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RNA silencing relies on specific and efficient processing of double-stranded RNA by Dicer, which yields microRNAs (miRNAs) and small interfering RNAs (siRNAs) 1 , 2 . However, our current knowledge of the specificity of Dicer is limited to the secondary structures of its substrates: a double-stranded RNA of approximately 22 base pairs with a 2-nucleotide 3′ overhang and a terminal loop 3 – 11 . Here we found evidence pointing to an additional sequence-dependent determinant beyond these structural properties. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with pre-miRNA variants and human DICER (also known as DICER1). Our analyses revealed a deeply conserved cis -acting element, termed the ‘GYM motif’ (paired G, paired pyrimidine and mismatched C or A), near the cleavage site. The GYM motif promotes processing at a specific position and can override the previously identified ‘ruler’-like counting mechanisms from the 5′ and 3′ ends of pre-miRNA 3 – 6 . Consistently, integrating this motif into short hairpin RNA or Dicer-substrate siRNA potentiates RNA interference. Furthermore, we find that the C-terminal double-stranded RNA-binding domain (dsRBD) of DICER recognizes the GYM motif. Alterations in the dsRBD reduce processing and change cleavage sites in a motif-dependent fashion, affecting the miRNA repertoire in cells. In particular, the cancer-associated R1855L substitution in the dsRBD strongly impairs GYM motif recognition. This study uncovers an ancient principle of substrate recognition by metazoan Dicer and implicates its potential in the design of RNA therapeutics. Massively parallel assays reveal a highly conserved sequence motif termed the GYM motif, which potentiates RNA interference by directing Dicer-mediated small RNA processing.

A parasitic plant produces microRNAs that target host messenger RNAs, causing them to be processed into small interfering RNAs. miRNAs in plant parasitism Dodders are parasitic plants that obtain water and nutrients from the stems of their host plants, and exchange other material with their hosts, through structures called haustoria. Michael Axtell and colleagues report how haustoria mediate dodders' parasitism. Dodders accumulate many microRNAs (miRNAs) in their haustoria while infesting a host plant. These miRNAs seem to then transfer to the host, where they silence target messenger RNAs (mRNAs) through the production of secondary small interfering RNAs and mRNA cleavage. The authors also identify host proteins that are targeted by dodder miRNAs and provide evidence that such regulation of host gene expression through inter-species transfer of miRNAs is not limited to one host. Dodders ( Cuscuta spp.) are obligate parasitic plants that obtain water and nutrients from the stems of host plants via specialized feeding structures called haustoria. Dodder haustoria facilitate bidirectional movement of viruses, proteins and mRNAs between host and parasite 1 , but the functional effects of these movements are not known. Here we show that Cuscuta campestris haustoria accumulate high levels of many novel microRNAs (miRNAs) while parasitizing Arabidopsis thaliana . Many of these miRNAs are 22 nucleotides in length. Plant miRNAs of this length are uncommon, and are associated with amplification of target silencing through secondary short interfering RNA (siRNA) production 2 . Several A. thaliana mRNAs are targeted by 22-nucleotide C. campestris miRNAs during parasitism, resulting in mRNA cleavage, secondary siRNA production, and decreased mRNA accumulation. Hosts with mutations in two of the loci that encode target mRNAs supported significantly higher growth of C. campestris . The same miRNAs that are expressed and active when C. campestris parasitizes A. thaliana are also expressed and active when it infects Nicotiana benthamiana . Homologues of target mRNAs from many other plant species also contain the predicted target sites for the induced C. campestris miRNAs. These data show that C. campestris miRNAs act as trans-species regulators of host-gene expression, and suggest that they may act as virulence factors during parasitism.

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.

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes 1 . Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation 2 , 3 , respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here we report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases— NIA1 and NIA2 —produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2 , which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, our research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses. Characterization of 22-nucleotide short interfering RNAs in plants finds that they accumulate in response to environmental stress, causing translational repression, inhibition of plant growth and enhanced stress responses.

Delivery of biomolecules to plants relies on Agrobacterium infection or biolistic particle delivery, the former of which is amenable only to DNA delivery. The difficulty in delivering functional biomolecules such as RNA to plant cells is due to the plant cell wall, which is absent in mammalian cells and poses the dominant physical barrier to biomolecule delivery in plants. DNA nanostructure-mediated biomolecule delivery is an effective strategy to deliver cargoes across the lipid bilayer of mammalian cells; however, nanoparticle-mediated delivery without external mechanical aid remains unexplored for biomolecule delivery across the cell wall in plants. Herein, we report a systematic assessment of different DNA nanostructures for their ability to internalize into cells of mature plants, deliver siRNAs, and effectively silence a constitutively expressed gene in Nicotiana benthamiana leaves. We show that nanostructure internalization into plant cells and corresponding gene silencing efficiency depends on the DNA nanostructure size, shape, compactness, stiffness, and location of the siRNA attachment locus on the nanostructure. We further confirm that the internalization efficiency of DNA nanostructures correlates with their respective gene silencing efficiencies but that the endogenous gene silencing pathway depends on the siRNA attachment locus. Our work establishes the feasibility of biomolecule delivery to plants with DNA nanostructures and both details the design parameters of importance for plant cell internalization and also assesses the impact of DNA nanostructure geometry for gene silencing mechanisms.

