A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA, we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using mutants that block small RNA (sRNA) biogenesis in either source or recipient tissue, we found that transgene-derived sRNA as well as a substantial proportion of the endogenous sRNA had moved across the graft union, and we provide evidence that 24-nucleotide mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defense and responses to external stimuli that have persistent effects in plants.

contributed equally to this work Temperature dramatically affects plant±virus interactions. Outbreaks of virus diseases are frequently associated with low temperature, while at high temperature viral symptoms are often attenuated (heat masking) and plants rapidly recover from virus diseases. However, the underlying mechanisms of these well-known observations are not yet understood. RNA silencing is a conserved defence system of eukaryotic cells, which operates against molecular parasites including viruses and transgenes. Here we show that at low temperature both virus and transgene triggered RNA silencing are inhibited. Therefore, in cold, plants become more susceptible to viruses, and RNA silencing-based phenotypes of transgenic plants are lost. Consistently, the levels of virus-and transgenederived small (21±26 nucleotide) interfering (si) RNAsÐthe central molecules of RNA silencing-mediated defence pathwaysÐare dramatically reduced at low temperature. In contrast, RNA silencing was activated and the amount of siRNAs gradually increased with rising temperature. However, temperature does not in¯uence the accumulation of micro (mi) RNAs, which play a role in developmental regulation, suggesting that the two classes of small (si and mi) RNAs are generated by different nuclease complexes.

MicroRNAs (miRNAs) in eukaryotes guide post-transcriptional regulation by means of targeted RNA degradation and translational arrest. They are released by a Dicer nuclease as a 21-24-nucleotide RNA duplex from a precursor in which an imperfectly matched inverted repeat forms a partly double-stranded region. One of the two strands is then recruited by an Argonaute nuclease that is the effector protein of the silencing mechanism. Short interfering RNAs (siRNAs), which are similar to miRNAs, are also produced by Dicer but the precursors are perfectly double-stranded RNA. These siRNAs guide post-transcriptional regulation, as with miRNAs, and epigenetic genome modification. Diverse eukaryotes including fungi, plants, protozoans and metazoans produce siRNAs but, until now, miRNAs have not been described in unicellular organisms and it has been suggested that they evolved together with multicellularity in separate plant and animal lineages. Here we show that the unicellular alga Chlamydomonas reinhardtii contains miRNAs, putative evolutionary precursors of miRNAs and species of siRNAs resembling those in higher plants. The common features of miRNAs and siRNAs in an alga and in higher plants indicate that complex RNA-silencing systems evolved before multicellularity and were a feature of primitive eukaryotic cells.

RNA silencing is conserved in a broad range of eukaryotes and includes the phenomena of RNA interference in animals and posttranscriptional gene silencing (PTGS) in plants. In plants, PTGS acts as an antiviral system; a successful virus infection requires suppression or evasion of the induced silencing response. Small interfering RNAs (siRNAs) accumulate in plants infected with positive-strand RNA viruses and provide specificity to this RNA-mediated defense. We present here the results of a survey of virus-specific siRNAs characterized by a sequence analysis of siRNAs from plants infected with Cymbidium ringspot tombusvirus (CymRSV). CymRSV siRNA sequences have a nonrandom distribution along the length of the viral genome, suggesting that there are hot spots for virus-derived siRNA generation. CymRSV siRNAs bound to the CymRSV p19 suppressor protein have the same asymmetry in strand polarity as the sequenced siRNAs and are imperfect double-stranded RNA duplexes. Moreover, an analysis of siRNAs derived from two other nonrelated positive-strand RNA viruses showed that they displayed the same asymmetry as CymRSV siRNAs. Finally, we show that Tobacco mosaic virus (TMV) carrying a short inverted repeat of the phytoene desaturase (PDS) gene triggered more accumulation of PDS siRNAs than the corresponding antisense PDS sequence. Taken together, these results suggest that virus-derived siRNAs originate predominantly by direct DICER cleavage of imperfect duplexes in the most folded regions of the positive strand of the viral RNA.Eukaryotic organisms have developed a highly adaptable and specific mechanism to protect their genomes against aberrant endogenous or exogenous RNA molecules. This phenomenon, referred to as RNA silencing, is an ancient defense mechanism induced by double-stranded RNAs (dsRNAs) that leads to homology-dependent degradation of target RNAs. RNA silencing is conserved across kingdoms and is manifested as quelling in fungi, RNA interference (RNAi) in animals, and cosuppression or posttranscriptional gene silencing (PTGS) in plants. The unifying feature of RNA silencing is the presence of 21-to 26-nucleotide (nt) small interfering RNAs (siRNAs) (17,18,40). In addition, biochemical and genetic analyses have shown that the core mechanisms of RNA silencing are shared among different eukaryotes (5,20,32,40,55,60). RNA silencing is induced by dsRNAs or structured single-stranded RNAs (ssRNAs) that are processed into siRNAs by RNase III-like enzymes such as DICER (6, 37). siRNAs guide the sequencespecific degradation of target mRNAs by the RNA-induced silencing complex (RISC) (19). The RISC mediates the cleavage of a target mRNA when there is perfect or nearly perfect base pairing between the mRNA and a short guide RNA and mediates translation repression when there is partial complementarity (3,10,13,23). siRNAs can also guide another effector complex, namely, the RNA-induced initiation of transcriptional gene silencing (RITS) complex, to direct the chromatin modification of homologous DNA sequences (53).In ...

