In planta dynamics, transport biases, and endogenous functions of mobile siRNAs in Arabidopsis.

In RNA interference (RNAi), small interfering RNAs (siRNAs) produced from double-stranded RNA guide ARGONAUTE (AGO) proteins to silence sequence-complementary RNA/DNA. RNAi can propagate locally and systemically in plants, but despite recent advances in our understanding of the underlying mechanisms, basic questions remain unaddressed. For instance, RNAi is inferred to diffuse through plasmodesmata (PDs), yet how its dynamics in planta compares with that of established symplastic diffusion markers remains unknown. Also is why select siRNA species, or size classes thereof, are apparently recovered in RNAi recipient tissues, yet only under some experimental settings. Shootward movement of endogenous RNAi in micro-grafted Arabidopsis is also yet to be achieved, while potential endogenous functions of mobile RNAi remain scarcely documented. Here, we show (i) that temporal, localized PD occlusion in source leaves' companion cells (CCs) suffices to abrogate all systemic manifestations of CC-activated mobile transgene silencing, including in sink leaves; (ii) that the presence or absence of specific AGOs in incipient/traversed/recipient tissues likely explains the apparent siRNA length selectivity observed upon vascular movement; (iii) that stress enhancement allows endo-siRNAs of a single inverted repeat (IR) locus to translocate against the shoot-to-root phloem flow; and (iv) that mobile endo-siRNAs generated from this locus have the potential to regulate hundreds of transcripts. Our results close important knowledge gaps, rationalize previously noted inconsistencies between mobile RNAi settings, and provide a framework for mobile endo-siRNA research.

A genetic framework for proximal secondary vein branching in the Arabidopsis thaliana embryo.

Over time, plants have evolved flexible self-organizing patterning mechanisms to adapt tissue functionality for continuous organ growth. An example of this process is the multicellular organization of cells into a vascular network in foliar organs. An important, yet poorly understood component of this process is secondary vein branching, a mechanism employed to extend vascular tissues throughout the cotyledon surface. Here, we uncover two distinct branching mechanisms during embryogenesis by analyzing the discontinuous vein network of the double mutant cotyledon vascular pattern 2 (cvp2) cvp2-like 1 (cvl1). Similar to wild-type embryos, distal veins in cvp2 cvl1 embryos arise from the bifurcation of cell files contained in the midvein, whereas proximal branching is absent in this mutant. Restoration of this process can be achieved by increasing OCTOPUS dosage as well as by silencing RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) expression. Although RPK2-dependent rescue of cvp2 cvl1 is auxin- and CLE peptide-independent, distal branching involves polar auxin transport and follows a distinct regulatory mechanism. Our work defines a genetic network that confers plasticity to Arabidopsis embryos to spatially adapt vascular tissues to organ growth.

HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement.

Plant microRNAs (miRNAs) guide cytosolic post-transcriptional gene silencing of sequence-complementary transcripts within the producing cells, as well as in distant cells and tissues. Here, we used an artificial miRNA-based system (amiRSUL) in Arabidopsis thaliana to explore the still elusive mechanisms of inter-cellular miRNA movement via forward genetics. This screen identified many mutant alleles of HASTY (HST), the ortholog of mammalian EXPORTIN5 (XPO5) with a recently reported role in miRNA biogenesis in Arabidopsis. In both epidermis-peeling and grafting assays, amiRSUL levels were reduced much more substantially in miRNA-recipient tissues than in silencing-emitting tissues. We ascribe this effect to HST controlling cell-to-cell and phloem-mediated movement of the processed amiRSUL, in addition to regulating its biogenesis. While HST is not required for the movement of free GFP or siRNAs, its cell-autonomous expression in amiRSUL-emitting tissues suffices to restore amiRSUL movement independently of its nucleo-cytosolic shuttling activity. By contrast, HST is dispensable for the movement and activity of amiRSUL within recipient tissues. Finally, HST enables movement of endogenous miRNAs that display mostly unaltered steady-state levels in hst mutant tissues. We discuss a role for HST as a hitherto unrecognized regulator of miRNA movement in relation to its recently assigned nuclear function at the nexus of MIRNA transcription and miRNA processing.

