Project description:This SuperSeries is composed of the following subset Series: GSE17162: Structural and Functional Analysis of Viral siRNAs using Solexa sequencing GSE17164: Structural and Functional Analysis of Viral siRNAs using 454 sequencing Refer to individual Series
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.
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.
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.
Project description:In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, very few of these siRNAs actually interfere with viral replication. A reliable approach to define the characteristics underlying the activity of these immunologically effective siRNAs (esiRNAs) has not been available so far. We developed a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. The approach is essentially based on the use of a cytoplasmic extract from Nicotiana tabacum BY-2 protoplasts (BY-2 lysate, BYL), that shows Dicer-like (DCL) activity and facilitates the assembly of active RNA-induced silencing complexes (RISC) with an in vitro-translated Argonaute (AGO) protein of choice. We exposed double-stranded (ds) RNA of Tomato bushy stunt virus (TBSV) to the BYL to generate viral siRNAs (DCL assay). Total RNA was isolated from the reactions and DCL-generated TBSV siRNAs were identified by NGS. In another approach, AGO1/RISC- or AGO2/RISC-associated siRNAs were isolated using FLAG-AGO immunoprecipitation (AGO-IP) and analyzed by NGS. Subsequently, the antiviral activity of siRNAs with high affinity to AGO proteins was characterized in vitro and in vivo.
Project description:We investigated the role of A. thaliana RDRs in the RNAi-mediated viral immunity by using a mutant of cucumber mosaic virus (CMV) that does not express the VSR protein 2b. CMV contains three positive-strand genomic RNAs and the 2b protein encoded by RNA2 is essential for infection by suppressing antiviral silencing initiated by either DCL4 or DCL2. Our results demonstrate an essential role for the amplification of viral siRNAs by either RDR1 or RDR6 in antiviral silencing. Further analyses, including Illumina sequencing of more than 3.5 million viral siRNAs, indicated target specificity of the two antiviral RDRs.