Limits...
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.

Show MeSH

Related in: MedlinePlus

Computational prediction and experimental validation of host genes targeted by TMV-Cg-siRNAs.(A) Scores of host genes predicted as targets for sense (left panel) and antisense (left panel) viral siRNAs. The library size-normalized reads for the associated viral siRNAs are shown. (B) Validation of TMV-Cg-induced cleavage of CPSF30 and TRAP α-like mRNAs by RLM-5′RACE. Gene-specific RACE products for SCL6-III, a known target for miR171, as well as those for CPSF30 and TRAP α-like were resolved on 1.5% agarose gel. M indicates DNA size markers. (C) Sequence alignment between the predicted targets and TMV-Cg siRNAs. Arrows indicate the mapped cleavage sites, and the number of 5′ RACE clones corresponding to each site is shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654919&req=5

pone-0004971-g005: Computational prediction and experimental validation of host genes targeted by TMV-Cg-siRNAs.(A) Scores of host genes predicted as targets for sense (left panel) and antisense (left panel) viral siRNAs. The library size-normalized reads for the associated viral siRNAs are shown. (B) Validation of TMV-Cg-induced cleavage of CPSF30 and TRAP α-like mRNAs by RLM-5′RACE. Gene-specific RACE products for SCL6-III, a known target for miR171, as well as those for CPSF30 and TRAP α-like were resolved on 1.5% agarose gel. M indicates DNA size markers. (C) Sequence alignment between the predicted targets and TMV-Cg siRNAs. Arrows indicate the mapped cleavage sites, and the number of 5′ RACE clones corresponding to each site is shown.

Mentions: Given the potential sequence complexity of viral siRNAs, it is possible that some of them may target the transcripts of host genes for posttranscriptional regulation. To explore this possibility, an informatics analysis to systemically identify the potential host targets was performed for each of the 21-nt computationally generated TMV-Cg siRNAs. A target prediction algorithm with a scoring stringency similar to those previously used for miRNA target prediction was applied in this procedure (a host gene with a score of 3.5 or lower for a specific viral siRNA was considered as a potential target; see materials and methods) [26]. Based on this procedure, a large set (4,784) of host genes were predicted as potential targets of TMV-Cg siRNAs (Supplementary Table S1). Some host genes were predicted to be targeted by multiple viral siRNAs at distinct locations within the gene. The predicted targets covered a wide range of functional categories, including transcription factors, RNA processing factors, and defense-related proteins (Supplementary Table S1). Intriguingly, we noticed that the TMV-Cg siRNAs with a low-scored predicted host target were in general found at low abundance (Fig. 5A). To test if TMV-Cg siRNAs direct the cleavage of predicted targets in vivo, a small subset of the predicted targets were selected for experimental validation by modified RNA ligase-mediated rapid amplification of cDNA ends (RLM-5′RACE) [27], a method that has been widely used for mapping the 5′ end of the 3′ cleavage product. Using the same RNA samples (Col-0; mock and TMV-Cg-infected, 3dpi) as those used for small RNA sequencing, 5′ RACE products were detected for only two of the 16 predicted targets tested. The two host genes yielding a positive 5′RACE product encode a cleavage and polyadenylation specificity factor (CPSF30, At1g30460) and an unknown protein similar to translocon-associated protein alpha (TRAP α), respectively (Fig. 5B). While the miR171-directed cleavage of SCL6-III (At3g60630) mRNA [27] was detected in both mock- and TMV-Cg-infected wild type Arabidopsis (Fig 5B, lanes 1 and 2), cleavage of the CPSF30 and the TRAP α -like mRNAs appeared to be specific to TMV-Cg-infection (Fig 5B, lanes 3–6). Sequencing of 5′RACE products revealed multiple cleavage sites within the predicted TMV-siRNA-interacting region in CPSF30 mRNA, with only one of them corresponded to the predicted canonical site for a cleavage directed by TMV-Cg-siR221(+), one of three viral siRNAs predicted to interact with the target (Fig 5C, upper panel). Sequencing of the TRAP α-like-specific 5′RACE products mapped a cleavage site corresponded to the predicted canonical site for a cleavage directed by TMV-Cg-siR118 (+), one of three viral siRNAs predicted to interact with this target (Fig 5C, lower panel).


