An atypical class of non-coding small RNAs is produced in rice leaves upon bacterial infection

Non-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant–microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20–22 nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant–microbe interactions.

Dysregulation of RNA interference components in COVID-19 patients

Objective Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus causing severe respiratory illness (COVID-19). This virus was initially identified in Wuhan city, a populated area of the Hubei province in China, and still remains one of the major global health challenges. RNA interference (RNAi) is a mechanism of post-transcriptional gene silencing that plays a crucial role in innate viral defense mechanisms by inhibiting the virus replication as well as expression of various viral proteins. Dicer, Drosha, Ago2, and DGCR8 are essential components of the RNAi system, which is supposed to be dysregulated in COVID-19 patients. This study aimed to assess the expression level of the mentioned mRNAs in COVID-19patients compared to healthy individuals. Results Our findings demonstrated that the expression of Dicer, Drosha, and Ago2 was statistically altered in COVID-19 patients compared to healthy subjects. Ultimately, the RNA interference mechanism as a crucial antiviral defense system was suggested to be dysregulated in COVID-19 patients.

Exogenous miRNAs induce post-transcriptional gene silencing in plants

Plants seem to take up exogenous RNA that was artificially designed to target specific genes, followed by activation of the RNA interference (RNAi) machinery. It is, however, not known whether plants use RNAs themselves as signalling molecules in plant-to-plant communication, other than evidence that an exchange of small RNAs occurs between parasitic plants and their hosts. Exogenous RNAs from the environment, if taken up by some living organisms, can indeed induce RNAi. This phenomenon has been observed in nematodes and insects, and host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver plant small RNAs into Botrytis cinerea. Here we show that micro-RNAs (miRNAs) produced by plants act as signalling molecules affecting gene expression in other, nearby plants. Exogenous miRNAs, such as miR156 and miR399, trigger RNAi via a mechanism requiring both AGO1 and RDR6. This emphasizes that the production of secondary small interfering RNAs is required. This evidence highlights the existence of a mechanism in which miRNAs represent signalling molecules that enable communication between plants.

small RNA can move long distances through plant vasculature to influence gene expression in shoot apical meristems

In plants, small RNA (sRNA) can regulate gene expression via post transcriptional gene silencing (PTGS) or through RNA-directed DNA methylation (RdDM) leading to transcriptional gene silencing (TGS). sRNA is mobile throughout the plant, with movement occurring short distances from cell-to-cell as well as long distances through the vasculature via phloem trafficking. The range of long-distance sRNA mediated signaling from the vasculature to the shoot apical meristem (SAM) is not clear. To investigate this, two independent transgenic approaches were used to examine trafficking of phloem-expressed sRNA to the SAM in Arabidopsis thaliana. First, the phloem companion-cell specific promoter SUC2 was used to drive expression of an inverted repeat complementary to FLOWERING LOCUS D (FD), a flowering time regulator expressed exclusively in the SAM. In a separate experiment, the SUC2 promoter was used to express an artificial microRNA (aMiR) designed to target a synthetic CLAVATA3 (CLV3) target in the SAM stem cells. Both systems provide evidence of a phloem-to-SAM sRNA communication axis connecting distal regions of the plant to the stem cells of the SAM, which ultimately gives rise to all shoot tissues, including gametes. Thus, phloem-to-SAM sRNA movement defines an important link between sRNA expressed in distal regions of the plant and the growing shoot. Importantly, phloem-to-SAM sRNA trafficking may allow somatic sRNA to direct SAM RdDM, fixing transient sRNA expression events into stable epigenetic changes.

Key Amino Acids for Pepper Vein Yellows Virus P0 Protein Pathogenicity, Gene Silencing, and Subcellular Localization

Pepper vein yellows virus (PeVYV) is a newly recognized Polerovirus extracted from Chinese pepper. The symptoms of PeVYV-infested pepper plants comprise intervein yellow staining, leaf curl formation and other malformations, and leaf internodal shrinkage, but the roles of the viral proteins remain undetermined. The P0 protein of the genus Polerovirus has established post-transcriptional gene silencing (PTGS) activity. This investigation focused on the PeVYV-encoded P0 protein and assessed its potential virulence capacity, PTGS activity, and tendencies to localize in the nucleus. This study revealed that P0 influenced the pathogenic properties of a specific heterologous potato virus X. In addition, P0 proteins impaired local gene silencing, although they did not regulate generalized gene silencing within Nicotiana benthamiana 16c plants. Furthermore, P0 proteins localized mainly in the nucleus, particularly in the nucleolus. P0 deletion mutagenesis demonstrated that the F-box motif (56–72 amino acids, AAs) of P0 was essential for symptom determination, inhibition of PTGS, and subcellular localization. Mutation analysis of the F-box motif of P0 protein indicated that AA 57 of the P0 protein was a pivotal site in symptom development and that AA 56 of the P0 protein was indispensable for inhibiting PTGS and subcellular localization. The outcomes obtained here suggest that further studies should be conducted on the molecular mechanisms of amino acids of the F-box domain of P0 protein in the interaction of PeVYV with plants.

