Transitivity-dependent and -independent cell-to-cell movement of RNA silencing

One manifestation of RNA silencing, known as post-transcriptional gene silencing (PTGS) in plants and RNA interference (RNAi) in animals, is a nucleotide sequence-specific RNA turnover mechanism with the outstanding property of propagating throughout the organism, most likely via movement of nucleic acids. Here, the cell-to-cell movement of RNA silencing in plants is investigated. We show that a short-distance movement process, once initiated from a small group of cells, can spread over a limited and nearly constant number of cells, independent of the presence of homologous transcripts. There is also a long-range cell-to-cell movement process that occurs as a relay amplification, which requires the combined activity of SDE1, a putative RNA-dependent RNA polymerase, and SDE3, a putative RNA helicase. Extensive and limited cell-to-cell movements of silencing are triggered by the same molecules, occur within the same tissues and likely recruit the same plasmodesmata channels. We propose that they are in fact manifestations of the same process, and that extensive cell-to-cell movement of RNA silencing results from re-iterated short-distance signalling events. The likely nature of the nucleic acids involved is presented

Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins

In plants, worms, and fungi, RNA-dependent RNA polymerases (RDRs) amplify the production of short-interfering RNAs (siRNAs) that mediate RNA silencing. In Arabidopsis, RDR6 is thought to copy endogenous and exogenous RNA templates into double-stranded RNAs (dsRNAs), which are subsequently processed into siRNAs by one or several of the four Dicer-like enzymes (DCL1→4). This reaction produces secondary siRNAs corresponding to sequences outside the primary targeted regions of a transcript, a phenomenon called transitivity. One recognized role of RDR6 is to strengthen the RNA silencing response mounted by plants against viruses. Accordingly, suppressor proteins deployed by viruses inhibit this defense. However, interactions between silencing suppressors and RDR6 have not yet been documented. Additionally, the mechanism underlying transitivity remains poorly understood. Here, we report how several viral silencing suppressors inhibit the RDR6-dependent amplification of virus-induced and transgene-induced gene silencing. Viral suppression of primary siRNA accumulation shows that transitivity can be initiated with minute amounts of DCL4-dependent 21-nucleotide (nt)-long siRNAs, whereas DCL3-dependent 24-nt siRNAs appear dispensable for this process. We further show that unidirectional (3→5′) transitivity requires the hierarchical and redundant functions of DCL4 and DCL2 acting downstream from RDR6 to produce 21- and 22-nt-long siRNAs, respectively. The 3→5′ transitive reaction is likely to be processive over >750 nt, with secondary siRNA production progressively decreasing as the reaction proceeds toward the 5′-proximal region of target transcripts. Finally, we show that target cleavage by a primary small RNA and 3→5′ transitivity can be genetically uncoupled, and we provide in vivo evidence supporting a key role for priming in this specific reaction

In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA

We show, with miR171, that plant miRNA genes are modular independent transcription units in which the fold-back pre-miRNA is sufficient for miRNA processing, and that the upstream region contains highly specific promoter elements. Processing depends on flanking sequences within the miRNA stem-loop precursor rather than the miRNA sequence itself, and mutations affecting target pairing at the center and 5′ but not 3′ region of the miRNA compromise its function in vivo. Inactivation of the SDE1 RNA-dependent-RNA-polymerase was mandatory for accurate representation of miRNA activity by sensor constructs in Arabidopsis. Work in sde1 background revealed a near-perfect spatial overlap between the patterns of miR171 transcription and activity, supporting the idea that plant miRNAs enable cell differentiation

An endogenous, systemic RNAi pathway in plants (Retracted article. See vol. 34, pg. 2596, 2015)

Recent work on metazoans has uncovered the existence of an endogenous RNA-silencing pathway that functionally recapitulates the effects of experimental RNA interference (RNAi) used for gene knockdown in organisms such as Caenorhabditis elegans and Drosophila. The endogenous short interfering (si) RNA involved in this pathway are processed by Dicer-like nucleases from genomic loci re-arranged to form extended inverted repeats (IRs) that produce perfect or near-perfect dsRNA molecules. Although such IR loci are commonly detected in plant genomes, their genetics, evolution and potential contribution to plant biology through endogenous silencing have remained largely unexplored. Through an exhaustive analysis performed using Arabidopsis, we provide here evidence that at least two such endogenous IRs are genetically virtually indistinguishable from the transgene constructs commonly used for RNAi in plants. We show how these loci can be useful probes of the cellular mechanism and fluidity of RNA-silencing pathways in plants, and provide evidence that they may arise and disappear on an ecotype scale, show highly cell-specific expression patterns and respond to various stresses. IR loci thus have the potential to act as molecular sensors of the local environments found within distinct ecological plant niches. We further show that the various siRNA size classes produced by at least one of these IR loci are functionally loaded into cognate effector proteins and mediate both post-transcriptional gene silencing and RNA-directed DNA methylation (RdDM) of endogenous as well as exogenous targets. Finally, and as previously reported during plant experimental RNAi, we provide evidence that endogenous IR-derived siRNAs of all size classes are not cell-autonomous and can be transported through graft junctions over long distances, in target tissues where they are functional, at least in mediating RdDM. Collectively, these results define the existence of a bona fide, endogenous and systemic RNAi pathway in plants that may have implications in adaptation, epiallelism and trans-generational memory. The EMBO Journal (2010) 29, 1699-1712. doi: 10.1038/ emboj. 2010.65; Published online 22 April 201

A cellular microRNA mediates antiviral defense in human cells.

International audienceIn eukaryotes, 21- to 24-nucleotide-long RNAs engage in sequence-specific interactions that inhibit gene expression by RNA silencing. This process has regulatory roles involving microRNAs and, in plants and insects, it also forms the basis of a defense mechanism directed by small interfering RNAs that derive from replicative or integrated viral genomes. We show that a cellular microRNA effectively restricts the accumulation of the retrovirus primate foamy virus type 1 (PFV-1) in human cells. PFV-1 also encodes a protein, Tas, that suppresses microRNA-directed functions in mammalian cells and displays cross-kingdom antisilencing activities. Therefore, through fortuitous recognition of foreign nucleic acids, cellular microRNAs have direct antiviral effects in addition to their regulatory functions