Movement and differential consumption of short interfering RNA duplexes underlie mobile RNA interference.

In RNA interference (RNAi), the RNase III Dicer processes long double-stranded RNA (dsRNA) into short interfering RNA (siRNA), which, when loaded into ARGONAUTE (AGO) family proteins, execute gene silencing1. Remarkably, RNAi can act non-cell autonomously2,3: it is graft transmissible4-7, and plasmodesmata-associated proteins modulate its cell-to-cell spread8,9. Nonetheless, the molecular mechanisms involved remain ill defined, probably reflecting a disparity of experimental settings. Among other caveats, these almost invariably cause artificially enhanced movement via transitivity, whereby primary RNAi-target transcripts are converted into further dsRNA sources of secondary siRNA5,10,11. Whether siRNA mobility naturally requires transitivity and whether it entails the same or distinct signals for cell-to-cell versus long-distance movement remains unclear, as does the identity of the mobile signalling molecules themselves. Movement of long single-stranded RNA, dsRNA, free/AGO-bound secondary siRNA or primary siRNA have all been advocated12-15; however, an entity necessary and sufficient for all known manifestations of plant mobile RNAi remains to be ascertained. Here, we show that the same primary RNAi signal endows both vasculature-to-epidermis and long-distance silencing movement from three distinct RNAi sources. The mobile entities are AGO-free primary siRNA duplexes spreading length and sequence independently. However, their movement is accompanied by selective siRNA depletion reflecting the AGO repertoires of traversed cell types. Coupling movement with this AGO-mediated consumption process creates qualitatively distinct silencing territories, potentially enabling unlimited spatial gene regulation patterns well beyond those granted by mere gradients.

Genome-scale, single-cell-type resolution of microRNA activities within a whole plant organ.

Loaded into ARGONAUTE(AGO) proteins, eukaryotic micro(mi)RNAs regulate gene expression via cleavage, translational repression, and/or accelerated decay of sequence-complementary target transcripts. Despite their importance in development, cell identity maintenance and stress responses, how individual miRNAs contribute to spatial gene regulation within the complex cell mosaics formed in tissues/organs has remained inaccessible in any organism to date. We have developed a non-invasive methodology to examine, at single-cell-type resolution, the AGO-loading and activity patterns of entire miRNA cohorts in intact organs, applied here to the Arabidopsis root tip. A dual miRNAome-targetome analytical interface allowing intuitive data integration/visualization was developed as the basis for in-depth investigations via single-cell-type experimentation. These uncovered an array of so far speculative or hitherto unknown types of spatial miRNA-mediated gene regulation schemes, including via widespread cell-to-cell movement between contiguous layers of distinct identities. This study provides the proof of principle that minimally invasive, genome-scale analysis of miRNA activities within and between single-cell types of whole organs is achievable.

Imbalanced Regulation of Fungal Nutrient Transports According to Phosphate Availability in a Symbiocosm Formed by Poplar, Sorghum, and Rhizophagus irregularis.

