Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs

Abstract

Eukaryotic RNA interference (RNAi) results in gene silencing upon the sequence-specific degradation of target transcripts by complementary small RNAs (sRNAs). In plants, RNAi-based tools have been optimized for high efficacy and high specificity, and are extensively used in gene function studies and for crop improvement. However, efficient methods for finely adjusting the degree of induced silencing are missing. Here, we present two different strategies based on artificial sRNAs for fine-tuning targeted RNAi efficacy in plants. First, the degree of silencing induced by synthetic-trans-acting small interfering RNAs (syn-tasiRNAs) can be adjusted by modifying the precursor position from which the syn-tasiRNA is expressed. The accumulation and efficacy of Arabidopsis TAS1c-based syn-tasiRNAs progressively decrease as the syn-tasiRNA is expressed from positions more distal to the trigger miR173 target site. And second, syn-tasiRNA activity can also be tweaked by modifying the degree of base-pairing between the 3′ end of the syn-tasiRNA and the 5′ end of the target RNA. Both strategies were used to finely modulate the degree of silencing of endogenous and exogenous target genes in Arabidopsis thaliana and Nicotiana benthamiana. New high-throughput syn-tasiRNA vectors were developed and functionally analyzed, and should facilitate the precise control of gene expression in multiple plant species.

Ligation bias is a major contributor to nonstoichiometric abundances of secondary siRNAs and impacts analyses of microRNAs

“…We observed that phasiRNA accumulation is extremely variable when comparing between kits (Supplemental Figure 4, Supplemental table 2). As described before (28,29), most phasiRNAs from a single PHAS locus accumulate to diverse levels (Supplemental Figure 4, Supplemental table 2). The biogenesis of phasiRNAs by processive activity of Dicer should yield phasiRNA duplexes from a given locus in stoichiometrically equal levels; yet, of tens of thousands of reproductive phasiRNAs only a small proportion are abundant and observed in nonstoichiometric abundances within a PHAS locus (28).…”

Secondary siRNAs in Plants: Biosynthesis, Various Functions, and Applications in Virology

“…Most of the contributions on antiviral strategies using SIGS have come from the lab of Carbonell et al (2019a , b) , Cisneros and Carbonell (2020) , Lopez-Dolz et al (2020) . Carbonell’s group has worked extensively on a deadly RNA virus, namely, TSWV.…”

“…Controlling the degree of induced gene silencing to fine-tune it, keeping the promoter of the expression of silencing sRNAs invariant, is a very desirable objective but not so easy to achieve. Carbonell’s group has revealed the strategies to achieve the same by playing with the order of positioning (with respect to the cleavage point of the initiator miRNA) of the expressed syn-tasiRNAs and tweaking the sequences of the same ( Lopez-Dolz et al, 2020 ). The investigators have shown that the level and activity of Arabidopsis TAS1c-based syn-tasiRNAs gradually reduce as the syn-tasiRNA is expressed from positions more distant to the trigger miR173 cleavage site.…”

Artificial Small RNA-Based Silencing Tools for Antiviral Resistance in Plants

“…On the other hand, TAS precursors possess a "natural" multiplexing capability allowing the insertion of multiple syn-tasiRNAs in a single construct (Figure 3). This feature combined with the availability of high-throughput syn-tasiRNA "B/c" vectors [7,49] allows for the efficient generation of antiviral syn-tasiRNA constructs. An interesting possibility is to combine in a single syn-tasiRNA construct a number of effective amiRNAs previously identified in large screens.…”

“…Although very popular, these approaches lacked high specificity as the large populations of sRNAs produced from these type of precursors favor the accidental targeting of complementary cellular transcripts [6]. This limitation was overcome with the development of a series of tools based on artificial sRNAs (art-sRNAs) [7], 21-nucleotide sRNAs expressed in planta from endogenous sRNA precursors and computationally designed to silence target RNAs with high specificity [8].…”