus increasing the difficulty in their separation and relative quantitation by LC/MS. It is possible that a specific isomer with a certain structural form induces toxicities. Therefore, characterization of each isomer separation is in high demand. In this study, we separated and characterized oligonucleotide isomers by employing a cyclic ion mobility mass spectrometry (cyclic IMS) system, which allows the separation of ions with the same m/z ratio based on their structural differences. Patisiran antisense and sense strands and their n - 1 and abasic isomers were used as sample sequences, and their ratio characterization was achieved by cyclic IMS. In addition, we evaluated the PS → PO conversion isomers of the antisense strand of givosiran, which originally contained four PS modification sites. The PS → PO isomers exhibited specific and distinguishable mobiligram patterns. We believe that cyclic IMS is a promising method for evaluating therapeutic oligonucleotide isomers.

Endogenous Bornavirus-like Elements in Bats: Evolutionary Insights from the Conserved Riboviral L-Gene in Microbats and Its Antisense Transcription in Myotis daubentonii.

Bats are ecologically diverse vertebrates characterized by their ability to host a wide range of viruses without apparent illness and the presence of numerous endogenous viral elements (EVEs). EVEs are well preserved, expressed, and may affect host biology and immunity, but their role in bat immune system evolution remains unclear. Among EVEs, endogenous bornavirus-like elements (EBLs) are bornavirus sequences integrated into animal genomes. Here, we identified a novel EBL in the microbat Myotis daubentonii, EBLL-Cultervirus.10-MyoDau (short name is CV.10-MyoDau) that shows protein-level conservation with the L-protein of a Cultervirus (Wuhan sharpbelly bornavirus). Surprisingly, we discovered a transcript on the antisense strand comprising three exons, which we named AMCR-MyoDau. The active transcription in Myotis daubentonii tissues of AMCR-MyoDau, confirmed by RNA-Seq analysis and RT-PCR, highlights its potential role during viral infections. Using comparative genomics comprising 63 bat genomes, we demonstrate nucleotide-level conservation of CV.10-MyoDau and AMCR-MyoDau across various bat species and its detection in 22 Yangochiropera and 12 Yinpterochiroptera species. To the best of our knowledge, this marks the first occurrence of a conserved EVE shared among diverse bat species, which is accompanied by a conserved antisense transcript. This highlights the need for future research to explore the role of EVEs in shaping the evolution of bat immunity.

CRISPR/Cas12a Collateral Cleavage-Driven Transcription Amplification for Direct Nucleic Acid Detection.

The clustered regularly interspaced short palindromic repeat/Cas (CRISPR/Cas) system is a powerful tool for nucleic acid detection owing to specific recognition as well as cis- and trans-cleavage capabilities. However, the sensitivity of CRISPR/Cas-based diagnostic approaches is determined by nucleic acid preamplification, which has several limitations. Here, we present a method for direct nucleic acid detection without preamplification, by combining the CRISPR/Cas12a system with signal enhancement based on light-up RNA aptamer transcription. We first designed two DNA templates to transcribe the light-up RNA aptamer and kleptamer (Kb) RNA: the first DNA template encodes a Broccoli RNA aptamer for fluorescence signal generation, and the Kb DNA template comprises a dsDNA T7 promoter sequence and an ssDNA sequence that encodes an antisense strand for the Broccoli RNA aptamer. Hepatitis B virus (HBV) target recognition activates a CRISPR/Cas12a complex, leading to the catalytic cleavage of the ssDNA sequence. Transcription of the added Broccoli DNA template can then produce several Broccoli RNA aptamer transcripts for fluorescence enhancement. The proposed strategy exhibited excellent sensitivity and specificity with 22.4 fM detection limit, good accuracy, and stability for determining the target HBV dsDNA in human serum samples. Overall, this newly designed signal enhancement strategy can be employed as a universal sensing platform for ultrasensitive nucleic acid detection.

2'-O-Alkyl-N 3-Methyluridine Functionalized Passenger Strand Improves RNAi Activity by Modulating the Thermal Stability.

Herein, we have demonstrated that the siRNA activity could be enhanced by incorporating the guide strand in the RISC complex through thermodynamic asymmetry caused by m3U-based destabilizing modifications. A nuclease stability study revealed that 2'-OMe-m3U and 2'-OEt-m3U modifications slightly improved the half-lives of siRNA strands in human serum. In the in vitro gene silencing assay, 2'-OMe-m3U modification at the 3'-overhang and cleavage site of the passenger strand in anti-renilla and anti-Bcl-2 siRNA duplexes were well-tolerated and exhibited improved gene silencing activity. However, gene silencing activity was attenuated when these modifications were incorporated at position 3 in the seed region of the antisense strand. The molecular modeling studies using these modifications at the seed region with the MID domain of hAGO2 explained that the 2'-alkoxy group makes steric interactions with the amino acid residues of the hAGO2 protein.