In arbuscular mycorrhizal (AM) symbiosis, key components of nutrient uptake and exchange are specialized transporters that facilitate nutrient transport across membranes. As phosphate is a nutrient and a regulator of nutrient exchanges, we investigated the effect of P availability to extraradical mycelium (ERM) on both plant and fungus transcriptomes and metabolomes in a symbiocosm system. By perturbing nutrient exchanges under the control of P, our objectives were to identify new fungal genes involved in nutrient transports, and to characterize in which extent the fungus differentially modulates its metabolism when interacting with two different plant species. We performed transportome analysis on the ERM and intraradical mycelium of the AM fungus Rhizophagus irregularis associated to Populus trichocarpa and Sorghum bicolor under high and low P availability in ERM, using quantitative RT-PCR and Illumina mRNA-sequencing. We observed that mycorrhizal symbiosis induces expression of specific phosphate and ammonium transporters in both plants. Furthermore, we identified new AM-inducible transporters and showed that a subset of phosphate transporters is regulated independently of symbiotic nutrient exchange. mRNA-Sequencing revealed that the fungal transportome was not similarly regulated in the two host plant species according to P availability. Mirroring this effect, many plant carbohydrate transporters were down-regulated in P. trichocarpa mycorrhizal root tissue. Metabolome analysis revealed further that AM root colonization led to a modification of root primary metabolism under low and high P availability and to a decrease of primary metabolite pools in general. Moreover, the down regulation of the sucrose transporters suggests that the plant limits carbohydrate long distance transport (i.e. from shoot to the mycorrhizal roots). By simultaneous uptake/reuptake of nutrients from the apoplast at the biotrophic interface, plant and fungus are both able to control reciprocal nutrient fluxes.

A Suppressor Screen for AGO1 Degradation by the Viral F-Box P0 Protein Uncovers a Role for AGO DUF1785 in sRNA Duplex Unwinding.

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCFP0) complex and subsequent degradation. The AGO1 P0 degron is conserved and confers P0-mediated degradation to other AGO proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening) compromises recognition by SCFP0 Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding, leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and the in vivo function of DUF1785 in RNA silencing.

Structural Flexibility Enables Alternative Maturation, ARGONAUTE Sorting and Activities of miR168, a Global Gene Silencing Regulator in Plants.

In eukaryotes, the RNase-III Dicer often produces length/sequence microRNA (miRNA) variants, called "isomiRs", owing to intrinsic structural/sequence determinants of the miRNA precursors (pre-miRNAs). In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 (AGO1). We identified a motif conserved among plant pre-miR168 orthologs, which enables flexible internal base-pairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via "transitive RNAi", a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGO1-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of pre-miRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.

Nucleo-cytosolic Shuttling of ARGONAUTE1 Prompts a Revised Model of the Plant MicroRNA Pathway.

Unlike in metazoans, plant microRNAs (miRNAs) undergo stepwise nuclear maturation before engaging cytosolic, sequence-complementary transcripts in association with the silencing effector protein ARGONAUTE1 (AGO1). Since their discovery, how and under which form plant miRNAs translocate to the cytosol has remained unclear, as has their sub-cellular AGO1 loading site(s). Here, we show that the N termini of all plant AGO1s contain a nuclear-localization (NLS) and nuclear-export signal (NES) that, in Arabidopsis thaliana (At), enables AtAGO1 nucleo-cytosolic shuttling in a Leptomycin-B-inhibited manner, diagnostic of CRM1(EXPO1)/NES-dependent nuclear export. Nuclear-only AtAGO1 contains the same 2'O-methylated miRNA cohorts as its nucleo-cytosolic counterpart, but it preferentially interacts with the miRNA loading chaperone HSP90. Furthermore, mature miRNA translocation and miRNA-mediated silencing both require AtAGO1 nucleo-cytosolic shuttling. These findings lead us to propose a substantially revised view of the plant miRNA pathway in which miRNAs are matured, methylated, loaded into AGO1 in the nucleus, and exported to the cytosol as AGO1:miRNA complexes in a CRM1(EXPO1)/NES-dependent manner.

A genome-wide transcriptome and translatome analysis of Arabidopsis transposons identifies a unique and conserved genome expression strategy for Ty1/Copia retroelements.

