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Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis.

Qi X, Bao FS, Xie Z - PLoS ONE (2009)

Bottom Line: Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis.Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays.Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, United States of America.

ABSTRACT
RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

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Effects of the rdr mutations on TMV-Cg siRNAs biogenesis in Arabidopsis thaliana.(A) A close view of a 200-nt TMV-Cg genomic segment showing the number of mapped unique viral siRNAs. A small RNA that is non-identical in sequence to any others within each size class was defined as “unique”. The number of unique siRNAs mapped to a specific genomic location, in either sense (above the X-axis) or antisense (below the X-axis) configuration was plotted in each single-nucleotide sliding window along the viral genome. Data from different source libraries are color-coded in black (Col-0; TMV-Cg 3dpi), red (rdr1-1; TMV-Cg 3dpi), and blue (rdr6-15; TMV-Cg 3dpi). (B) A close view of the same genomic segment as in (A), showing the abundance of mapped viral siRNAs, which was calculated and plotted as in Fig. 2B. Data from different source libraries are color-coded as in (A). (C) A survey on phased 21-nt TMV-Cg siRNAs in the wild type and rdr mutant libraries. Sense and antisense siRNAs are shown separately. The frequencies for phased siRNAs with a defined number (2 to 19) of continued phases are shown.
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pone-0004971-g004: Effects of the rdr mutations on TMV-Cg siRNAs biogenesis in Arabidopsis thaliana.(A) A close view of a 200-nt TMV-Cg genomic segment showing the number of mapped unique viral siRNAs. A small RNA that is non-identical in sequence to any others within each size class was defined as “unique”. The number of unique siRNAs mapped to a specific genomic location, in either sense (above the X-axis) or antisense (below the X-axis) configuration was plotted in each single-nucleotide sliding window along the viral genome. Data from different source libraries are color-coded in black (Col-0; TMV-Cg 3dpi), red (rdr1-1; TMV-Cg 3dpi), and blue (rdr6-15; TMV-Cg 3dpi). (B) A close view of the same genomic segment as in (A), showing the abundance of mapped viral siRNAs, which was calculated and plotted as in Fig. 2B. Data from different source libraries are color-coded as in (A). (C) A survey on phased 21-nt TMV-Cg siRNAs in the wild type and rdr mutant libraries. Sense and antisense siRNAs are shown separately. The frequencies for phased siRNAs with a defined number (2 to 19) of continued phases are shown.

Mentions: To gain further insights on viral small RNA biogenesis, the sequence complexity and normalized abundance of TMV-Cg-derived small RNAs were compared for different size classes. Viral small RNAs in each of the 21- to 24-nt size classes were mapped onto the viral genome separately and the small RNA sequence complexity was measured by counting the number of unique small RNA sequences in each of the single nucleotide sliding windows. As shown in a representative 200-nt viral genomic segment, the highest overall sequence complexity of viral small RNAs in the wild type library was found in the 21-nt size class, followed by the 22-nt size class (Fig. 4A, black traces). Notably, in the 21-nt size class, maximum (i.e. 21) or near-maximum sequence complexity was observed for both sense and antisense viral small RNAs at multiple genomic locations, a strong indication that these siRNAs arose from a dsRNA precursor and that DCL4 processing occurred in most, if not all, possible phases (Fig. 4A, black traces). At least at certain locations, comparable sequence complexity for both sense and antisense 22-nt viral siRNAs was observed, suggesting suboptimal processing of the dsRNA precursors by DCL2. This is consistent with the notion that DCL2 functions as a partially redundant secondary dicing activity in antiviral defense. Nonetheless, the fact that many viral siRNAs were found at a very low level and that only a few accumulated to high abundance, mostly the sense siRNAs (Fig. 4B, black traces), suggests the existence of selection mechanism(s) that is not only sequence-dependent but also strand-specific. The low sequence complexity and low abundance of the 23- and 24-nt viral siRNAs were also indicative of their origin from marginal DCL3 activity (Fig. 4A, black traces). A general reduction in sequence complexity across all size classes of viral siRNAs was observed in both the rdr1 and rdr6 mutant libraries, with a much more profound reduction found for the most part in the rdr1 library (Fig. 4A; red and blue traces). Since a general reduction in the abundance of TMV-Cg-derived small RNAs in each size class was also seen in the rdr1 and rdr6 mutants (Fig. 4B), we conclude that the reduced total viral small RNA reads in the rdr mutants resulted from reduced sequence complexity as well as abundance of the small RNAs.


Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis.

Qi X, Bao FS, Xie Z - PLoS ONE (2009)

Effects of the rdr mutations on TMV-Cg siRNAs biogenesis in Arabidopsis thaliana.(A) A close view of a 200-nt TMV-Cg genomic segment showing the number of mapped unique viral siRNAs. A small RNA that is non-identical in sequence to any others within each size class was defined as “unique”. The number of unique siRNAs mapped to a specific genomic location, in either sense (above the X-axis) or antisense (below the X-axis) configuration was plotted in each single-nucleotide sliding window along the viral genome. Data from different source libraries are color-coded in black (Col-0; TMV-Cg 3dpi), red (rdr1-1; TMV-Cg 3dpi), and blue (rdr6-15; TMV-Cg 3dpi). (B) A close view of the same genomic segment as in (A), showing the abundance of mapped viral siRNAs, which was calculated and plotted as in Fig. 2B. Data from different source libraries are color-coded as in (A). (C) A survey on phased 21-nt TMV-Cg siRNAs in the wild type and rdr mutant libraries. Sense and antisense siRNAs are shown separately. The frequencies for phased siRNAs with a defined number (2 to 19) of continued phases are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654919&req=5

