DCL4 targets Cucumber mosaic virus satellite RNA at novel secondary structures
ABSTRACT 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.
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ABSTRACT: Symptoms on virus-infected plants are often very specific to the given virus. The molecular mechanisms involved in viral symptom induction have been extensively studied, but are still poorly understood. Cucumber mosaic virus (CMV) Y satellite RNA (Y-sat) is a non-coding subviral RNA and modifies the typical symptom induced by CMV in specific hosts; Y-sat causes a bright yellow mosaic on its natural host Nicotiana tabacum. The Y-sat-induced yellow mosaic failed to develop in the infected Arabidopsis and tomato plants suggesting a very specific interaction between Y-sat and its host. In this study, we revealed that Y-sat produces specific short interfering RNAs (siRNAs), which interfere with a host gene, thus inducing the specific symptom. We found that the mRNA of tobacco magnesium protoporphyrin chelatase subunit I (ChlI, the key gene involved in chlorophyll synthesis) had a 22-nt sequence that was complementary to the Y-sat sequence, including four G-U pairs, and that the Y-sat-derived siRNAs in the virus-infected plant downregulate the mRNA of ChlI by targeting the complementary sequence. ChlI mRNA was also downregulated in the transgenic lines that express Y-sat inverted repeats. Strikingly, modifying the Y-sat sequence in order to restore the 22-nt complementarity to Arabidopsis and tomato ChlI mRNA resulted in yellowing symptoms in Y-sat-infected Arabidopsis and tomato, respectively. In 5'-RACE experiments, the ChlI transcript was cleaved at the expected middle position of the 22-nt complementary sequence. In GFP sensor experiments using agroinfiltration, we further demonstrated that Y-sat specifically targeted the sensor mRNA containing the 22-nt complementary sequence of ChlI. Our findings provide direct evidence that the identified siRNAs derived from viral satellite RNA directly modulate the viral disease symptom by RNA silencing-based regulation of a host gene.PLoS Pathogens 05/2011; 7(5):e1002021. · 8.14 Impact Factor
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ABSTRACT: Cellular organisms have evolved related pathways for the biogenesis and function of small interfering RNAs (siRNAs), microRNAs and PIWI-interacting RNAs (piRNAs). These distinct classes of small RNAs guide specific gene silencing at both transcriptional and posttranscriptional levels by serving as specificity determinants. Small RNAs of virus and host origins have been found to modulate virus–host interactions by RNA interference (RNAi), leading to antiviral immunity or viral pathogenesis. Deep sequencing-based profiling of virus-derived small RNAs as products of host immune recognition not only allowed us to gain insight into the expansion and functional specialization of host factors involved in the antiviral immunity but also made it possible to identify new viruses in a culture-independent manner. Here we review recent developments on the characterization and function of virus-derived siRNAs and piRNAs in eukaryotic hosts.Current opinion in virology. 12/2011; 1(6):533-44.
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ABSTRACT: A large amount of short interfering RNA (vsiRNA) is generated from plant viruses during infection, but the function, structure and biogenesis of these is not understood. We profiled vsiRNAs using two different high-throughput sequencing platforms and also developed a hybridisation based array approach. The profiles obtained through the Solexa platform and by hybridisation were very similar to each other but different from the 454 profile. Both deep sequencing techniques revealed a strong bias in vsiRNAs for the positive strand of the virus and identified regions on the viral genome that produced vsiRNA in much higher abundance than other regions. The hybridisation approach also showed that the position of highly abundant vsiRNAs was the same in different plant species and in the absence of RDR6. We used the Terminator 5'-Phosphate-Dependent Exonuclease to study the 5' end of vsiRNAs and showed that a perfect control duplex was not digested by the enzyme without denaturation and that the efficiency of the Terminator was strongly affected by the concentration of the substrate. We found that most vsiRNAs have 5' monophosphates, which was also confirmed by profiling short RNA libraries following either direct ligation of adapters to the 5' end of short RNAs or after replacing any potential 5' ends with monophosphates. The Terminator experiments also showed that vsiRNAs were not perfect duplexes. Using a sensor construct we also found that regions from the viral genome that were complementary to non-abundant vsiRNAs were targeted in planta just as efficiently as regions recognised by abundant vsiRNAs. Different high-throughput sequencing techniques have different reproducible sequence bias and generate different profiles of short RNAs. The Terminator exonuclease does not process double stranded RNA, and because short RNAs can quickly re-anneal at high concentration, this assay can be misleading if the substrate is not denatured and not analysed in a dilution series. The sequence profiles and Terminator digests suggest that CymRSV siRNAs are produced from the structured positive strand rather than from perfect double stranded RNA or by RNA dependent RNA polymerase.PLoS Pathogens 01/2010; 6(4):e1000838. · 8.14 Impact Factor