Retroelements, the prevalent class of plant transposons, have major impacts on host genome integrity and evolution. They produce multiple proteins from highly compact genomes and, similar to viruses, must have evolved original strategies to optimize gene expression, although this aspect has been seldom investigated thus far. Here, we have established a high-resolution transcriptome/translatome map for the near-entirety of Arabidopsis thaliana transposons, using two distinct DNA methylation mutants in which transposon expression is broadly de-repressed. The value of this map to study potentially intact and transcriptionally active transposons in A. thaliana is illustrated by our comprehensive analysis of the cotranscriptional and translational features of Ty1/Copia elements, a family of young and active retroelements in plant genomes, and how such features impact their biology. Genome-wide transcript profiling revealed a unique and widely conserved alternative splicing event coupled to premature termination that allows for the synthesis of a short subgenomic RNA solely dedicated to production of the GAG structural protein and that preferentially associates with polysomes for efficient translation. Mutations engineered in a transgenic version of the Arabidopsis EVD Ty1/Copia element further show how alternative splicing is crucial for the appropriate coordination of full-length and subgenomic RNA transcription. We propose that this hitherto undescribed genome expression strategy, conserved among plant Ty1/Copia elements, enables an excess of structural versus catalytic components, mandatory for mobilization.

A single miR390 targeting event is sufficient for triggering TAS3-tasiRNA biogenesis in Arabidopsis.

In plants, tasiRNAs form a class of endogenous secondary siRNAs produced through the action of RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6) upon microRNA-mediated cleavage of non-coding TAS RNAs. In Arabidopsis thaliana, TAS1, TAS2 and TAS4 tasiRNA production proceeds via a single cleavage event mediated by 22nt-long or/and asymmetric miRNAs in an ARGONAUTE-1 (AGO1)-dependent manner. By contrast, tasiRNA production from TAS3 seems to follow the so-called 'two-hit' process, where dual targeting of TAS3, specifically mediated by the 21nt-long, symmetric miR390, initiates AGO7-dependent tasiRNA production. Interestingly, features for TAS3 tasiRNA production differ in other plant species and we show here that such features also enable TAS3 tasiRNA biogenesis in Arabidopsis, and that a single miR390 targeting event is, in fact, sufficient for this process, suggesting that the 'one-hit' model underpins all the necessary rudiments of secondary siRNA biogenesis from plant TAS transcripts. Further results suggest that the two-hit configuration likely enhances the fidelity of tasiRNA production and, hence, the accuracy of downstream gene regulation. Finally, we show that a 'non-cleavable one-hit' process allows tasiRNA production from both TAS1 and TAS3 transcripts, indicating that RDR6 recruitment does not require miRNA cleavage, nor does the recruitment, as we further show, of SUPRRESSOR-OF-GENE-SILENCING-3, indispensable for tasiRNA generation.

Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays.

Epigenetic response to stress in plants involves changes in DNA methylation, histone modifications, and expression of small noncoding RNAs (sRNA). Here we present the method of analysis of differential expression of sRNA populations using DNA tiling arrays. sRNA extracted from Arabidopsis thaliana plants exposed to pathogen elicitor or control plants were reverse-transcribed into cDNAs, and subsequently hybridized after labeling to a custom-made DNA tiling array covering Arabidopsis chromosome 4. We first designed a control experiment with eight cDNA clones corresponding to sequences located on chromosome 4 and obtained robust and specific hybridization signals. Furthermore, hybridization signals along chromosome 4 were in good agreement with sRNA abundance as previously determined by massive parallel sequence signature (MPSS) in the case of untreated plants, but differed substantially after stress treatment. These results demonstrate the utility of hybridization to DNA tiling arrays to detect major changes in sRNA abundance.

DNA Methylation Influences the Expression of DICER-LIKE4 Isoforms, Which Encode Proteins of Alternative Localization and Function.

