title: Deep sequencing of small RNAs specifically associated with Arabidopsis AGO1 and AGO4 uncovers new AGO functions.
abstract: As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate the interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in microRNA (miRNA) and small-interfering RNA (siRNA) pathways for post-transcriptional gene silencing (PTGS), whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-nucleotide (nt) smRNAs. To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina technology. Besides recovering most previously annotated smRNAs, we also identified some additional miRNAs, phased smRNA clusters and small-interfering RNAs derived from the overlapping region of natural antisense transcript pairs (NAT) (nat-siRNAs). We also identified a smRNA distribution feature on miRNA precursors which may help to identify authentic miRNAs. Organ-specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. The presence and conservation of collateral miRNAs on known miRNA precursors were also investigated. Intriguingly, about 30% of AGO1-associated smRNAs were 24-nt long and unrelated to the 21-nt species. Further analysis showed that DNA-dependent RNA polymerase IV (Pol IV)-dependent smRNAs were mainly 24 nt and associated with AGO4, whereas the majority of the potential Pol V-dependent ones were 21-nt smRNAs and bound to AGO1, suggesting the potential involvement of AGO1 in Pol V-related pathways.
authors: Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA.
authors: Huan
authors: H
authors: Wang
authors: Xiuren
authors: X
authors: Zhang
authors: Jun
authors: J
authors: Liu
authors: Takatoshi
authors: T
authors: Kiba
authors: Jongchan
authors: J
authors: Woo
authors: Tolulope
authors: T
authors: Ojo
authors: Markus
authors: M
authors: Hafner
authors: Thomas
authors: T
authors: Tuschl
authors: Nam-Hai
authors: NH
authors: Chua
authors: Xiu-Jie
authors: XJ
authors: Wang
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D060565:Argonaute Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D047468:Immunoprecipitation; D035683:MicroRNAs / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D058977:Molecular Sequence Annotation; D034622:RNA Interference; D018749:RNA, Plant / Q000235:genetics* / Q000302:isolation & purification
chemical_list: C117457:AGO1 protein, Arabidopsis; C471209:AGO4 protein, Arabidopsis; D029681:Arabidopsis Proteins; D060565:Argonaute Proteins; D035683:MicroRNAs; D018749:RNA, Plant; D012321:DNA-Directed RNA Polymerases