The residues forming the catalytic DDH motif are shown in red

The residues forming the catalytic DDH motif are shown in red. cataloged the sRNAs in each AGO1 complex by deep sequencing and found that all three AGO1s predominantly bound known miRNAs. Most of the miRNAs were evenly distributed in the three AGO1 complexes, suggesting a redundant role for the AGO1s. Intriguingly, a subset of miRNAs were specifically incorporated into or excluded from one of the AGO1s, suggesting functional specialization among the AGO1s. Furthermore, we identified rice miRNA targets at a global level. The validated targets include transcription factors that control major stages of Glycyl-H 1152 2HCl development and also genes involved in a variety of physiological processes, indicating a broad regulatory role for miRNAs in rice. == INTRODUCTION == In plants, small RNA (sRNA)-based gene silencing systems play important roles in developmental regulation, responses to biotic and abiotic stresses, and epigenetic control of transposable elements (Baulcombe, 2004;Vaucheret, 2006). Central to the sRNA pathways are proteins in the Dicer and Argonaute (AGO) families. Dicer or Dicer-like (DCL) proteins contain two RNaseIII domains and process double-stranded RNAs into 21- to 25-nucleotide sRNA duplexes. Upon dicing, a selected strand of the sRNA duplex is bound by an AGO protein to form the core of the effector complex termed RNA-induced silencing complex (RISC). RISC is guided by the sRNA to act on its target (mRNA or chromatin), resulting in mRNA degradation, translational repression, or chromatin modifications (Hannon, 2002;Baulcombe, 2004). Our understanding of the mechanism and biology of plant sRNA pathways comes mainly from studies usingArabidopsis thalianaand rice (Oryza sativa) as model systems. Thus far, five classes of sRNAs have been discovered in plants: miRNAs and four types of small interfering RNAs (siRNAs), including trans-acting siRNAs (ta-siRNAs), natural antisense transcript-derived siRNAs (nat-siRNAs), repeat-associated siRNAs (ra-siRNAs), and long siRNA (lsiRNA). Most miRNA loci are encoded by independent transcriptional units in intergenic regions that are transcribed by RNA polymerase II (Xie et al., 2005b). The primary transcript of a miRNA contains a stem-loop structure that can be recognized and processed by DCL1 into a miRNA precursor (pre-miRNA) and further processed by DCL1 into a miRNA/miRNA* duplex (Voinnet, 2009). Rice also encodes natural antisense transcript miRNAs (nat-miRNAs) (Lu et al., 2008). nat-miRNAs are derived from overlapping transcripts antisense to their targets; the maturation of nat-miRNAs resembles that of canonical miRNAs and requires DCL1. Plant miRNAs have near-perfect pairing to their targets and repress target gene expression primarily through mRNA cleavage (Llave et al., 2002). However, there are evidences supporting the notion that miRNA-mediated gene regulation also operates through repression of translation (Aukerman and Sakai, 2003;Chen, 2004;Brodersen et al., 2008;Lanet et al., 2009). ta-siRNAs are a plant-specific class of siRNAs, the production of which is initiated by miRNA-mediated cleavage of noncoding transcripts. The cleavage products serve as substrates for an RNA-dependent RNA polymerase (RDR6 inArabidopsis) to generate double-stranded RNAs that are further processed by DCL4 into phased siRNAs (Peragine et al., 2004;Vazquez et al., 2004;Allen et al., 2005;Xie et al., 2005a;Yoshikawa et al., 2005;Axtell et al., 2006).Arabidopsista-siRNAs regulate the juvenile-to-adult Rabbit Polyclonal to BRI3B vegetative phase change (Adenot et al., 2006;Fahlgren et al., 2006;Garcia et al., 2006;Hunter et al., 2006). nat-siRNAs are derived from naturalcis-antisense transcript pairs and are involved in cellular Glycyl-H 1152 2HCl responses to salt stress and pathogen attack (Borsani et al., 2005;Katiyar-Agarwal et al., 2006). lsiRNAs are 30 to 40 nucleotides in length and are induced by pathogen infection or under specific growth conditions; the mechanism of lsiRNA biogenesis remains elusive (Katiyar-Agarwal et al., 2007). Plants also contain an abundant class of 24-nucleotide ra-siRNAs derived from transposons and repetitive elements. The biogenesis of ra-siRNAs requires activities of DCL3, RDR2, and Pol IV, a plant-specific DNA-dependent RNA polymerase. ra-siRNAs play a role in the methylation and silencing of many transposons Glycyl-H 1152 2HCl and also some genes that are adjacent to repeats (Henderson and Jacobsen, 2007;Zaratiegui et al., 2007). All types of sRNAs interact with AGO proteins to exert their functions. AGOs contain three characteristic domains: PAZ, MID, and PIWI (Song and Joshua-Tor, 2006). The PAZ domain binds to the 3 end of sRNAs (Ma et al., 2004), whereas the MID domain provides a binding pocket for the 5 end (Ma et al., 2005) and is thought to confer the binding specificity of an AGO to an sRNA with a particular 5 end nucleotide (Mi et al., 2008). PIWI domain adopts the structure of RNase H that contains the catalytic site formed by three residues (Asp, Asp,.