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.

Plant Polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains

Heterochromatin is the predominant architectural feature of genomes that ensures genomic stability across eukaryotes. It mostly functions in restricting expression of repeats, mobile elements such as transposons and other regions. The establishment, maintenance and spreading of heterochromatin requires several factors including chromatin modifiers. However, how exactly heterochromatin formation is avoided in protein-coding domains is poorly understood. Here we show that a plant specific paralogue of RNA polymerase (pol) II, named pol IV, is involved in avoidance of facultative heterochromatic marks in protein coding genes, in addition to silencing the repeats and transposons forming constitutive heterochromatin. In its absence, H3K27 trimethylation mark intrudes the protein coding genes, more profoundly in genes embedded with repeats. In a subset of genes that lack the compensatory silencing, spurious transcriptional activity results in small(s)RNA production leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with larger genome with distributed heterochromatin when compared to Arabidopsis. Our results indicate the surprising division of labour among plant-specific polymerases, not just in establishing effective silencing via small RNAs and epigenetics, but also in influencing chromatin boundaries.

siRNA Mediate RNA Interference Concordant with Early On-Target Transient Transcriptional Interference

Exogenous siRNAs are commonly used to regulate endogenous gene expression levels for gene function analysis, genotype–phenotype association studies and for gene therapy. Exogenous siRNAs can target mRNAs within the cytosol as well as nascent RNA transcripts within the nucleus, thus complicating siRNA targeting specificity. To highlight challenges in achieving siRNA target specificity, we targeted an overlapping gene set that we found associated with a familial form of multiple synostosis syndrome type 4 (SYSN4). In the affected family, we found that a previously unknown non-coding gene TOSPEAK/C8orf37AS1 was disrupted and the adjacent gene GDF6 was downregulated. Moreover, a conserved long-range enhancer for GDF6 was found located within TOSPEAK which in turn overlapped another gene which we named SMALLTALK/C8orf37. In fibroblast cell lines, SMALLTALK is transcribed at much higher levels in the opposite (convergent) direction to TOSPEAK. siRNA targeting of SMALLTALK resulted in post transcriptional gene silencing (PTGS/RNAi) of SMALLTALK that peaked at 72 h together with a rapid early increase in the level of both TOSPEAK and GDF6 that peaked and waned after 24 h. These findings indicated the following sequence of events: Firstly, the siRNA designed to target SMALLTALK mRNA for RNAi in the cytosol had also caused an early and transient transcriptional interference of SMALLTALK in the nucleus; Secondly, the resulting interference of SMALLTALK transcription increased the transcription of TOSPEAK; Thirdly, the increased transcription of TOSPEAK increased the transcription of GDF6. These findings have implications for the design and application of RNA and DNA targeting technologies including siRNA and CRISPR. For example, we used siRNA targeting of SMALLTALK to successfully restore GDF6 levels in the gene therapy of SYNS4 family fibroblasts in culture. To confidently apply gene targeting technologies, it is important to first determine the transcriptional interference effects of the targeting reagent and the targeted gene.

Detection of SARS-CoV-2 Derived Small RNAs and Changes in Circulating Small RNAs Associated with COVID-19

Cleavage of double-stranded RNA is described as an evolutionary conserved host defense mechanism against viral infection. Small RNAs are the product and triggers of post transcriptional gene silencing events. Up until now, the relevance of this mechanism for SARS-CoV-2-directed immune responses remains elusive. Herein, we used high throughput sequencing to profile the plasma of active and convalescent COVID-19 patients for the presence of small circulating RNAs. The existence of SARS-CoV-2 derived small RNAs in plasma samples of mild and severe COVID-19 cases is described. Clusters of high siRNA abundance were discovered, homologous to the nsp2 3’-end and nsp4 virus sequence. Four virus-derived small RNA sequences have the size of human miRNAs, and a target search revealed candidate genes associated with ageusia and long COVID symptoms. These virus-derived small RNAs were detectable also after recovery from the disease. The additional analysis of circulating human miRNAs revealed differentially abundant miRNAs, discriminating mild from severe cases. A total of 29 miRNAs were reduced or absent in severe cases. Several of these are associated with JAK-STAT response and cytokine storm.