SummaryMicroRNAs (miRNAs) are small RNAs, 21 to 22 nucleotides long, with important regulatory roles. They are processed from longer RNA molecules with imperfectly matched foldback regions and they function in modulating the stability and translation of mRNA. Recently, we and others have demonstrated that the unicellular alga Chlamydomonas reinhardtii, like diverse multicellular organisms, contains miRNAs. These RNAs resemble the miRNAs of land plants in that they direct site-specific cleavage of target mRNA with miRNA-complementary motifs and, presumably, act as regulatory molecules in growth and development. Utilizing these findings we have developed a novel artificial miRNA system based on ligation of DNA oligonucleotides that can be used for specific high-throughput gene silencing in green algae.

In most eukaryotes, double-stranded RNA is processed into small RNAs that are potent regulators of gene expression. This gene silencing process is known as RNA silencing or RNA interference (RNAi) and, in plants and nematodes, it is associated with the production of a mobile signal that can travel from cell-to-cell and over long distances. The sequence-specific nature of systemic RNA silencing indicates that a nucleic acid is a component of the signalling complex. Recent work has shed light on the mobile RNA species, the genes involved in the production and transport of the signal. This review discusses the advances in systemic RNAi and presents the current challenges and questions in this rapidly evolving field.

We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3′ and/or 5′ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5′ differences and in support of this we report that a 5′ isomiR-9–1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5′ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes.

Post-transcriptional gene silencing (PTGS) is INTRODUCTIONPost-transcriptional gene silencing (PTGS), first identified in plants, is now thought to be an ancient self-defense mechanism acting against molecular parasites (Waterhouse et al., 2001). Introduction of double-stranded RNA (dsRNA) into plant cells triggers PTGS, resulting in the degradation of dsRNA and cognate mRNAs (Schweizer et al., 2000). A similar mechanism appears to operate in a wide variety of organisms, including filamentous fungi, nematodes, Drosophila , mice, and cultured HeLa cells, and generally is referred to as RNA interference (RNAi) (Cogoni and Macino, 1999a;Fire, 1999;Grant, 1999;Sharp and Zamore, 2000;Elbashir et al., 2001a;Svoboda et al., 2000). Recently, homologous genes required for PTGS were identified from different organisms, demonstrating the conservation of the gene-silencing machinery Macino, 1999a, 1999b;Ketting et al., 1999;Tabara et al., 1999;Catalanotto et al., 2000;Dalmay et al., 2000Dalmay et al., , 2001Domeier et al., 2000;Fagard et al., 2000;Mourrain et al., 2000;Smardon et al., 2000;Wu-Scharf et al., 2000). The accumulation of 21-to 25-nucleotide RNAs corresponding to both sense and antisense strands of target RNA occurs during PTGS in plant and animal cells (Hamilton and Baulcombe, 1999;Hammond et al., 2000;Parrish et al., 2000). These 21-to 25-nucleotide RNAs are generated by an RNase III-like enzyme (DICER) as the initiation step of RNAi, providing the specificity of a second RNase complex (RISC) that targets the cognate single-stranded (ss) RNAs (Bernstein et al., 2001).In plants, PTGS has evolved as an antiviral system. PTGS is triggered efficiently by dsRNA intermediates of cytoplasmically replicating viruses. The RNA genome of the invading virus is targeted and eliminated specifically when this natural antiviral mechanism is activated (Waterhouse et al., 1998(Waterhouse et al., , 1999Baulcombe, 1999;Smith et al., 2000;. In higher plants, PTGS is not limited to the cells in which it is activated, because mobile signals produced by PTGS can spread and confer sequence-specific RNA degradation in distant tissues (Palauqui et al., 1997;Voinnet and Baulcombe, 1997).Consistent with the importance of PTGS as an antiviral response, many viruses encode gene-silencing suppressor proteins (Anandalakshmi et al., 1998;Beclin et al., 1998;Brigneti et al., 1998;Kasschau and Carrington, 1998;Voinnet et al., 1999Voinnet et al., , 2000. However, not all viruses are able to suppress PTGS, and some virus-infected plants recover after the development of the first systemic viral symptoms (e.g., 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed. E-mail burgyan@ abc.hu; fax 36-28-430-416. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010366. 360The Plant Cell nepovirus-infected tobacco plants; Ratcliff et al., 1997). Upper leaves of recovered plants lack symptoms (or show attenuated symptoms), and the virus content in these leav...