Structural and Functional Analysis of Viral siRNAs
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ABSTRACT:
ORGANISM(S): Nicotiana benthamiana
SUBMITTER: Simon Moxon
PROVIDER: E-GEOD-17278 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description: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 were the same in different plant species and in the absence of RDR6. We used the TerminatorTM 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 not complementary to highly 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. Size-fractionated (19-24 nt) small RNA from early systemic leaves of CymRSV-infected Nicotiana benthamiana total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification, the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.
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Project description:Small RNA expression from Nicotiana benthamiana leaves was profiled with the primary goal of ascertaining microRNA isoform diversity for known, conserved families. A secondary goal was to provide a baseline small RNA expression atlas for this species and tissue. Two biological replicates of leaves from growth-room grown plants. The two libraries were each run twice on different runs, so there are a total of four datasets. For most analyses, it will be appropriate to combine the run1 and run2 versions for the libraries.
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Project description:The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense PTGS induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably. Two samples were analyzed. The control sample has no presence of miR173.
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