Plant RNA silencing operates via RNA-directed DNA-methylation (RdDM) to repress transcription or by targeting mRNAs via posttranscriptional gene silencing (PTGS). These pathways rely on distinct Dicer-like (DCL) proteins that process double-stranded RNA (dsRNA) into small-interfering RNAs (siRNAs). Here, we explored the expression and subcellular localization of Arabidopsis thaliana DCL4. DCL4 expression predominates as a transcription start site isoform encoding a cytoplasmic protein, which also represents the ancestral form in plants. A longer DCL4 transcript isoform encoding a nuclear localization signal, DCL4NLS, is present in Arabidopsis, but DNA methylation normally suppresses its expression. Hypomethylation caused by mutation, developmental reprogramming, and biotic stress correlates with enhanced DCL4NLS expression, while hypermethylation of a DCL4 transgene causes a reduction in DCL4NLS expression. DCL4NLS functions in a noncanonical siRNA pathway, producing a unique set of 21-nucleotide-long "disiRNAs," for DCL4NLS isoform-dependent siRNAs, through the nuclear RdDM dsRNA synthesis pathway. disiRNAs originate mostly from transposable elements (TEs) and TE-overlapping/proximal genes, load into the PTGS effector ARGONAUTE1 (AGO1), and display a subtle effect on transcript accumulation together with overlapping 24-nucleotide siRNAs. We propose that, via PTGS, disiRNAs could help to tighten the expression of epigenetically activated TEs and genes using the methylation-state-responsive DCL4NLS.

Correction: The Arabidopsis miR472-RDR6 silencing pathway modulates PAMP- and effector-triggered immunity through the post-transcriptional control of disease resistance genes.

[This corrects the article DOI: 10.1371/journal.ppat.1003883.].

Dominance hierarchy arising from the evolution of a complex small RNA regulatory network.

The prevention of fertilization through self-pollination (or pollination by a close relative) in the Brassicaceae plant family is determined by the genotype of the plant at the self-incompatibility locus (S locus). The many alleles at this locus exhibit a dominance hierarchy that determines which of the two allelic specificities of a heterozygous genotype is expressed at the phenotypic level. Here, we uncover the evolution of how at least 17 small RNA (sRNA)-producing loci and their multiple target sites collectively control the dominance hierarchy among alleles within the gene controlling the pollen S-locus phenotype in a self-incompatible Arabidopsis species. Selection has created a dynamic repertoire of sRNA-target interactions by jointly acting on sRNA genes and their target sites, which has resulted in a complex system of regulation among alleles.

The diversity of small non-coding RNAs in the diatom Phaeodactylum tricornutum.

BACKGROUND: Marine diatoms constitute a major component of eukaryotic phytoplankton and stand at the crossroads of several evolutionary lineages. These microalgae possess peculiar genomic features and novel combinations of genes acquired from bacterial, animal and plant ancestors. Furthermore, they display both DNA methylation and gene silencing activities. Yet, the biogenesis and regulatory function of small RNAs (sRNAs) remain ill defined in diatoms. RESULTS: Here we report the first comprehensive characterization of the sRNA landscape and its correlation with genomic and epigenomic information in Phaeodactylum tricornutum. The majority of sRNAs is 25 to 30 nt-long and maps to repetitive and silenced Transposable Elements marked by DNA methylation. A subset of this population also targets DNA methylated protein-coding genes, suggesting that gene body methylation might be sRNA-driven in diatoms. Remarkably, 25-30 nt sRNAs display a well-defined and unprecedented 180 nt-long periodic distribution at several highly methylated regions that awaits characterization. While canonical miRNAs are not detectable, other 21-25 nt sRNAs of unknown origin are highly expressed. Besides, non-coding RNAs with well-described function, namely tRNAs and U2 snRNA, constitute a major source of 21-25 nt sRNAs and likely play important roles under stressful environmental conditions. CONCLUSIONS: P. tricornutum has evolved diversified sRNA pathways, likely implicated in the regulation of largely still uncharacterized genetic and epigenetic processes. These results uncover an unexpected complexity of diatom sRNA population and previously unappreciated features, providing new insights into the diversification of sRNA-based processes in eukaryotes.

Exploring new models of easiRNA biogenesis.

