Diverse Regulations of Viral and Host Genes in Tomato Germplasms Responding to Tomato Yellow Leaf Curl Virus Inoculation

Tomato yellow leaf curl virus (TYLCV) is the dominating pathogen of tomato yellow leaf curl disease that caused severe loss to tomato production in China. In this study, we found that a TYLCV-resistant tomato line drastically reduced the accumulation of viral complementary-sense strand mRNAs but just moderately inhibit that of viral DNA and virion-sense strand mRNAs. However, two other resistant lines did not have such virus inhibition pattern. Analysis of differential expressed genes showed that the potential host defense-relevant processes varied in different resistant tomatoes, as compared to the susceptible line, suggesting a diversity of tomato TYLCV-resistance mechanisms.

Self-assembling short immunostimulatory duplex RNAs with broad spectrum antiviral activity

The current COVID-19 pandemic highlights the need for broad-spectrum antiviral therapeutics. Here we describe a new class of self-assembling immunostimulatory short duplex RNAs that potently induce production of type I and type III interferon (IFN-I and IFN-III), in a wide range of human cell types. These RNAs require a minimum of 20 base pairs, lack any sequence or structural characteristics of known immunostimulatory RNAs, and instead require a unique conserved sequence motif (sense strand: 5'-C, antisense strand: 3'-GGG) that mediates end-to-end dimer self-assembly of these RNAs by Hoogsteen G-G base-pairing. The presence of terminal hydroxyl or monophosphate groups, blunt or overhanging ends, or terminal RNA or DNA bases did not affect their ability to induce IFN. Unlike previously described immunostimulatory siRNAs, their activity is independent of TLR7/8, but requires the RIG-I/IRF3 pathway that induces a more restricted antiviral response with a lower proinflammatory signature compared with poly(I:C). Immune stimulation mediated by these duplex RNAs results in broad spectrum inhibition of infections by many respiratory viruses with pandemic potential, including SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza A, as well as the common cold virus HCoV-NL63 in both cell lines and human Lung Chips that mimic organ-level lung pathophysiology. These short dsRNAs can be manufactured easily, and thus potentially could be harnessed to produce broad-spectrum antiviral therapeutics at low cost.

Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing

In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using ‘click’ chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5′-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

Beyond identity: Understanding the contribution of the 5’ nucleotide of the antisense strand to RNAi activity

In both the pharmaceutical and agricultural fields, RNA-based products have capitalized upon the mechanism of RNA interference for targeted reduction of gene expression to improve phenotypes and traits. Reduction in gene expression by RNAi is the result of a small interfering RNA (siRNA) molecule binding to an ARGONAUTE (AGO) protein and directing the effector complex to a homologous region of a target gene’s mRNA. siRNAs properties that govern RNA-AGO association have been studied in detail. The siRNA 5’ nucleotide (nt) identity has been demonstrated in plants to be an important property responsible for directing association of endogenous small RNAs with different AGO effector proteins. However, it has not been investigated whether the 5’ nt identity is an efficacious determinant for topically-applied chemically synthesized siRNAs. In this study, we employed a sandpaper abrasion method to study the silencing efficacies of topically-applied 21 base-pair siRNA duplexes. The MAGNESIUM CHELATASE and GREEN FLUORESCENT PROTEIN genes were selected as endogenous and transgenic gene targets, respectively, to assess the molecular and phenotypic effects of gene silencing. Collections of siRNA variants with different 5’ nt identities and different pairing states between the 5’ antisense nt and its match in the sense strand of the siRNA duplex were tested for their silencing efficacy. Our results suggest a flexibility in the 5’ nt requirement for topically applied siRNA duplexes in planta and highlight the similarity of 5’ thermodynamic rules governing topical siRNA efficacy across plants and animals.

Analysis of gene expression and mutation data points on contribution of transcription to the mutagenesis by APOBEC enzymes

Since the discovery of the role of the APOBEC enzymes in human cancers, the mechanisms of this type of mutagenesis remain little understood. Theoretically, targeting of single-stranded DNA by the APOBEC enzymes could occur during cellular processes leading to the unwinding of DNA double-stranded structure. Some evidence points to the importance of replication in the APOBEC mutagenesis, while the role of transcription is still underexplored. Here, we analyzed gene expression and whole genome sequencing data from five types of human cancers with substantial APOBEC activity to estimate the involvement of transcription in the APOBEC mutagenesis and compare its impact with that of replication. Using the TCN motif as the mutation signature of the APOBEC enzymes, we observed a correlation of active APOBEC mutagenesis with gene expression, confirmed the increase of APOBEC-induced mutations in early-replicating regions and estimated the relative impact of transcription and replication on the APOBEC mutagenesis. We also found that the known effect of higher density of APOBEC-induced mutations on the lagging strand was highest in middle-replicating regions and observed higher APOBEC mutation density on the sense strand, the latter bias positively correlated with the gene expression level.

