Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species

Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5′-terminus, and changes in the expression of certain miRNAs, most of which were downregulated.

Plant Small RNA Species Direct Gene Silencing in Pathogenic Bacteria as well as Disease Protection

Plant small RNAs (sRNAs) and/or double-stranded RNAs (dsRNAs) trigger RNA interference (RNAi) in interacting eukaryotic pathogens or parasites. However, it is unknown whether this phenomenon could operate in bacterial phytopathogens, which lack a eukaryotic-like RNAi machinery. Here, we first show that Arabidopsis-encoded inverted repeat transgenes trigger silencing of Pseudomonas syringae heterologous reporter and endogenous virulence-associated genes during infection. Antibacterial Gene Silencing (AGS) of the latter was associated with a reduced pathogenesis, which was also observed upon application of corresponding plant-derived RNAs onto wild-type plants prior to infection. We additionally demonstrate that sRNAs directed against virulence factor transcripts were causal for silencing and pathogenesis reduction, while cognate long dsRNAs were inactive. Overall, this study provides the first evidence that plant sRNAs can directly reprogram gene expression in a phytopathogenic bacterium and may have wider implications in the understanding of how plants regulate transcriptome, community composition and genome evolution of associated bacteria.

Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols

BACKGROUND Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria. METHODS We compared 4 different manipulations of RNA for library preparation: (a) a ligation-based procedure allowing only 5′-mono-phosphorylated RNA to enter the library, (b) a ligation-based procedure allowing additional 5′-triphosphates and Cap structures, (c) a ligation-independent, template-switch-based library preparation, and (d) a template-switch-based library preparation allowing 3′-phosphorylated RNAs to enter the library. RESULTS Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species. CONCLUSIONS Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.

Sequencing-based quantitative mapping of the cellular small RNA landscape

Current next-generation RNA sequencing methods cannot provide accurate quantification of the population of small RNAs within a sample due to strong sequence-dependent biases in capture, ligation, and amplification during library preparation. We report the development of an RNA sequencing method – AQRNA-seq – that minimizes biases and enables absolute quantification of all small RNA species in a sample mixture. Validation of AQRNA-seq library preparation and data mining algorithms using a 963-member microRNA reference library, RNA oligonucleotide standards of varying lengths, and northern blots demonstrated a direct, linear correlation between sequencing read count and RNA abundance. Application of AQRNA-seq to bacterial tRNA pools, a traditionally hard-to-sequence class of RNAs, revealed 80-fold variation in tRNA isoacceptor copy numbers, patterns of site-specific tRNA fragmentation caused by stress, and quantitative maps of ribonucleoside modifications, all in a single AQRNA-seq experiment. AQRNA-seq thus provides a means to quantitatively map the small RNA landscape in cells and tissues.

Telomeric small RNAs in the genus Caenorhabditis

Telomeric DNA is composed of simple tandem repeat sequences and has a G-rich strand that runs 5’ to 3’ towards the chromosome terminus. Small RNAs with homology to telomeres have been observed in several organisms and could originate from telomeres or from interstitial telomere sequences (ITSs), which are composites of degenerate and perfect telomere repeat sequences found on chromosome arms. We identified C. elegans small RNAs composed of the Caenorhabditis telomere sequence (TTAGGC)n with up to three mismatches, which might interact with telomeres. We rigorously defined ITSs for genomes of C. elegans and for two closely related nematodes, C. briggsae and C. remanei. We found that most telomeric small RNAs with mismatches originated from ITSs, which were depleted from mRNAs and but were enriched in introns whose genes often displayed hallmarks of genomic silencing. C. elegans small RNAs composed of perfect telomere repeats were very rare but were increased by several orders of magnitude in C. briggsae and C. remanei. Major small RNA species in C. elegans begin with a 5’ guanine nucleotide, which was strongly depleted from perfect telomeric small RNAs of all three Caenorhabditis species. Perfect telomeric small RNAs corresponding to the G-rich strand of the telomere commonly began with 5’ UAGGCU and 5’UUAGGC, whereas C-rich strand RNAs commonly begin with 5’CUAAGC. In contrast, telomeric small RNAs with mismatches had a mixture of all four 5’ nucleotides. Together, our results imply that perfect telomeric small RNAs have a mechanism of biogenesis that is distinct from known classes of small RNAs and that a dramatic change in their regulation occurred during recent Caenorhabditis evolution.

Plant Extracellular Vesicles Contain Diverse Small RNA Species and Are Enriched in 10 to 17 Nucleotide “Tiny” RNAs

ABSTRACTSmall RNAs (sRNAs) that are 21 to 24 nucleotides (nt) in length are found in most eukaryotic organisms and regulate numerous biological functions, including transposon silencing, development, reproduction, and stress responses, typically via control of the stability and/or translation of target mRNAs. Major classes of sRNAs in plants include microRNAs (miRNAs) and small interfering RNAs (siRNAs); sRNAs are known to travel as a silencing signal from cell to cell, root to shoot, and even between host and pathogen. In mammals, sRNAs are transported inside extracellular vesicles (EVs), which are mobile lipid compartments that participate in intercellular communication. In addition to sRNAs, EVs carry proteins, lipids, metabolites, and potentially other types of nucleic acids. Here we report that plant EVs also contain diverse species of sRNA. We found that specific miRNAs and siRNAs are preferentially loaded into plant EVs. We also report a previously overlooked class of “tiny RNAs” (10 to 17 nt) that are highly enriched in EVs. This new RNA category of unknown function has a broad and very diverse genome origin and might correspond to degradation products.