Although silent transposons in plants can be reactivated by stress or during development, their potential deleterious effects are prevented by transposon-derived epigenetically activated small interfering RNAs (easiRNAs). A new study shows how serendipitous interactions between reactivated transposons and endogenous microRNAs might initiate easiRNA biogenesis, establishing an unexpected link between these two classes of silencing small RNAs.

The Arabidopsis miR472-RDR6 silencing pathway modulates PAMP- and effector-triggered immunity through the post-transcriptional control of disease resistance genes.

RNA-DEPENDENT RNA POLYMERASE 6 (RDR6) is a key RNA silencing factor initially characterized in transgene silencing and virus resistance. This enzyme also contributes to the biosynthesis of endogenous short interfering RNAs (siRNAs) from non-coding RNAs, transposable elements and protein-coding transcripts. One class of protein-coding transcripts that have recently emerged as major sources of RDR6-dependent siRNAs are nucleotide-binding leucine-rich repeat (NB-LRR) proteins, a family of immune-receptors that perceive specific pathogen effector proteins and mount Effector-Triggered Immunity (ETI). Nevertheless, the dynamic post-transcriptional control of NB-LRR transcripts during the plant immune response and the functional relevance of NB-LRRs in signaling events triggered by Pathogen-Associated Molecular Patterns (PAMPs) remain elusive. Here, we show that PTI is constitutive and sensitized in the Arabidopsis rdr6 loss-of-function mutant, implicating RDR6 as a novel negative regulator of PTI. Accordingly, rdr6 mutant exhibits enhanced basal resistance towards a virulent Pseudomonas syringae strain. We further provide evidence that dozens of CC-NB-LRRs (CNLs), including the functionally characterized RPS5 gene, are post-transcriptionally controlled by RDR6 both constitutively and during PTI. These CNL transcripts are also regulated by the Arabidopsis microRNA miR472 and knock-down of this miRNA recapitulates the PTI and basal resistance phenotypes observed in the rdr6 mutant background. Furthermore, both miR472 and rdr6 mutants were more resistant to Pto DC3000 expressing AvrPphB, a bacterial effector recognized by the disease resistance protein RPS5, whereas transgenic plants overexpressing miR472 were more susceptible to this bacterial strain. Finally, we show that the enhanced basal and RPS5-mediated resistance phenotypes observed in the rdr6 mutant are dependent on the proper chaperoning of NB-LRR proteins, and might therefore be due to the enhanced accumulation of CNL proteins whose cognate mRNAs are no longer controlled by RDR6-dependent siRNAs. Altogether, this study supports a model whereby the miR472- and RDR6-mediated silencing pathway represents a key regulatory checkpoint modulating both PTI and ETI responses through the post-transcriptional control of disease resistance genes.

RNAi-dependent and independent control of LINE1 accumulation and mobility in mouse embryonic stem cells.

In most mouse tissues, long-interspersed elements-1 (L1s) are silenced via methylation of their 5'-untranslated regions (5'-UTR). A gradual loss-of-methylation in pre-implantation embryos coincides with L1 retrotransposition in blastocysts, generating potentially harmful mutations. Here, we show that Dicer- and Ago2-dependent RNAi restricts L1 accumulation and retrotransposition in undifferentiated mouse embryonic stem cells (mESCs), derived from blastocysts. RNAi correlates with production of Dicer-dependent 22-nt small RNAs mapping to overlapping sense/antisense transcripts produced from the L1 5'-UTR. However, RNA-surveillance pathways simultaneously degrade these transcripts and, consequently, confound the anti-L1 RNAi response. In Dicer(-/-) mESC complementation experiments involving ectopic Dicer expression, L1 silencing was rescued in cells in which microRNAs remained strongly depleted. Furthermore, these cells proliferated and differentiated normally, unlike their non-complemented counterparts. These results shed new light on L1 biology, uncover defensive, in addition to regulatory roles for RNAi, and raise questions on the differentiation defects of Dicer(-/-) mESCs.