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DCL4 Targets Cucumber Mosaic Virus Satellite RNA at Novel Secondary Structures

Quan-Sheng Du,^1,3, Cheng-Guo Duan,^1,3, Zhong-Hui Zhang,^2,3, Yuan-Yuan Fang,¹ Rong-Xiang Fang,¹ Qi Xie,² and Hui-Shan Guo^1*

State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China,¹ State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China,² Graduate University of Chinese Academy of Sciences, Beijing 100049, China³

Received 31 December 2006/ Accepted 22 June 2007

It has been reported that plant virus-derived small interfering RNAs (vsiRNAs) originated predominantly from structured single-stranded viral RNA of a positive single-stranded RNA virus replicating in the cytoplasm and from the nuclear stem-loop 35S leader RNA of a double-stranded DNA (dsDNA) virus. Increasing lines of evidence have also shown that hierarchical actions of plant Dicer-like (DCL) proteins are required in the biogenesis process of small RNAs, and DCL4 is the primary producer of vsiRNAs. However, the structures of such single-stranded viral RNA that can be recognized by DCLs remain unknown. In an attempt to determine these structures, we have cloned siRNAs derived from the satellite RNA (satRNA) of Cucumber mosaic virus (CMV-satRNA) and studied the relationship between satRNA-derived siRNAs (satsiRNAs) and satRNA secondary structure. satsiRNAs were confirmed to be derived from single-stranded satRNA and are primarily 21 (64.7%) or 22 (22%) nucleotides (nt) in length. The most frequently cloned positive-strand satsiRNAs were found to derive from novel hairpins that differ from the structure of known DCL substrates, miRNA and siRNA precursors, which are prevalent stem-loop-shaped or dsRNAs. DCL4 was shown to be the primary producer of satsiRNAs. In the absence of DCL4, only 22-nt satsiRNAs were detected. Our results suggest that DCL4 is capable of accessing flexibly structured single-stranded RNA substrates (preferably T-shaped hairpins) to produce satsiRNAs. This result reveals that viral RNA of diverse structures may stimulate antiviral DCL activities in plant cells.

Published ahead of print on 3 July 2007.

Q.-S.D., C.-G.D., and Z.-H.Z. contributed equally to this work.

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