Asymmetric bulges within hairpin RNA transgenes influence small RNA size, secondary siRNA production and viral defence.

Small RNAs (sRNAs) are essential for normal plant development and range in size classes of 21-24 nucleotides. The 22nt small interfering RNAs (siRNAs) and miRNAs are processed by Dicer-like 2 (DCL2) and DCL1 respectively and can initiate secondary siRNA production from the target transcript. 22nt siRNAs are under-represented due to competition between DCL2 and DCL4, while only a small number of 22nt miRNAs exist. Here we produce abundant 22nt siRNAs and other siRNA size classes using long hairpin RNA (hpRNA) transgenes. By introducing asymmetric bulges into the antisense strand of hpRNA, we shifted the dominant siRNA size class from 21nt of the traditional hpRNA to 22, 23 and 24nt of the asymmetric hpRNAs. The asymmetric hpRNAs effectively silenced a β-glucuronidase (GUS) reporter transgene and the endogenous ethylene insensitive-2 (EIN2) and chalcone synthase (CHS) genes. Furthermore, plants containing the asymmetric hpRNA transgenes showed increased amounts of 21nt siRNAs downstream of the hpRNA target site compared to plants with the traditional hpRNA transgenes. This indicates that these asymmetric hpRNAs are more effective at inducing secondary siRNA production to amplify silencing signals. The 22nt asymmetric hpRNA constructs enhanced virus resistance in plants compared to the traditional hpRNA constructs.

Strand displacement-triggered FRET nanoprobe tracking TK1 mRNA in living cells for ratiometric fluorimetry of nucleic acid biomarker.

Aprecisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

Ago4-piRNA complex is a key component of genomic immune system against transposon expression in Penaeus monodon

Argonaute proteins are key constituents of small RNA-guided regulatory pathways. In crustaceans, members of the AGO subfamily of Argonaute proteins that play vital roles in immune defense are well studied, while proteins of the PIWI subfamily are less established. PmAgo4 of the black tiger shrimp, Penaeus monodon, though phylogenetically clustered with the AGO subfamily, has distinctive roles of the PIWI subfamily in safeguarding the genome from transposon invasion and controlling germ cell development. This study explored a molecular mechanism by which PmAgo4 regulates transposon expression in the shrimp germline. PmAgo4-associated small RNAs were co-immunoprecipitated from shrimp testis lysate using a PmAgo4-specific polyclonal antibody. RNA-seq revealed a majority of 26–27 nt long small RNAs in the PmAgo4-IP fraction suggesting that PmAgo4 is predominantly associated with piRNAs. Mapping of these piRNAs on nucleotide sequences of two gypsy and a mariner-like transposons of P. monodon suggested that most piRNAs were originated from the antisense strand of transposons. Suppression of PmAgo4 expression by a specific dsRNA elevated the expression levels of the three transposons while decreasing the levels of transposon-related piRNAs. Taken together, these results imply that PmAgo4 exerts its suppressive function on transposons by controlling the biogenesis of transposon-related piRNAs and thus, provides a defense mechanism against transposon invasion in shrimp germline cells.

Increased Brucella abortus asRNA_0067 expression under intraphagocytic stressors is associated with enhanced virB2 transcription

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.

Identification and Characterization of Three Novel Solemo-like Viruses in the White-Backed Planthopper, Sogatella furcifera.

Agricultural insects play a crucial role in transmitting plant viruses and host a considerable number of insect-specific viruses (ISVs). Among these insects, the white-backed planthoppers (WBPH; Sogatella furcifera, Hemiptera: Delphacidae) are noteworthy rice pests and are responsible for disseminating the southern rice black-streaked dwarf virus (SRBSDV), a significant rice virus. In this study, we analyzed WBPH transcriptome data from public sources and identified three novel viruses. These newly discovered viruses belong to the plant-associated viral family Solemoviridae and were tentatively named Sogatella furcifera solemo-like virus 1-3 (SFSolV1-3). Among them, SFSolV1 exhibited a prevalent existence in different laboratory populations, and its complete genome sequence was obtained using rapid amplification of cDNA ends (RACE) approaches. To investigate the antiviral RNA interference (RNAi) response in WBPH, we conducted an analysis of virus-derived small interfering RNAs (vsiRNAs). The vsiRNAs of SFSolV1 and -2 exhibited typical patterns associated with the host's siRNA-mediated antiviral immunity, with a preference for 21- and 22-nt vsiRNAs derived equally from both the sense and antisense genomic strands. Furthermore, we examined SFSolV1 infection and distribution in WBPH, revealing a significantly higher viral load of SFSolV1 in nymphs' hemolymph compared to other tissues. Additionally, in adult insects, SFSolV1 exhibited higher abundance in male adults than in female adults.

