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Black color in flowers is a highly attractive trait in the floricultural industry, but its underlying mechanisms are largely unknown. This study was performed to identify the bases of the high accumulation of anthocyanidins in black cultivars and to determine whether the high accumulation of total anthocyanidins alone leads to the black appearance. Our approach was to compare black dahlia (Dahlia variabilis) cultivars with purple cultivars and a purple flowering mutant of a black cultivar, using pigment and molecular analyses. Black cultivars characteristically exhibited low lightness, high petal accumulation of cyanidin and total anthocyanidins without flavones, and marked suppression of flavone synthase (DvFNS) expression. A comparative study using black and purple cultivars revealed that neither the absence of flavones nor high accumulation of total anthocyanidins is solely sufficient for black appearance, but that cyanidin content in petals is also an important factor in the phenotype. A study comparing the black cultivar 'Kokucho' and its purple mutant showed that suppression of DvFNS abolishes the competition between anthocyanidin and flavone synthesis and leads to accumulation of cyanidin and total anthocyanidins that produce a black appearance. Surprisingly, in black cultivars the suppression of DvFNS occurred in a post-transcriptional manner, as determined by small RNA mapping.

RNA interference (RNAi) has been widely adopted to repress specific gene expression and is easily achieved by designing small interfering RNAs (siRNAs) with perfect sequence complementarity to the intended target mRNAs. Although siRNAs direct Argonaute (Ago), a core component of the RNA-induced silencing complex (RISC), to recognize and silence target mRNAs, they also inevitably function as microRNAs (miRNAs) and suppress hundreds of off-targets. Such miRNA-like off-target repression is potentially detrimental, resulting in unwanted toxicity and phenotypes. Despite early recognition of the severity of miRNA-like off-target repression, this effect has often been overlooked because of difficulties in recognizing and avoiding off-targets. However, recent advances in genome-wide methods and knowledge of Ago–miRNA target interactions have set the stage for properly evaluating and controlling miRNA-like off-target repression. Here, we describe the intrinsic problems of miRNA-like off-target effects caused by canonical and noncanonical interactions. We particularly focus on various genome-wide approaches and chemical modifications for the evaluation and prevention of off-target repression to facilitate the use of RNAi with secured specificity.

Key Points MicroRNAs (miRNAs) have important roles in many aspects of human diseases, and their targeted inhibition may have substantial therapeutic impact. Inhibition of miRNAs can be achieved through a variety of methods and chemically modified antisense oligonucleotides (anti-miRs) have shown the most prominent effects. Targeted delivery of anti-miRs is crucial to achieve intended therapeutic effects, and further efforts are warranted to develop more efficient delivery systems. MicroRNAs (miRNAs) — 21- to 23-nucleotide single-stranded RNAs that regulate gene expression — have roles in numerous diseases, and are therefore attractive therapeutic targets. Li and Rana discuss strategies in the design of miRNA-targeting oligonucleotides with increased efficacy and improved in vivo delivery characteristics, and highlight some of the challenges that lie ahead in the clinical development of these therapeutics. MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that have crucial roles in regulating gene expression. Increasing evidence supports a role for miRNAs in many human diseases, including cancer and autoimmune disorders. The function of miRNAs can be efficiently and specifically inhibited by chemically modified antisense oligonucleotides, supporting their potential as targets for the development of novel therapies for several diseases. In this Review we summarize our current knowledge of the design and performance of chemically modified miRNA-targeting antisense oligonucleotides, discuss various in vivo delivery strategies and analyse ongoing challenges to ensure the specificity and efficacy of therapeutic oligonucleotides in vivo . Finally, we review current progress on the clinical development of miRNA-targeting therapeutics.

The finding that sequence-specific gene silencing occurs in response to the presence of double-stranded RNAs has had an enormous impact on biology, uncovering an unsuspected level of regulation of gene expression. This process, known as RNA interference (RNAi) or RNA silencing, involves small non-coding RNAs, which associate with nuclease-containing regulatory complexes and then pair with complementary messenger RNA targets, thereby preventing the expression of these mRNAs. Remarkable progress has been made towards understanding the underlying mechanisms of RNAi, raising the prospect of deciphering the 'RNAi code' that, like transcription factors, allows the fine-tuning and networking of complex suites of gene activity, thereby specifying cellular physiology and development.

