No abstract

The Arabidopsis genome encodes four Dicer-like (DCL) proteins, two of which contain putative nuclear localization signals. This suggests one or more nuclear pathways for processing double-stranded (ds) RNA in plants. To study the subcellular location of processing of nuclear-encoded dsRNA involved in transcriptional silencing, we examined short interfering (si) RNA and micro (mi) RNA accumulation in transgenic Arabidopsis expressing nuclear and cytoplasmic variants of P19, a viral protein that suppresses posttranscriptional gene silencing. P19 binds specifically to DCL-generated 21-to 25-nucleotide (nt) dsRNAs with 2-nt 3Ј overhangs and reportedly suppresses the accumulation of all size classes of siRNA. Nuclear P19 resulted in a significant reduction of 21-to 22-nt siRNAs and a 21-nt miRNA, but had a lesser effect on 24-nt siRNAs. Cytoplasmic P19 did not decrease the quantity but resulted in a 2-nt truncation of siRNAs and miRNA. This suggests that the direct products of DCL cleavage of dsRNA precursors of 21-to 22-nt siRNAs and miRNA are present in the nucleus, where their accumulation is partially repressed, and in the cytoplasm, where both normal sized and truncated forms accumulate. DCL1, which contains two putative nuclear localization signals, is required for miRNA production but not siRNA production. DCL1-green fluorescent protein fusion proteins localize to nuclei in transient expression assays, indicating that DCL1 is a nuclear protein. The results are consistent with a model in which dsRNA precursors of miRNAs and at least some 21-to 22-nt siRNAs are processed in the nucleus, the former by nuclear DCL1 and the latter by an unknown nuclear DCL."RNA silencing" is the suppression of gene expression through nucleotide (nt) sequence-specific interactions that are mediated by RNA (Voinnet, 2002). RNA silencing is triggered by double-stranded (ds) RNA that is processed by an RNase III activity termed Dicer into short RNAs 21 to 25 nt in length (Hannon, 2002;Zamore, 2002). In plants, RNA silencing can act at the posttranscriptional and transcriptional levels (Cerutti, 2003). The 21-to 22-nt short interfering (si) RNAs and micro (mi) RNAs silence genes posttranscriptionally by targeting cognate mRNAs for degradation by an endonuclease complex (Llave et al., 2002a;Tang et al., 2003). A longer class of siRNAs in plants (24-26 nt) has been implicated in directing homologous DNA methylation and in systemic silencing (Hamilton et al., 2002). RNA-directed DNA methylation (RdDM) can lead to transcriptional gene silencing (TGS) if promoter sequences are targeted by homologous RNA (Mette et al., 1999(Mette et al., , 2000Jones et al., 1999Jones et al., , 2001Sijen et al., 2001;Aufsatz et al., 2002aAufsatz et al., , 2002b.The length and functional diversity of short RNAs in plants are reflected in the multiplicity of Dicer-like (DCL) activities. In contrast to genomes of other organisms, which encode one (human, mouse, fission yeast [Schizosaccharomyces pombe], and Caenorhabditis elegans) or two (fruitfly [Drosophila mel...

The importance of maternal cells in controlling early embryogenesis is well understood in animal development, yet in plants the precise role of maternal cells in embryogenesis is unclear. We demonstrated previously that maternal activity of the SIN1 (SHORT INTEGUMENTS1) gene of Arabidopsis is essential for embryo pattern formation and viability, and that its postembryonic activity is required for several processes in reproductive development, including flowering time control and ovule morphogenesis. Here, we report the cloning of SIN1, and demonstrate its identity to the CAF (CARPEL FACTORY) gene important for normal flower morphogenesis and to the SUS1 (SUSPENSOR1) gene essential for embryogenesis. SIN1/SUS1/CAF has sequence similarity to the Drosophila melanogaster gene Dicer, which encodes a multidomain ribonuclease specific for double-stranded RNA, first identified by its role in RNA silencing. The Dicer protein is essential for temporal control of development in animals, through the processing of small RNA hairpins that in turn inhibit the translation of target mRNAs. Structural modeling of the wild-type and sin1 mutant proteins indicates that the RNA helicase domain of SIN1/SUS1/CAF is important for function. The mRNA was detected in floral meristems, ovules, and early embryos, consistent with the mutant phenotypes. A 3.3-kb region 5Ј of the SIN1/SUS1/CAF gene shows asymmetric parent-of-origin activity in the embryo: It confers transcriptional activation of a reporter gene in early embryos only when transmitted through the maternal gamete. These results suggest that maternal SIN1/SUS1/CAF functions early in Arabidopsis development, presumably through posttranscriptional regulation of specific mRNA molecules.Molecular details behind genetic regulation in the early development of plants are beginning to emerge (for a recent review, see Chaudhury et al., 2001;Baroux et al., 2002). In Arabidopsis, only two genes have been identified whose activities are required in the maternal sporophyte (or female somatic cells) for normal pattern formation during embryo development (Ray et al., 1996b;Prigge and Wagner, 2000). Of these, SERRATE encodes a zinc finger protein presumably involved in chromatin structure modulation, and is required maternally for normal cotyledon initiation (Prigge and Wagner, 2000). At least one wild-type allele of SIN1 (SHORT INTEGUMENTS1), the only other gene identified in this class, is required in the maternal sporophyte for normal patterning in the embryo (Ray et al., 1996b). Plant strains homozygous for the hypomorphic sin1-2 mutant allele show a strong maternal effect on embryo development, producing developmentally arrested embryos with loss of apical, basal, or radial symmetry elements regardless of embryo genotype. Most of these form defective plants with no differentiation of shoot or root apical meristems (Ray et al., 1996b).The exact role of the mother plant in regulating embryogenesis is the subject of some debate. The discussion so far has focused on the role of gene products cont...