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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.

In plants, the vascular tissue contains the enucleated sieve tubes facilitating long-distance transport of nutrients, hormones, and proteins. In addition, several mRNAs and small interfering RNAs/microRNAs were shown to be delivered via sieve tubes whose content is embodied by the phloem sap (PS). A number of these phloem transcripts are transported from source to sink tissues and function at targeted tissues. To gain additional insights into phloem-delivered RNAs and their potential role in signaling, we isolated and characterized PS RNA molecules distinct from microRNAs/small interfering RNAs with a size ranging from 30 to 90 bases. We detected a high number of full-length and phloem-specific fragments of noncoding RNAs such as tRNAs, ribosomal RNAs, and spliceosomal RNAs in the PS of pumpkin (Cucurbita maxima). In vitro assays show that small quantities of PS RNA molecules efficiently inhibit translation in an unspecific manner. Proof of concept that PS-specific tRNA fragments may interfere with ribosomal activity was obtained with artificially produced tRNA fragments. The results are discussed in terms of a functional role for long distance delivered noncoding PS RNAs.

Epigenetics is a discipline that studies heritable changes in gene expression that do not involve altering the DNA sequence. Over the past decade, researchers have shown that epigenetic regulation plays a momentous role in cell growth, differentiation, autoimmune diseases, and cancer. The main epigenetic mechanisms include the well-understood phenomenon of DNA methylation, histone modifications, and regulation by non-coding RNAs, a mode of regulation that has only been identified relatively recently and is an area of intensive ongoing investigation. It is generally known that the majority of human transcripts are not translated but a large number of them nonetheless serve vital functions. Non-coding RNAs are a cluster of RNAs that do not encode functional proteins and were originally considered to merely regulate gene expression at the post-transcriptional level. However, taken together, a wide variety of recent studies have suggested that miRNAs, piRNAs, endogenous siRNAs, and long non-coding RNAs are the most common regulatory RNAs, and, significantly, there is a growing body of evidence that regulatory non-coding RNAs play an important role in epigenetic control. Therefore, these non-coding RNAs (ncRNAs) highlight the prominent role of RNA in the regulation of gene expression. Herein, we summarize recent research developments with the purpose of coming to a better understanding of non-coding RNAs and their mechanisms of action in cells, thus gaining a preliminary understanding that non-coding RNAs feed back into an epigenetic regulatory network.

High complementarity between plant microRNAs (miRNAs) and their messenger RNA targets is thought to cause silencing, prevalently by endonucleolytic cleavage. We have isolated Arabidopsis mutants defective in miRNA action. Their analysis provides evidence that plant miRNA-guided silencing has a widespread translational inhibitory component that is genetically separable from endonucleolytic cleavage. We further show that the same is true of silencing mediated by small interfering RNA (siRNA) populations. Translational repression is effected in part by the ARGONAUTE proteins AGO1 and AGO10. It also requires the activity of the microtubule-severing enzyme katanin, implicating cytoskeleton dynamics in miRNA action, as recently suggested from animal studies. Also as in animals, the decapping component VARICOSE (VCS)/Ge-1 is required for translational repression by miRNAs, which suggests that the underlying mechanisms in the two kingdoms are related.

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.

Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNAdependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets.

Dissection of the genetic pathways and mechanisms by which anther development occurs in grasses is crucial for both a basic understanding of plant development and for examining traits of agronomic importance such as male sterility. In rice, MULTIPLE SPOROCYTES1 (MSP1), a leucine-rich-repeat receptor kinase, plays an important role in anther development by limiting the number of sporocytes. OsTDL1a (a TPD1-like gene in rice) encodes a small protein that acts as a cofactor of MSP1 in the same regulatory pathway. In this study, we analyzed small RNA and mRNA changes in different stages of spikelets from wild-type rice, and from msp1 and ostdl1a mutants. Analysis of the small RNA data identified miRNAs demonstrating differential abundances. miR2275 was depleted in the two rice mutants; this miRNA is specifically enriched in anthers and functions to trigger the production of 24-nt phased secondary siRNAs (phasiRNAs) from PHAS loci. We observed that the 24-nt phasiRNAs as well as their precursor PHAS mRNAs were also depleted in the two mutants. An analysis of co-expression identified three Argonaute-encoding genes (OsAGO1d, OsAGO2b, and OsAGO18) that accumulate transcripts coordinately with phasiRNAs, suggesting a functional relationship. By mRNA in situ analysis, we demonstrated a strong correlation between the spatiotemporal pattern of these OsAGO transcripts and phasiRNA accumulations.

Pachytene PIWI-interacting RNAs (piRNAs), which comprise >80% of small RNAs in the adult mouse testis, have been proposed to bind and regulate target RNAs like microRNAs, cleave targets like short interfering RNAs or lack biological function altogether. Although piRNA pathway protein mutants are male sterile, no biological function has been identified for any mammalian piRNA-producing locus. Here, we report that males lacking piRNAs from a conserved mouse pachytene piRNA locus on chromosome 6 ( pi6 ) produce sperm with defects in capacitation and egg fertilization. Moreover, heterozygous embryos sired by pi6 −/− fathers show reduced viability in utero. Molecular analyses suggest that pi6 piRNAs repress gene expression by cleaving messenger RNAs encoding proteins required for sperm function. pi6 also participates in a network of piRNA–piRNA precursor interactions that initiate piRNA production from a second piRNA locus on chromosome 10, as well as pi6 itself. Our data establish a direct role for pachytene piRNAs in spermiogenesis and embryo viability. Male mice lacking piRNAs from a conserved locus on chromosome 6 ( pi6 ) produce sperm with defects in capacitation and egg fertilization. pi6 piRNAs repress mRNAs required for sperm function.

Small RNA guides--microRNAs, small interfering RNAs, and repeat-associated small interfering RNAs, 21 to 30 nucleotides in length--shape diverse cellular pathways, from chromosome architecture to stem cell maintenance. Fifteen years after the discovery of RNA silencing, we are only just beginning to understand the depth and complexity of how these RNAs regulate gene expression and to consider their role in shaping the evolutionary history of higher eukaryotes.

Targeted delivery of RNA-based therapeutics for cancer therapy remains a challenge. We have developed a LPH (liposome-polycation-hyaluronic acid) nanoparticle formulation modified with tumor-targeting single-chain antibody fragment (scFv) for systemic delivery of small interfering RNA (siRNA) and microRNA (miRNA) into experimental lung metastasis of murine B16F10 melanoma. The siRNAs delivered by the scFv targeted nanoparticles efficiently downregulated the target genes (c-Myc/MDM2/VEGF) in the lung metastasis. Two daily intravenous injections of the combined siRNAs in the GC4-targeted nanoparticles significantly reduced the tumor load in the lung. miRNA-34a (miR-34a) induced apoptosis, inhibited survivin expression, and downregulated MAPK pathway in B16F10 cells. miR-34a delivered by the GC4-targeted nanoparticles significantly downregulated the survivin expression in the metastatic tumor and reduced tumor load in the lung. When miR-34a and siRNAs were co-formulated in GC4-targeted nanoparticles, an enhanced anticancer effect was observed.