The effect of RNA silencing in plants can be amplified if the production of secondary small interfering RNAs (siRNAs) is triggered by the interaction of microRNAs (miRNAs) or siRNAs with a long target RNA. miRNA and siRNA interactions are not all equivalent, however; most of them do not trigger secondary siRNA production. Here we use bioinformatics to show that the secondary siRNA triggers are miRNAs and transacting siRNAs of 22 nt, rather than the more typical 21-nt length. Agrobacterium-mediated transient expression in Nicotiana benthamiana confirms that the siRNA-initiating miRNAs, miR173 and miR828, are effective as triggers only if expressed in a 22-nt form and, conversely, that increasing the length of miR319 from 21 to 22 nt converts it to an siRNA trigger. We also predicted and validated that the 22-nt miR771 is a secondary siRNA trigger. Our data demonstrate that the function of small RNAs is influenced by size, and that a length of 22 nt facilitates the triggering of secondary siRNA production.

In some organisms, miRNA-mediated regulation leads to RNA-dependent RNA polymerase based amplification, involving generation of secondary siRNAs that target the same transcript, but why this happens for some miRNAs and not others has been unclear. It is now shown that Arabidopsis miRNAs that trigger this amplified response tend to be 22 nt rather than 21 nt in length, and that generation of 22 nt miRNAs this length seems to depend on the foldback structure of the precursor. RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans -acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in the production of 21-nt forms of miR173, miR472 and miR828. Both 21- and 22-nt forms associated with AGO1 and guided accurate slicer activity, but only 22-nt forms were competent to trigger RDR6-dependent siRNA production from target RNA. These data suggest that AGO1 functions differentially with 21- and 22-nt miRNAs to engage the RDR6-associated amplification apparatus.

In contrast to microRNAs and Piwi-associated RNAs, short interfering RNAs (siRNAs) are seemingly dispensable for host-directed gene regulation in Drosophila. This notion is based on the fact that mutants lacking the core siRNA-generating enzyme Dicer-2 or the predominant siRNA effector Argonaute 2 are viable, fertile and of relatively normal morphology. Moreover, endogenous Drosophila siRNAs have not yet been identified. Here we report that siRNAs derived from long hairpin RNA genes (hpRNAs) programme Slicer complexes that can repress endogenous target transcripts. The Drosophila hpRNA pathway is a hybrid mechanism that combines canonical RNA interference factors (Dicer-2, Hen1 (known as CG12367) and Argonaute 2) with a canonical microRNA factor (Loquacious) to generate 21-nucleotide siRNAs. These novel regulatory RNAs reveal unexpected complexity in the sorting of small RNAs, and open a window onto the biological usage of endogenous RNA interference in Drosophila.

In mammals, the acquisition of the germline from the soma provides the germline with an essential challenge: the need to erase and reset genomic methylation 1 . In the male germline, RNA-directed DNA methylation silences young, active transposable elements 2 – 4 . The PIWI protein MIWI2 (PIWIL4) and its associated PIWI-interacting RNAs (piRNAs) instruct DNA methylation of transposable elements 3 , 5 . piRNAs are proposed to tether MIWI2 to nascent transposable element transcripts; however, the mechanism by which MIWI2 directs the de novo methylation of transposable elements is poorly understood, although central to the immortality of the germline. Here we define the interactome of MIWI2 in mouse fetal gonocytes undergoing de novo genome methylation and identify a previously unknown MIWI2-associated factor, SPOCD1, that is essential for the methylation and silencing of young transposable elements. The loss of Spocd1 in mice results in male-specific infertility but does not affect either piRNA biogenesis or the localization of MIWI2 to the nucleus. SPOCD1 is a nuclear protein whose expression is restricted to the period of de novo genome methylation. It co-purifies in vivo with DNMT3L and DNMT3A, components of the de novo methylation machinery, as well as with constituents of the NURD and BAF chromatin remodelling complexes. We propose a model whereby tethering of MIWI2 to a nascent transposable element transcript recruits repressive chromatin remodelling activities and the de novo methylation apparatus through SPOCD1. In summary, we have identified a previously unrecognized and essential executor of mammalian piRNA-directed DNA methylation. Newly identified protein SPOCD1 is crucial in de novo DNA methylation directed by PIWI proteins and piRNAs, helping to control DNA silencing in mouse male germline.

Drosophila have two pathways for PIWI-interacting RNA (piRNA) 3′-end formation—depending on which pathway is used, piRNA biogenesis is directed towards either cytoplasmic or nuclear PIWI protein effectors, which balances post-transcriptional versus transcriptional transposon silencing. Twin track of piRNA 3′-end formation Unlike microRNAs and short interfering RNAs, PIWI-interacting RNAs (piRNAs) are not generated by Dicer cleavage. The process leading to the 5′ ends of piRNAs is known, but mediating the formation of the 3′-ends has been difficult. Julius Brennecke and colleagues now show that there are two pathways in Drosophila capable of 3′-end formation. Depending on which pathway is used, the piRNA product is directed to either a cytoplasmic or a nuclear PIWI protein effector, which determines whether the piRNA functions in post-transcriptional or transcriptional events. Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing 1 . Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)—the 22–30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated independently of Dicer from single-stranded precursors 2 , 3 . piRNA 5′ ends are defined either by Zucchini, the Drosophila homologue of mitoPLD—a mitochondria-anchored endonuclease 4 , 5 , or by piRNA-guided target cleavage 6 , 7 . Formation of piRNA 3′ ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3′ ends in Drosophila . The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3′ ends 8 , 9 , Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3′-to-5′ exoribonuclease Nibbler ( Drosophila homologue of Mut-7) 10 , 11 , 12 , 13 . The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5′ and 3′ ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3′-end formation.