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)

Computational prediction and experimental validation of host genes targeted by TMV-Cg-siRNAs.(A) Scores of host genes predicted as targets for sense (left panel) and antisense (left panel) viral siRNAs. The library size-normalized reads for the associated viral siRNAs are shown. (B) Validation of TMV-Cg-induced cleavage of CPSF30 and TRAP α-like mRNAs by RLM-5′RACE. Gene-specific RACE products for SCL6-III, a known target for miR171, as well as those for CPSF30 and TRAP α-like were resolved on 1.5% agarose gel. M indicates DNA size markers. (C) Sequence alignment between the predicted targets and TMV-Cg siRNAs. Arrows indicate the mapped cleavage sites, and the number of 5′ RACE clones corresponding to each site is shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654919&req=5

pone-0004971-g005: Computational prediction and experimental validation of host genes targeted by TMV-Cg-siRNAs.(A) Scores of host genes predicted as targets for sense (left panel) and antisense (left panel) viral siRNAs. The library size-normalized reads for the associated viral siRNAs are shown. (B) Validation of TMV-Cg-induced cleavage of CPSF30 and TRAP α-like mRNAs by RLM-5′RACE. Gene-specific RACE products for SCL6-III, a known target for miR171, as well as those for CPSF30 and TRAP α-like were resolved on 1.5% agarose gel. M indicates DNA size markers. (C) Sequence alignment between the predicted targets and TMV-Cg siRNAs. Arrows indicate the mapped cleavage sites, and the number of 5′ RACE clones corresponding to each site is shown.
Mentions: Given the potential sequence complexity of viral siRNAs, it is possible that some of them may target the transcripts of host genes for posttranscriptional regulation. To explore this possibility, an informatics analysis to systemically identify the potential host targets was performed for each of the 21-nt computationally generated TMV-Cg siRNAs. A target prediction algorithm with a scoring stringency similar to those previously used for miRNA target prediction was applied in this procedure (a host gene with a score of 3.5 or lower for a specific viral siRNA was considered as a potential target; see materials and methods) [26]. Based on this procedure, a large set (4,784) of host genes were predicted as potential targets of TMV-Cg siRNAs (Supplementary Table S1). Some host genes were predicted to be targeted by multiple viral siRNAs at distinct locations within the gene. The predicted targets covered a wide range of functional categories, including transcription factors, RNA processing factors, and defense-related proteins (Supplementary Table S1). Intriguingly, we noticed that the TMV-Cg siRNAs with a low-scored predicted host target were in general found at low abundance (Fig. 5A). To test if TMV-Cg siRNAs direct the cleavage of predicted targets in vivo, a small subset of the predicted targets were selected for experimental validation by modified RNA ligase-mediated rapid amplification of cDNA ends (RLM-5′RACE) [27], a method that has been widely used for mapping the 5′ end of the 3′ cleavage product. Using the same RNA samples (Col-0; mock and TMV-Cg-infected, 3dpi) as those used for small RNA sequencing, 5′ RACE products were detected for only two of the 16 predicted targets tested. The two host genes yielding a positive 5′RACE product encode a cleavage and polyadenylation specificity factor (CPSF30, At1g30460) and an unknown protein similar to translocon-associated protein alpha (TRAP α), respectively (Fig. 5B). While the miR171-directed cleavage of SCL6-III (At3g60630) mRNA [27] was detected in both mock- and TMV-Cg-infected wild type Arabidopsis (Fig 5B, lanes 1 and 2), cleavage of the CPSF30 and the TRAP α -like mRNAs appeared to be specific to TMV-Cg-infection (Fig 5B, lanes 3–6). Sequencing of 5′RACE products revealed multiple cleavage sites within the predicted TMV-siRNA-interacting region in CPSF30 mRNA, with only one of them corresponded to the predicted canonical site for a cleavage directed by TMV-Cg-siR221(+), one of three viral siRNAs predicted to interact with the target (Fig 5C, upper panel). Sequencing of the TRAP α-like-specific 5′RACE products mapped a cleavage site corresponded to the predicted canonical site for a cleavage directed by TMV-Cg-siR118 (+), one of three viral siRNAs predicted to interact with this target (Fig 5C, lower panel).

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