Molecular and gene expression analyses of chicken oncomodulin and their association with breast myopathies in broilers

Oncomodulins (OCMs), also known as non-α-parvalbumins, are small molecules known for their high-affinity binding of Ca2+ ions. They play crucial roles as Ca2+ buffers and participate in signaling pathways within muscle and neuron cells. In chickens, 3 oncomodulin molecules have been identified at the protein level and are named chicken oncomodulin 1 (OCM1), -3 (OCM3), and alpha-parvalbumin (PVALB). OCM4 was newly assigned by genome annotation. A gene cluster containing OCM1, OCM3, and OCM4 is located in chromosome 14, while a single gene of PVALB is on chromosome 1. The Ca2+ signaling pathway may be a potential contributor to the onset of chicken breast myopathies. However, chicken OCMs have not been extensively studied in muscle tissues. In this study, the genetic specifications, tissue-specific and differential expression of OCM1, OCM3, OCM4, and PVALB in the context of chicken breast myopathies were investigated. OCM1 exhibited moderate expression in the liver, intestine, and kidney. OCM3 was highly expressed in thymus and breast muscle. A long noncoding RNA (lncRNA) transcribed from the antisense strand of the OCM3 gene was found to be expressed in liver, lung, heart, intestine, and kidney tissues. OCM4 was barely expressed in thymus, thigh-, and breast muscle. PVALB exhibited high expression across all tissues examined. Results of quantitative PCR (qPCR) indicated that the expression of OCM3 was significantly increased (4.4 ± 0.7 fold; P-value = 0.03) in woody breast (WB) muscle and even greater (8.5 ± 0.6 fold; P-value = 0.004) in WB/white striping (WS) muscles. The expression of PVALB showed no difference in WB muscle, but it was notably higher (4.6 ± 0.7 fold; P-value = 0.054) in WB/WS muscle, although statistical significance was not reached. These findings suggest that increased expression of OCM3 and PVALB may be linked to chicken breast myopathies with regard to disruption of Ca2+ buffering.

The RNAi Machinery in the Fungus Fusarium fujikuroi Is Not Very Active in Synthetic Medium and Is Related to Transposable Elements.

Small RNAS (sRNAs) participate in regulatory RNA interference (RNAi) mechanisms in a wide range of eukaryotic organisms, including fungi. The fungus Fusarium fujikuroi, a model for the study of secondary metabolism, contains a complete set of genes for RNAi pathways. We have analyzed by high-throughput sequencing the content of sRNAs in total RNA samples of F. fujikuroi grown in synthetic medium in the dark or after 1 h of illumination, using libraries below 150 nt, covering sRNAs and their precursors. For comparison, a parallel analysis with Fusarium oxysporum was carried out. The sRNA reads showed a higher proportion of 5' uracil in the RNA samples of the expected sizes in both species, indicating the occurrence of genuine sRNAs, and putative miRNA-like sRNAs (milRNAS) were identified with prediction software. F. fujikuroi carries at least one transcriptionally expressed Ty1/copia-like retrotransposable element, in which sRNAs were found in both sense and antisense DNA strands, while in F. oxysporum skippy-like elements also show sRNA formation. The finding of sRNA in these mobile elements indicates an active sRNA-based RNAi pathway. Targeted deletion of dcl2, the only F. fujikuroi Dicer gene with significant expression under the conditions tested, did not produce appreciable phenotypic or transcriptomic alterations.

C9orf72-Associated Dipeptide Repeat Expansions Perturb ER-Golgi Vesicular Trafficking, Inducing Golgi Fragmentation and ER Stress, in ALS/FTD

AbstractHexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both are debilitating neurodegenerative conditions affecting either motor neurons (ALS) in the brain and spinal cord or neurons in the frontal and/or temporal cortical lobes (FTD). HREs undergo repeat-associated non-ATG (RAN) translation on both sense and anti-sense strands, generating five distinct dipeptide repeat proteins (DPRs), poly-GA, -GR, -GP, -PA and -PR. Perturbed proteostasis is well-recognised in ALS pathogenesis, including processes affecting the endoplasmic reticulum (ER) and Golgi compartments. However, these mechanisms have not been well characterised for C9orf72-mediated ALS/FTD. In this study we demonstrate that C9orf72 DPRs polyGA, polyGR and polyGP (× 40 repeats) disrupt secretory protein transport from the ER to the Golgi apparatus in neuronal cells. Consistent with this finding, these DPRs also induce fragmentation of the Golgi apparatus, activate ER stress, and inhibit the formation of the omegasome, the precursor of the autophagosome that originates from ER membranes. We also demonstrate Golgi fragmentation in cells undergoing RAN translation that express polyGP. Furthermore, dysregulated ER-Golgi transport was confirmed in C9orf72 patient dermal fibroblasts. Evidence of aberrant ER-derived vesicles in spinal cord motor neurons from C9orf72 ALS patients compared to controls was also obtained. These data thus confirm that ER proteostasis and ER-Golgi transport is perturbed in C9orf72-ALS in the absence of protein over-expression. Hence this study identifies novel molecular mechanisms associated with the ER and Golgi compartments induced by the C9orf72 HRE.