An Antisense Circular RNA Regulates Expression of RuBisCO Small Subunit Genes in Arabidopsis

Circular RNA (circRNA) is a novel class of endogenous long non-coding RNA (lncRNA) and participates in diverse physiological process in plants. From the dataset obtained by high-throughput RNA sequencing, we identified a circRNA encoded by the sense strand of the exon regions spanning two RuBisCO small subunit genes, RBCS2B and RBCS3B, in Arabidopsis thaliana. We further applied the single specific primer-polymerase chain reaction (PCR) and Sanger sequencing techniques to verify this circRNA and named it ag-circRBCS (antisense and across genic-circular RNA RBCS). Using quantitative real-time PCR (qRT-PCR), we found that ag-circRBCS shares a similar rhythmic expression pattern with other RBCS genes. The expression level of ag-circRBCS is 10–40 times lower than the expression levels of RBCS genes in the photosynthetic organs in Arabidopsis, whereas the Arabidopsis root lacked ag-circRBCS expression. Furthermore, we used the delaminated layered double hydroxide lactate nanosheets (LDH-lactate-NS) to deliver in vitro synthesized ag-circRBCS into Arabidopsis seedlings. Our results indicate that ag-circRBCS could significantly depress the expression of RBCS. Given that ag-circRBCS was expressed at low concentration in vivo, we suggest that ag-circRBCS may represent a fine-tuning mechanism to regulating the expression of RBCS genes and protein content in Arabidopsis.

Antisense Oligonucleotides as a Novel Therapy for Cushing’s Disease

Background: Cushing’s disease (CD) is caused by high levels of blood cortisol resulting from excess secretion of ACTH from a corticotroph adenoma in the anterior pituitary gland. Clinical features include hypertension, diabetes, osteoporosis, and depression. If untreated CD has an increased mortality of five-fold owing to cardiovascular comorbidities, stroke or raised vulnerability to infection. Transsphenoidal surgery is considered the first-line treatment but remission is achieved in only 65% of cases and the relapse rate is high. Furthermore, medical treatments are often accompanied by unpleasant side-effects. Antisense therapy is a technique for suppressing gene expression at the level of translation using antisense oligonucleotides (ASOs) against the mRNA of interest. Aims: To investigate antisense therapy as a treatment for CD by targeting ASOs against ACTH-encoding POMC mRNA thereby reducing secretion of the hormone. To transfect mouse AtT20 cells (cells that secrete high levels of ACTH) with ASOs against POMC at varying doses to determine which is the most effective at reducing ACTH secretion. Methods: AtT-20 cells that secrete high levels of ACTH were used as the model system. ASOs were designed to specifically target exon 3 of the POMC gene. Transfection of AtT-20 was carried out using Lipofectamine. FACS was used to determine transfection efficiency. ACTH levels secreted by AtT-20 cells were determined by immunoassay. Statistical analysis was done using ANOVA with P values < 0.05 considered significant. Results: ASOs that targeted POMC exon 3 (ASO-2 and ASO-3) were transfected into AtT-20 cells at 10 and 100 nM. Control ASOs were ASO-1 (matched to POMC sense strand) and ASO-4 (a scrambled version of ASO-3). Experiments included untreated AtT-20 cells and AtT-20 cells treated with transfection reagent or ASOs alone. The results of six experiments indicated that ACTH secretion from AtT-20 cells was reduced after transfection with ASO-2 and ASO-3 at 100 nM (ANOVA, P = < 0.05) and 10 nM (ANOVA, P < 0.05) when compared with untreated AtT20 cells. ASO-1 and ASO-4 had no effect on ACTH secretion by AtT-20 cells (ANOVA, P > 0.05). Conclusions: Initial experiments have shown that ASOs against POMC can reduce ACTH secretion from AtT-20 cells and may be useful as a novel therapy for CD.

