In Drosophila, repeat-associated small interfering RNAs (rasiRNAs) are produced in the germ line by a Dicer-independent pathway and function through the PIWI subfamily of Argonautes to ensure silencing of retrotransposons. We sequenced small RNAs associated with the PIWI subfamily member AGO3. Although other members of PIWI, Aubergine (Aub) and Piwi, associated with rasiRNAs derived mainly from the antisense strand of retrotransposons, AGO3-associated rasiRNAs arose mainly from the sense strand. Aub- and Piwi-associated rasiRNAs showed a strong preference for uracil at their 5' ends, and AGO3-associated rasiRNAs showed a strong preference for adenine at nucleotide 10. Comparisons between AGO3- and Aub-associated rasiRNAs revealed pairs of rasiRNAs showing complementarities in their first 10 nucleotides. Aub and AGO3 exhibited Slicer activity in vitro. These data support a model in which formation of a 5' terminus within rasiRNA precursors is guided by rasiRNAs originating from transcripts of the other strand in concert with the Slicer activity of PIWI.

In Drosophila, Piwi (P-element-induced wimpy testis), which encodes a protein of the Argonaute family, is essential for germ stem cell self-renewal. Piwi has recently been shown to be a nuclear protein involved in gene silencing of retrotransposons and controlling their mobilization in the male germline. However, little is known about the molecular mechanisms of Piwi-dependent gene silencing. Here we show that endogenous Piwi immunopurified from ovary specifically associates with small RNAs of 25-29 nucleotides in length. Piwi-associated small RNAs were identified by cloning and sequencing as repeat-associated small interfering RNAs (rasiRNAs) derived from repetitive regions, such as retrotransposon and heterochromatic regions, in the Drosophila genome. Northern blot analyses revealed that in vivo Piwi does not associate with microRNAs (miRNAs) and that guide siRNA was not loaded onto Piwi when siRNA duplex was added to ovary lysate. In vitro, recombinant Piwi exhibits target RNA cleavage activity. These data together imply that Piwi functions in nuclear RNA silencing as Slicer by associating specifically with rasiRNAs originating from repetitive targets.[Keywords: Piwi; retrotransposon; rasiRNA; Slicer; Drosophila] Supplemental material is available at http://www.genesdev.org.

Argonaute proteins play important yet distinct roles in RNA silencing. Human Argonaute2 (hAgo2) was shown to be responsible for target RNA cleavage ("Slicer") activity in RNA interference (RNAi), whereas other Argonaute subfamily members do not exhibit the Slicer activity in humans. In Drosophila, AGO2 was shown to possess the Slicer activity. Here we show that AGO1, another member of the Drosophila Argonaute subfamily, immunopurified from Schneider2 (S2) cells associates with microRNA (miRNA) and cleaves target RNA completely complementary to the miRNA. Slicer activity is reconstituted with recombinant full-length AGO1. Thus, in Drosophila, unlike in humans, both AGO1 and AGO2 have Slicer functions. Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2 demonstrates that other regions in the Argonautes are not strictly necessary for small interfering RNA (siRNA)-binding and cleavage activities. It has been shown that in circumstances with AGO2-lacking, the siRNA duplex is not unwound and consequently an RNA-induced silencing complex (RISC) is not formed. We show that upon addition of an siRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner, and nuclease-resistant modification of the passenger strand impairs RISC formation. These findings give rise to a new model in which AGO2 is directly involved in RISC formation as "Slicer" of the passenger strand of the siRNA duplex.[Keywords: RNAi; Argonaute; Slicer, RISC; Drosophila] Supplemental material is available at http://www.genesdev.org.

The function of the novel gene MSP1 ( MULTIPLE SPOROCYTE ), which controls early sporogenic development, was elucidated by characterizing a retrotransposon-tagged mutation of rice. The MSP1 gene encoded a Leu-rich repeat receptorlike protein kinase. The msp1 mutation gave rise to an excessive number of both male and female sporocytes. In addition, the formation of anther wall layers was disordered and the tapetum layer was lost completely. Although the mutation never affected homologous chromosome pairing and chiasma maintenance, the development of pollen mother cells was arrested at various stages of meiotic prophase I, which resulted in complete male sterility. Meanwhile, plural megaspore mother cells in a mutant ovule generated several megaspores, underwent gametogenesis, and produced germinable seeds when fertilized with wild-type pollen despite disorganized female gametophytes. In situ expression of MSP1 was detected in surrounding cells of male and female sporocytes and some flower tissues, but never in the sporocytes themselves. These results suggest that the MSP1 product plays crucial roles in restricting the number of cells entering into male and female sporogenesis and in initiating anther wall formation in rice.