Therapeutic efficacy of a novel self-assembled immunostimulatory siRNA combining apoptosis promotion with RIG-I activation in gliomas

BackgroundCurrent cancer therapies often fall short in addressing the complexities of malignancies, underscoring the urgent need for innovative treatment strategies. RNA interference technology, which specifically suppresses gene expression, offers a promising new approach in the fight against tumors. Recent studies have identified a novel immunostimulatory small-interfering RNA (siRNA) with a unique sequence (sense strand, 5’-C; antisense strand, 3’-GGG) capable of activating the RIG-I/IRF3 signaling pathway. This activation induces the release of type I and III interferons, leading to an effective antiviral immune response. However, this class of immunostimulatory siRNA has not yet been explored in cancer therapy.MethodsIsiBCL-2, an innovative immunostimulatory siRNA designed to suppress the levels of B-cell lymphoma 2 (BCL-2), contains a distinctive motif (sense strand, 5’-C; antisense strand, 3’-GGG). Glioblastoma cells were subjected to 100 nM isiBCL-2 treatment in vitro for 48 h. Morphological changes, cell viability (CCK-8 assay), proliferation (colony formation assay), migration/invasion (scratch test and Transwell assay), apoptosis rate, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) were evaluated. Western blotting and immunofluorescence analyses were performed to assess RIG-I and MHC-I molecule levels, and ELISA was utilized to measure the levels of cytokines (IFN-β and CXCL10). In vivo heterogeneous tumor models were established, and the anti-tumor effect of isiBCL-2 was confirmed through intratumoral injection.ResultsIsiBCL-2 exhibited significant inhibitory effects on glioblastoma cell growth and induced apoptosis. BCL-2 mRNA levels were significantly decreased by 67.52%. IsiBCL-2 treatment resulted in an apoptotic rate of approximately 51.96%, accompanied by a 71.76% reduction in MMP and a 41.87% increase in ROS accumulation. Western blotting and immunofluorescence analyses demonstrated increased levels of RIG-I, MAVS, and MHC-I following isiBCL-2 treatment. ELISA tests indicated a significant increase in IFN-β and CXCL10 levels. In vivo studies using nude mice confirmed that isiBCL-2 effectively impeded the growth and progression of glioblastoma tumors.ConclusionsThis study introduces an innovative method to induce innate signaling by incorporating an immunostimulatory sequence (sense strand, 5’-C; antisense strand, 3’-GGG) into siRNA, resulting in the formation of RNA dimers through Hoogsteen base-pairing. This activation triggers the RIG-I signaling pathway in tumor cells, causing further damage and inducing a potent immune response. This inventive design and application of immunostimulatory siRNA offer a novel perspective on tumor immunotherapy, holding significant implications for the field.

Rational design of prodrug-type apoB-targeted siRNA for nuclease resistance improvement without compromising gene silencing potency

Synthetic siRNA molecules without chemical modifications are easily degraded in the body, and 2'-O-modifications are frequently introduced to enhance stability. However, such chemical modifications tend to impact the gene knockdown potency of siRNA negatively. To circumvent this problem, we previously developed a prodrug-type siRNA bearing 2'-O-methyldithiomethyl (MDTM) groups, which can be converted into unmodified siRNA under the reductive environment in cells. In this study, we developed a nuclease-resistant prodrug-type 2'-O-MDTM siRNA for deployment in future animal experiments. To rationally design siRNA modified with a minimal number of 2'-O-MDTM nucleotide residues, we identified the sites susceptible to nuclease digestion and tolerant to 2'-O-methyl (2'-OMe) modification in the antisense strand of apolipoprotein B-targeted siRNA. Subsequently, we optimized the positions where the 2'-OMe and 2'-O-MDTM groups should be incorporated. siRNA bearing the 2'-O-MDTM and 2'-OMe groups at their respective optimized positions exhibited efficient knockdown potency in vitro and enhanced stability in serum.