Figure 1.
Virus-derived vsRNA abundance varies as a function of virus type & strain, host type & genotype, time post-infection, and cloning method used.
Abundance of vsRNAs in various host systems infected with (1A) Dengue Virus, Vesicular Stomatitis Virus, or Polio Virus, and (1B) Hepatitis C Virus, West Nile Virus, or Flock House Virus. Samples sequenced on the Solexa platform are prefixed with ‘S-,’ while samples sequenced on the GS-20/GS-FLX are pre-fixed with ‘4-.’ The asterisks indicate vsRNAs from RNA pools captured using the 5′-P-INDependent cloning protocol. Samples that had no detectable vsRNAs were not plotted. Levels of vsRNAs in these samples (sense ‘BLUE’ or antisense ‘RED’ relative to the mRNA of the virus) are represented as a ratio relative to the count of all miRNAs (i.e. v/miR). miRNA sequences are defined in species-specific miRNA databases obtained from miRBase ver9.2. Note: v/miR values are represented on a logarithmic scale.
Figure 2.
Flock House Virus-derived vsRNAs are more abundant in RNAi-competent worms, and exist as both 5′-monophosphorylated, and 5′-triphosphorylated species.
The incidence, strandedness and lengths of vsRNAs are drawn as a function of their position along the viral genome. Each filled box represents one instance of a captured vsRNA, with the lengths of the boxes proportional to the lengths of the vsRNAs. vsRNAs from the positive and negative strands are shaded black and red respectively. All samples were sequenced on Illumina's platform. (2A) Sequence counts for all small RNAs, miRNAs, vsRNAs (Y-axis: log scale). 5′-P vsRNAs from wild-type Bristol N2 (2B; Sol-73), rde-1 (2C; Sol-72), rde-4 (2D; Sol-71), and rrf-1 (2E; Sol-74) worms, 24 hours post-heat-shock. 5′-xP vsRNAs from wild-type Bristol N2 (2F; Sol-52), rde-4 (2G; Sol-50), rde-1 (2H; Sol-51), and rrf-1 (2I; Sol-53) worms, 24 hours post-heat-shock.
Figure 3.
Poliovirus- and Vesicular Stomatitis Virus-derived vsRNAs are more abundant in MEFs deficient in Argonaute-2.
Samples sequenced on the Solexa platform are prefixed with ‘Sol-,’ while samples sequenced on the GS-20/GS-FLX are pre-fixed with ‘454-.’ (3A) Sequence count: all RNAs, miRNAs, vsRNAs (Y-axis: log scale). vsRNAs with a 5′-monophosphate moiety from ago-2+/+ MEFs (3B; Sample: Sol-82) and ago-2−/− MEFs (3C; Sample: Sol-83), transfected with a plasmid encoding for full-length, self-replicating Poliovirus RNA. vsRNAs with a 5′-monophosphate moiety from ago-2+/+ MEFs (3D; Sample: 454-87) and ago-2−/− MEFs (3E; Sample: 454-88) infected with Vesicular Stomatitis Virus.
Figure 4.
vsRNAs are abundant in infected hosts that do not have a functional Interferon-αβ Receptor.
(4A) Sequence count: all RNAs, miRNAs, vsRNAs (Y-axis: log scale). vsRNAs from leg muscle of an IFNαβR+/+; PVR+/+ (4B; 454-163), or IFNαβR−/−; PVR+/+ (4C; Sol-1) mouse infected with poliovirus (4 d.p.i; 5′-Phosphate-dependent capture). vsRNAs with a 5′ monophosphate from the spleen of an IFNαβR+/+ (4D; 454-131), or IFNαβR−/− (4E; 454-143) mouse infected with West Nile Virus (3 d.p.i).
Figure 5.
vsRNAs are detectable in different models of Hepatitis C Virus infection.
(5A) Sequence count: all RNAs, miRNAs, vsRNAs (Y-axis: log scale). vsRNAs from: (5B) Huh7 cells with HCV replicon (Sample: Sol-4); (5C) Huh7.5 cells infected with HCV virions, harvested 3 d.p.i (Sample: Sol-92).
Figure 6.
Sub-populations of sense and antisense vsRNAs exist in duplexes with canonical 1–2 nt 3′ overhangs.
The assumption inherent in this analysis is that both passenger and guide strands of an siRNA duplex are accessible for capture. All sense (positive strand) and antisense (negative strand) vsRNAs were considered potential partners for this analysis. X-axis: range of overhangs (+24 to −24); Y-axis: percent of duplexes that fall into each overhang category. Overhangs formed from overlapping sets of HCVrep-derived vsRNAs (Sol-176) after size segregation, represented as a percent of total number of overlapping instances in the +24 to −24 bp window: (6A) 20,21-mer vsRNAs; (6B) 24,25,26-mers; (6C) all size-classes of vsRNAs. (6D) Lagging overhangs are computed as: End position of antisense vsRNA – Start position of sense vsRNA; Leading overhangs are computed as: Start position of antisense vsRNA – End position of sense vsRNA. Thus, a 2 base 3′ overhang will have a value of −2, while a two base 5′ overhang will have a value of +2.
Figure 7.
Only one strand of the vsRNA duplex is incorporated into Argonaute complexes.
(7A) Percent enrichment for various RNAs in Ago-IPs, compared to Mock-IPs, computed as: [(xRNA/totSeq)IP/(xRNA/totSeq)MockIP]; xRNA = vsRNA, miRNA, miRNA*, or rRNA; totSeq = total number of sequences. The number of vsRNAs varied from 86 to 2472, and the number of total sequences varied from 126,022 to 2,147,467 in these samples. Fractionation of any specific RNA with Argonaute-bound complexes is evidenced in this analysis by retention of representation (compared to miRNAs) and enrichment (beyond that observed for rRNA-derived segments) in the immunoprecipitated pool. (7B) Comparison between leading and lagging overhangs formed by HCVrep-derived vsRNAs that either associate with an Argonaute (IP), or are present in cell lysates (totalRNA). All detected sense (positive strand) and antisense (negative strand) vsRNAs were considered potential partners for this analysis.