Secondary structural choice of DNA and RNA associated with CGG/CCG trinucleotide repeat expansion rationalizes the RNA misprocessing in FXTAS

CGG tandem repeat expansion in the 5′-untranslated region of the fragile X mental retardation-1 (FMR1) gene leads to unusual nucleic acid conformations, hence causing genetic instabilities. We show that the number of G…G (in CGG repeat) or C…C (in CCG repeat) mismatches (other than A…T, T…A, C…G and G…C canonical base pairs) dictates the secondary structural choice of the sense and antisense strands of the FMR1 gene and their corresponding transcripts in fragile X-associated tremor/ataxia syndrome (FXTAS). The circular dichroism (CD) spectra and electrophoretic mobility shift assay (EMSA) reveal that CGG DNA (sense strand of the FMR1 gene) and its transcript favor a quadruplex structure. CD, EMSA and molecular dynamics (MD) simulations also show that more than four C…C mismatches cannot be accommodated in the RNA duplex consisting of the CCG repeat (antisense transcript); instead, it favors an i-motif conformational intermediate. Such a preference for unusual secondary structures provides a convincing justification for the RNA foci formation due to the sequestration of RNA-binding proteins to the bidirectional transcripts and the repeat-associated non-AUG translation that are observed in FXTAS. The results presented here also suggest that small molecule modulators that can destabilize FMR1 CGG DNA and RNA quadruplex structures could be promising candidates for treating FXTAS.

Borf: Improved ORF prediction in de-novo assembled transcriptome annotation

ABSTRACTORF prediction in de-novo assembled transcriptomes is a critical step for RNA-Seq analysis and transcriptome annotation. However, current approaches do not appropriately account for factors such as strand-specificity and incompletely assembled transcripts. Strand-specific RNA-Seq libraries should produce assembled transcripts in the correct orientation, and therefore ORFs should only be annotated on the sense strand. Additionally, start site selection is more complex than appreciated as sequences upstream of the first start codon need to be correctly annotated as 5’ UTR in completely assembled transcripts, or part of the main ORF in incomplete transcripts. Both of these factors influence the accurate annotation of ORFs and therefore the transcriptome as a whole. We generated four de-novo transcriptome assemblies of well annotated species as a gold-standard dataset to test the impact strand specificity and start site selection have on ORF prediction in real data. Our results show that prediction of ORFs on the antisense strand in data from stranded RNA libraries results in false-positive ORFs with no or very low similarity to known proteins. In addition, we found that up to 23% of assembled transcripts had no stop codon upstream and in-frame of the first start codon, instead comprising a sequence of upstream codons. We found the optimal length cutoff of these upstream sequences to accurately classify these transcripts as either complete (upstream sequence is 5’ UTR) or 5’ incomplete (transcript is incompletely assembled and upstream sequence is part of the ORF). Here, we present Borf, the better ORF finder, specifically designed to minimise false-positive ORF prediction in stranded RNA-Seq data and improve annotation of ORF start-site prediction accuracy. Borf is written in Python3 and freely available at https://github.com/betsig/borf.

Analysis of gene expression and mutation data points on contribution of transcription to the mutagenesis by APOBEC enzymes

Since the discovery of the role of the APOBEC enzymes in human cancers, the mechanisms of this type of mutagenesis remain little understood. Theoretically, targeting of single-stranded DNA by the APOBEC enzymes could occur during cellular processes leading to the unwinding of DNA double-stranded structure. Some evidence points to the importance of replication in the APOBEC mutagenesis, while the role of transcription is still underexplored. Here, we analyzed gene expression and whole genome sequencing data from five types of human cancers with substantial APOBEC activity to estimate the involvement of transcription in the APOBEC mutagenesis and compare its impact with that of replication. Using the TCN motif as the mutation signature of the APOBEC enzymes, we observed a correlation of active APOBEC mutagenesis with gene expression, confirmed the increase of APOBEC-induced mutations in early-replicating regions, and estimated the relative impact of transcription and replication on the APOBEC mutagenesis, which turned out to be approximately equal in transcribed regions. We also found that the known effect of higher density of APOBEC-induced mutations on the lagging strand was highest in middle-replicating regions, and observed higher APOBEC mutation density on the sense strand, the latter bias positively correlated with the gene expression level.