RNAi pathways have evolved as important modulators of gene expression that act in the cytoplasm by degrading RNA target molecules via the activity of short (21-30nt) RNAs1-6 RNAi components have been reported to play a role in the nucleus as they are involved in epigenetic regulation and heterochromatin formation7-10. However, although RNAi-mediated post-transcriptional silencing (PTGS) is well documented, mechanisms of RNAi-mediated transcriptional gene silencing (TGS) and in particular the role of RNAi components in chromatin, especially in higher eukaryotes, are still elusive. Here we show that key RNAi components Dicer-2 (Dcr2) and and Argonaute-2 (AGO2) AGO2 associate with chromatin, with strong preference for euchromatic, transcriptionally active loci and interact with core transcription machinery. Notably Dcr2 and AGO2 loss of function show that transcriptional defects are accompanied by perturbation of Pol II positioning on promoters. Further, both Dcr2 and Ago2 null mutations as well as missense mutations compromising the RNAi activity impair global Pol II dynamics upon heat shock. Finally, AGO2 RIP-seq experiments reveal that, AGO2 is strongly enriched in small-RNAs encompassing promoter as well as other parts of heat shock and other gene loci on both sense and antisense, with a strong bias for antisense, particularly after heat shock. Taken together our results reveal a new scenario in which Dcr2 and AGO2 are globally associated with transcriptionally active loci and may play a pivotal role in shaping the transcriptome by controlling RNA Pol II processivity.

During postembryonic development of higher plants, the shoot apical meristem produces lateral organs in a regular spacing (phyllotaxy) and a regular timing (plastochron). Molecular analysis of mutants associated with phyllotaxy and plastochron would greatly increase understanding of the developmental mechanism of plant architecture because phyllotaxy and plastochron are fundamental regulators of plant architecture. pla1 of rice is not only a plastochron mutant showing rapid leaf initiation without affecting phyllotaxy, but also a heterochronic mutant showing ectopic shoot formation in the reproductive phase. Thus, pla1 provides a tool for analyzing the molecular basis of temporal regulation in leaf development. In this work, we isolated the PLA1 gene by map-based cloning. The identified PLA1 gene encodes a cytochrome P450, CYP78A11, which potentially catalyzes substances controlling plant development. PLA1 is expressed in developing leaf primordia, bracts of the panicle, and elongating internodes, but not in the shoot apical meristem. The expression pattern and mutant phenotype suggest that the PLA1 gene acting in developing leaf primordia affects the timing of successive leaf initiation and the termination of vegetative growth.

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that collectively regulate the expression of a large number of mRNAs by either promoting destabilization or repressing translation, or both. Therefore, they play a major role in shaping the transcriptomes and proteomes of eukaryotic organisms. Typically, animal miRNAs are produced from long primary transcripts with one or more of hairpin structures by two sequential processing reactions: one by Drosha in the nucleus and the other by Dicer in the cytoplasm. However, deviations from this paradigm have been observed: subclasses of miRNAs, which only partially meet the classical definition of a miRNA, are derived by alternative biogenesis pathways, thereby providing an additional level of complexity to miRNA-dependent regulation of gene expression.

We describe a boron (B) transporter, Os BOR1, in rice (Oryza sativa). Os BOR1 is a plasma membrane-localized efflux transporter of B and is required for normal growth of rice plants under conditions of limited B supply (referred to as -B). Disruption of Os BOR1 reduced B uptake and xylem loading of B. The accumulation of Os BOR1 transcripts was higher in roots than that in shoots and was not affected by B deprivation; however, Os BOR1 was detected in the roots of wild-type plants under -B conditions, but not under normal conditions, suggesting regulation of protein accumulation in response to B nutrition. Interestingly, tissue specificity of Os BOR1 expression is affected by B treatment. Transgenic rice plants containing an Os BOR1 promoter-b-glucuronidase (GUS) fusion construct grown with a normal B supply showed the strongest GUS activity in the steles, whereas after 3 d of -B treatment, GUS activity was elevated in the exodermis. After 6 d of -B treatment, GUS activity was again strong in the stele. Our results demonstrate that Os BOR1 is required both for efficient B uptake and for xylem loading of B. Possible roles of the temporal changes in tissue-specific patterns of Os BOR1 expression in response to B condition are discussed.