• Open Access

Deep sequencing of small RNAs specifically associated with Arabidopsis AGO1 and AGO4 uncovers new AGO functions

Authors

  • Huan Wang,

    1. Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA
    2. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
    3. Graduate University of the Chinese Academy of Sciences, Beijing, 100101, China
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    • These two authors contributed equally to this work.

  • Xiuren Zhang,

    1. Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA
    2. Department of Biochemistry and Biophysics, Institute for Plant Genomics and Biotechnology, Norman Borlaug Center 112A, 2123 TAMU, Texas A&M University, College Station, TX 77843, USA
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    • These two authors contributed equally to this work.

  • Jun Liu,

    1. Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA
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  • Takatoshi Kiba,

    1. Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA
    2. RIKEN Plant Science Center, Suehiro 1-7-22, Tsurumi, Yokohama 230-0045, Japan
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  • Jongchan Woo,

    1. Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA
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  • Tolulope Ojo,

    1. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
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  • Markus Hafner,

    1. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
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  • Thomas Tuschl,

    1. Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
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  • Nam-Hai Chua,

    Corresponding author
    1. Laboratory of Plant Molecular Biology, The Rockefeller University, New York, NY 10065, USA
      (fax +212 327 8327; e-mail chua@mail.rockefeller.edu or fax +86 10 64873428; e-mail xjwang@genetics.ac.cn).
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  • Xiu-Jie Wang

    Corresponding author
    1. State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
      (fax +212 327 8327; e-mail chua@mail.rockefeller.edu or fax +86 10 64873428; e-mail xjwang@genetics.ac.cn).
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(fax +212 327 8327; e-mail chua@mail.rockefeller.edu or fax +86 10 64873428; e-mail xjwang@genetics.ac.cn).

Summary

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.

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