Structural and Functional Analysis of Viral siRNAs

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.

The role of virus-derived small interfering RNAs in RNA silencing in plants

“…(vsiRNAs) is the most remarkable feature of virus-induced gene silencing [4,[8][9][10][11][12][13]. Current research indicates that vsiRNAs are derived either from the dsRNAs formed during the virus replication stage or from single-stranded viral RNAs (ssRNAs) with a hairpin-like fold-back structure [8,10,[14][15][16].…”

“…Current research indicates that vsiRNAs are derived either from the dsRNAs formed during the virus replication stage or from single-stranded viral RNAs (ssRNAs) with a hairpin-like fold-back structure [8,10,[14][15][16]. Both of these forms of viral RNA can be processed into vsiRNAs of a specific size by distinct DCLs proteins.…”

“…Other key components of silencing, such as Ago and RNA-dependent RNA polymerase (RDR), are also required for effective virus silencing [17,18]. It has been demonstrated that VIGS-mediated viral RNA inactivation is likely based on the vsiRNA-mediated cleavage of viral RNA [8,12] or of viral satellite RNA [19]. However, it cannot be excluded that the antiviral silencing mechanism also acts at the level of translational regulation.…”

“…These targeted recombinant RNA for degradation, as manifested by a decreased virus titer over time [11]. Viral genomes possess hot spots for generating vsiRNAs, as shown by high-throughput deep sequencing techniques [8].…”

“…Their results show that vsiRNAs generated from hotspots, despite their much greater abundance, do not show a greater efficiency than those from non-hotspots regions [8]. It has been shown that most of the Cymbidium ringspot virus (CymRSV) vsiRNAs (including those derived from hot spot regions) are primary vsiRNAs.…”