RNA silencing functions as an antiviral defense through the action of DICER-like (DCL) and ARGONAUTE (AGO) proteins. In turn, plant viruses have evolved strategies to counteract this defense mechanism, including the expression of suppressors of RNA silencing. Potato virus X (PVX) does not systemically infect Arabidopsis thaliana Columbia-0, but is able to do so effectively in mutants lacking at least two of the four Arabidopsis DCL proteins. PVX can also infect Arabidopsis ago2 mutants, albeit less effectively than double DCL mutants, suggesting that additional AGO proteins may mediate anti-viral defenses. Here we show, using functional assays, that all Arabidopsis AGO proteins have the potential to target PVX lacking its viral suppressor of RNA silencing (VSR), P25, but that only AGO2 and AGO5 are able to target wild-type PVX. However, P25 directly affects only a small subset of AGO proteins, and we present evidence indicating that its protective effect is mediated by precluding AGO proteins from accessing viral RNA, as well as by directly inhibiting the RNA silencing machinery. In agreement with functional assays, we show that Potexvirus infection induces AGO5 expression and that both AGO2 and AGO5 are required for full restriction of PVX infection in systemic tissues of Arabidopsis.

The latest advances in the field exogenous application of RNA molecules in plants help to protect and modify them through RNA interference (RNAi).

The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss-of-function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that microRNA (miRNA)-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a consensus gene signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 single guide RNAs (sgRNAs) in 6 cells lines and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.

The genome of the cellulase-producing fungus Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) was screened for a potent DNA ligase IV gene (ligD homologue). Homologous recombination efficiency in T. cellulolyticus is very low. Therefore, suppression of a non-homologous end-joining system was attempted to enable specific gene knockouts for molecular breeding. The transcript levels of ligD homologue were 0.037 of those of the parental YP-4 strain in the Li20 transformant carrying the RNAi construct targeting the ligD homologue. Transformation of the hairpin-type RNAi vector into T. cellulolyticus could be useful in fungal gene knockdown experiments. Cellulase production and protein secretion were similar in the parental YP-4 strain and the Li20 transformant. Knockout transformation of ligD homologue using the Li20 transformant led to 23.1 % double crossover gene targeting. Our results suggest that the potent DNA ligase IV gene of T. cellulolyticus is related to non-homologous end joining and that the knockdown of the ligD homologue is useful in gene targeting.

The approval of the first small interfering RNA (siRNA) drug Patisiran by FDA in 2018 marks a new era of RNA interference (RNAi) therapeutics. MicroRNAs (miRNA), an important post-transcriptional gene regulator, are also the subject of both basic research and clinical trials. Both siRNA and miRNA mimics are ~21 nucleotides RNA duplexes inducing mRNA silencing. Given the well performance of siRNA, researchers ask whether miRNA mimics are unnecessary or developed siRNA technology can pave the way for the emergence of miRNA mimic drugs. Through comprehensive comparison of siRNA and miRNA, we focus on (1) the common features and lessons learnt from the success of siRNAs; (2) the unique characteristics of miRNA that potentially offer additional therapeutic advantages and opportunities; (3) key areas of ongoing research that will contribute to clinical application of miRNA mimics. In conclusion, miRNA mimics have unique properties and advantages which cannot be fully matched by siRNA in clinical applications. MiRNAs are endogenous molecules and the gene silencing effects of miRNA mimics can be regulated or buffered to ameliorate or eliminate off-target effects. An in-depth understanding of the differences between siRNA and miRNA mimics will facilitate the development of miRNA mimic drugs.

Geminiviruses are plant viruses with limited coding capacity. Geminivirus-encoded proteins are traditionally identified by applying a 10-kDa arbitrary threshold; however, it is increasingly clear that small proteins play relevant roles in biological systems, which calls for the reconsideration of this criterion. Here, we show that geminiviral genomes contain additional ORFs. Using tomato yellow leaf curl virus, we demonstrate that some of these small ORFs are expressed during the infection, and that the encoded proteins display specific subcellular localizations. We prove that the largest of these additional ORFs, which we name V3, is required for full viral infection, and that the V3 protein localizes in the Golgi apparatus and functions as an RNA silencing suppressor. These results imply that the repertoire of geminiviral proteins can be expanded, and that getting a comprehensive overview of the molecular plant-geminivirus interactions will require the detailed study of small ORFs so far neglected. Geminiviruses are small plant-infecting viruses. Here the authors screen the geminivirus genome for small open reading frames that may have been previously overlooked and show that one encodes a protein named V3 that localizes to the Golgi and acts as an RNA silencing suppressor.