RNA polymerase V-dependent small RNAs in Arabidopsis originate from small, intergenic loci including most SINE repeats.

In plants, heterochromatin is maintained by a small RNA-based gene silencing mechanism known as RNA-directed DNA methylation (RdDM). RdDM requires the non-redundant functions of two plant-specific DNA-dependent RNA polymerases (RNAP), RNAP IV and RNAP V. RNAP IV plays a major role in siRNA biogenesis, while RNAP V may recruit DNA methylation machinery to target endogenous loci for silencing. Although small RNA-generating regions that are dependent on both RNAP IV and RNAP V have been identified previously, the genomic loci targeted by RNAP V for siRNA accumulation and silencing have not been described extensively. To characterize the RNAP V-dependent, heterochromatic siRNA-generating regions in the Arabidopsis genome, we deeply sequenced the small RNA populations of wild-type and RNAP V null mutant (nrpe1) plants. Our results showed that RNAP V-dependent siRNA-generating loci are associated predominately with short repetitive sequences in intergenic regions. Suppression of small RNA production from short repetitive sequences was also prominent in RdDM mutants including dms4, drd1, dms3 and rdm1, reflecting the known association of these RdDM effectors with RNAP V. The genomic regions targeted by RNAP V were small, with an estimated average length of 238 bp. Our results suggest that RNAP V affects siRNA production from genomic loci with features dissimilar to known RNAP IV-dependent loci. RNAP V, along with RNAP IV and DRM1/2, may target and silence a set of small, intergenic transposable elements located in dispersed genomic regions for silencing. Silencing at these loci may be actively reinforced by RdDM

Planting the Seeds of a New Paradigm

RNA-mediated gene silencing has emerged in recent years as an important mechanism for regulating gene expression. Some of the key discoveries have been made in plant

AGO6 Functions in RNA-Mediated Transcriptional Gene Silencing in Shoot and Root Meristems in Arabidopsis thaliana

RNA-directed DNA methylation (RdDM) is a small interfering RNA (siRNA)-mediated epigenetic modification that contributes to transposon silencing in plants. RdDM requires a complex transcriptional machinery that includes specialized RNA polymerases, named Pol IV and Pol V, as well as chromatin remodelling proteins, transcription factors, RNA binding proteins, and other plant-specific proteins whose functions are not yet clarified. In Arabidopsis thaliana, DICER-LIKE3 and members of the ARGONAUTE4 group of ARGONAUTE (AGO) proteins are involved, respectively, in generating and using 24-nt siRNAs that trigger methylation and transcriptional gene silencing of homologous promoter sequences. AGO4 is the main AGO protein implicated in the RdDM pathway. Here we report the identification of the related AGO6 in a forward genetic screen for mutants defective in RdDM and transcriptional gene silencing in shoot and root apical meristems in Arabidopsis thaliana. The identification of AGO6, and not AGO4, in our screen is consistent with the primary expression of AGO6 in shoot and root growing points

Early Examples of Gene Silencing in Transgenic Plants

<p>TGS: Normally when two plants harboring separate transgenes encoding resistance to kanamycin (kan) or hygromycin (hyg), respectively, are crossed, 50% of the progeny are resistant to the individual antibiotics and 25% are resistant to a combination of both (top). In cases of silencing, expression of the KAN marker is extinguished in the presence of the HYG marker, as indicated by only 25% kan resistance and no double resistance (middle). PTGS: Transformation of wild-type petunia (bottom left) with a transgene encoding a pigment protein can lead to loss of pigment (white areas) owing to cosuppression of the transgene and homologous endogenous plant gene. (Photos on the left and in the middle were provided by Jan Kooter and on the right were provided by Natalie Doetsch and Rich Jorgensen.)</p

RNA-Mediated Silencing

<p>Short RNAs derived from Dicer cleavage of dsRNA are incorporated into multiprotein effector complexes, such as RISC and RITS (RNA-induced initiation of TGS) (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020133#pbio-0020133-Verdel1" target="_blank">Verdel et al. 2004</a>) to target mRNA degradation (RNAi/PTGS), translation inhibition, or TGS and genome modifications. ARGONAUTE (AGO) proteins (the name comes from a plant mutant [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020133#pbio-0020133-Bohmert1" target="_blank">Bohmert et al. 1998</a>]) bind short RNAs and ‘shepherd’ them to appropriate effector complexes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020133#pbio-0020133-Carmell1" target="_blank">Carmell et al. 2002</a>). siRNAs originate from perfect RNA duplexes, which can be produced by RDR activity on ssRNA templates; miRNAs originate from imperfect RNA hairpins that are encoded in intergenic regions of plant and animal genomes. Functions are shown at the bottom. In addition to roles in transgene silencing, both TGS and RNAi/PTGS control genome parasites called transposons (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020133#pbio-0020133-Flavell1" target="_blank">Flavell 1994</a>; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020133#pbio-0020133-Plasterk1" target="_blank">Plasterk 2002</a>). Genome modifications (DNA and histone methylation) can potentially be targeted by short RNAs that basepair to DNA or to nascent RNA synthesized from the target gene (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020133#pbio-0020133-Grewal1" target="_blank">Grewal and Moazed 2003</a>). Target nucleic acids are shown in blue, short RNAs in red, proteins and enzyme complexes as ovals.</p