pone-0004971-g004: Effects of the rdr mutations on TMV-Cg siRNAs biogenesis in Arabidopsis thaliana.(A) A close view of a 200-nt TMV-Cg genomic segment showing the number of mapped unique viral siRNAs. A small RNA that is non-identical in sequence to any others within each size class was defined as “unique”. The number of unique siRNAs mapped to a specific genomic location, in either sense (above the X-axis) or antisense (below the X-axis) configuration was plotted in each single-nucleotide sliding window along the viral genome. Data from different source libraries are color-coded in black (Col-0; TMV-Cg 3dpi), red (rdr1-1; TMV-Cg 3dpi), and blue (rdr6-15; TMV-Cg 3dpi). (B) A close view of the same genomic segment as in (A), showing the abundance of mapped viral siRNAs, which was calculated and plotted as in Fig. 2B. Data from different source libraries are color-coded as in (A). (C) A survey on phased 21-nt TMV-Cg siRNAs in the wild type and rdr mutant libraries. Sense and antisense siRNAs are shown separately. The frequencies for phased siRNAs with a defined number (2 to 19) of continued phases are shown.
Mentions: To gain further insights on viral small RNA biogenesis, the sequence complexity and normalized abundance of TMV-Cg-derived small RNAs were compared for different size classes. Viral small RNAs in each of the 21- to 24-nt size classes were mapped onto the viral genome separately and the small RNA sequence complexity was measured by counting the number of unique small RNA sequences in each of the single nucleotide sliding windows. As shown in a representative 200-nt viral genomic segment, the highest overall sequence complexity of viral small RNAs in the wild type library was found in the 21-nt size class, followed by the 22-nt size class (Fig. 4A, black traces). Notably, in the 21-nt size class, maximum (i.e. 21) or near-maximum sequence complexity was observed for both sense and antisense viral small RNAs at multiple genomic locations, a strong indication that these siRNAs arose from a dsRNA precursor and that DCL4 processing occurred in most, if not all, possible phases (Fig. 4A, black traces). At least at certain locations, comparable sequence complexity for both sense and antisense 22-nt viral siRNAs was observed, suggesting suboptimal processing of the dsRNA precursors by DCL2. This is consistent with the notion that DCL2 functions as a partially redundant secondary dicing activity in antiviral defense. Nonetheless, the fact that many viral siRNAs were found at a very low level and that only a few accumulated to high abundance, mostly the sense siRNAs (Fig. 4B, black traces), suggests the existence of selection mechanism(s) that is not only sequence-dependent but also strand-specific. The low sequence complexity and low abundance of the 23- and 24-nt viral siRNAs were also indicative of their origin from marginal DCL3 activity (Fig. 4A, black traces). A general reduction in sequence complexity across all size classes of viral siRNAs was observed in both the rdr1 and rdr6 mutant libraries, with a much more profound reduction found for the most part in the rdr1 library (Fig. 4A; red and blue traces). Since a general reduction in the abundance of TMV-Cg-derived small RNAs in each size class was also seen in the rdr1 and rdr6 mutants (Fig. 4B), we conclude that the reduced total viral small RNA reads in the rdr mutants resulted from reduced sequence complexity as well as abundance of the small RNAs.

Bottom Line: Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis.Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays.Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Texas Tech University, Lubbock, Texas, United States of America.

ABSTRACT
RNA silencing functions as an important antiviral defense mechanism in a broad range of eukaryotes. In plants, biogenesis of several classes of endogenous small interfering RNAs (siRNAs) requires RNA-dependent RNA Polymerase (RDR) activities. Members of the RDR family proteins, including RDR1and RDR6, have also been implicated in antiviral defense, although a direct role for RDRs in viral siRNA biogenesis has yet to be demonstrated. Using a crucifer-infecting strain of Tobacco Mosaic Virus (TMV-Cg) and Arabidopsis thaliana as a model system, we analyzed the viral small RNA profile in wild-type plants as well as rdr mutants by applying small RNA deep sequencing technology. Over 100,000 TMV-Cg-specific small RNA reads, mostly of 21- (78.4%) and 22-nucleotide (12.9%) in size and originating predominately (79.9%) from the genomic sense RNA strand, were captured at an early infection stage, yielding the first high-resolution small RNA map for a plant virus. The TMV-Cg genome harbored multiple, highly reproducible small RNA-generating hot spots that corresponded to regions with no apparent local hairpin-forming capacity. Significantly, both the rdr1 and rdr6 mutants exhibited globally reduced levels of viral small RNA production as well as reduced strand bias in viral small RNA population, revealing an important role for these host RDRs in viral siRNA biogenesis. In addition, an informatics analysis showed that a large set of host genes could be potentially targeted by TMV-Cg-derived siRNAs for posttranscriptional silencing. Two of such predicted host targets, which encode a cleavage and polyadenylation specificity factor (CPSF30) and an unknown protein similar to translocon-associated protein alpha (TRAP alpha), respectively, yielded a positive result in cleavage validation by 5'RACE assays. Our data raised the interesting possibility for viral siRNA-mediated virus-host interactions that may contribute to viral pathogenicity and host specificity.

Show MeSH
Related in: MedlinePlus