Deep sequencing of small RNAs specifically associated with Arabidopsis AGO1 and AGO4 uncovers new AGO functions
| 作者： |
Huan Wang,Xiuren Zhang,Jun Liu,Takatoshi Kiba,Jongchan Woo,Tolulope Ojo,Markus Hafner,Thomas Tuschl,Nam-Hai Chua,Xiu-Jie Wang |
| 刊物名称： |
The Plant Journal |
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| 发表年度： |
2011-04-06 |
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| 摘要： |
As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in miRNA and siRNA pathways for post-transcriptional gene silencing (PTGS) whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-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’s technology. Besides recovering most previously annotated small RNAs, we also identified some additional miRNAs, phased smRNA clusters and 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 Pol IV-dependent smRNAs were mainly 24-nt and associated with AGO4 whereas 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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