Limits...
Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance.

Zhao M, San León D, Mesel F, García JA, Simón-Mateo C - PLoS ONE (2015)

Bottom Line: In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction.The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present.The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049, Madrid, Spain.

ABSTRACT
The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.

No MeSH data available.


Related in: MedlinePlus

Effect of miR173 expression in size distribution profiles of syn-tasiRNAs produced in N. benthamiana transient assays.Size distribution of small RNAs (18–26 nt) identified by high-throughput sequencing is shown. A) Small RNAs mapping to N. benthamiana genome. B) Small RNAs mapping to the common region of pMDC32-derived plasmids. C) Small RNAs mapping to PPV-derived regions from syn-tasiR-3NCR (3NCR) and syn-tasiR-CP (CP) constructs.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492489&req=5

pone.0132281.g003: Effect of miR173 expression in size distribution profiles of syn-tasiRNAs produced in N. benthamiana transient assays.Size distribution of small RNAs (18–26 nt) identified by high-throughput sequencing is shown. A) Small RNAs mapping to N. benthamiana genome. B) Small RNAs mapping to the common region of pMDC32-derived plasmids. C) Small RNAs mapping to PPV-derived regions from syn-tasiR-3NCR (3NCR) and syn-tasiR-CP (CP) constructs.

Mentions: The size distribution profiles of the-miR173 and +miR173 sRNA libraries (Fig 3) confirmed previous reports that 24-nt is the major species of endogenous sRNAs of N. benthamiana [37] and showed that expression of miR173 had no significant effect in the size distribution of endogenous populations of sRNAs (Fig 3A). In contrast, the size distribution profile of exogenous sRNAs, derived from pMDC-based plasmids showed peaks of similar height at 21 nt and 24 nt (Fig 3B and 3C). Moreover, expression of miR173 did not significantly alter the size distribution of sRNAs derived from pMDC-shared regions (Fig 3B). With respect to the size distribution of the sRNAs from PPV specific regions the pattern was very similar to those of pMDC-shared regions in the absence of miR173 (Fig 3C). However miR173 expression caused a large increase in the proportion of PPV 3’NCR-specific 21-nt sRNAs, which was much less pronounced for PPV CP-specific siRNAs. These data suggest that cleavage at a miR173 target site enhances the accumulation of 21-nt sRNAs derived from downstream sequences in the syn-tasiRNA constructs.


Assorted Processing of Synthetic Trans-Acting siRNAs and Its Activity in Antiviral Resistance.

Zhao M, San León D, Mesel F, García JA, Simón-Mateo C - PLoS ONE (2015)

Effect of miR173 expression in size distribution profiles of syn-tasiRNAs produced in N. benthamiana transient assays.Size distribution of small RNAs (18–26 nt) identified by high-throughput sequencing is shown. A) Small RNAs mapping to N. benthamiana genome. B) Small RNAs mapping to the common region of pMDC32-derived plasmids. C) Small RNAs mapping to PPV-derived regions from syn-tasiR-3NCR (3NCR) and syn-tasiR-CP (CP) constructs.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492489&req=5

pone.0132281.g003: Effect of miR173 expression in size distribution profiles of syn-tasiRNAs produced in N. benthamiana transient assays.Size distribution of small RNAs (18–26 nt) identified by high-throughput sequencing is shown. A) Small RNAs mapping to N. benthamiana genome. B) Small RNAs mapping to the common region of pMDC32-derived plasmids. C) Small RNAs mapping to PPV-derived regions from syn-tasiR-3NCR (3NCR) and syn-tasiR-CP (CP) constructs.
Mentions: The size distribution profiles of the-miR173 and +miR173 sRNA libraries (Fig 3) confirmed previous reports that 24-nt is the major species of endogenous sRNAs of N. benthamiana [37] and showed that expression of miR173 had no significant effect in the size distribution of endogenous populations of sRNAs (Fig 3A). In contrast, the size distribution profile of exogenous sRNAs, derived from pMDC-based plasmids showed peaks of similar height at 21 nt and 24 nt (Fig 3B and 3C). Moreover, expression of miR173 did not significantly alter the size distribution of sRNAs derived from pMDC-shared regions (Fig 3B). With respect to the size distribution of the sRNAs from PPV specific regions the pattern was very similar to those of pMDC-shared regions in the absence of miR173 (Fig 3C). However miR173 expression caused a large increase in the proportion of PPV 3’NCR-specific 21-nt sRNAs, which was much less pronounced for PPV CP-specific siRNAs. These data suggest that cleavage at a miR173 target site enhances the accumulation of 21-nt sRNAs derived from downstream sequences in the syn-tasiRNA constructs.

Bottom Line: In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction.The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present.The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma de Madrid, 28049, Madrid, Spain.

ABSTRACT
The use of syn-tasiRNAs has been proposed as an RNA interference technique alternative to those previously described: hairpin based, virus induced gene silencing or artificial miRNAs. In this study we engineered the TAS1c locus to impair Plum pox virus (PPV) infection by replacing the five native siRNAs with two 210-bp fragments from the CP and the 3´NCR regions of the PPV genome. Deep sequencing analysis of the small RNA species produced by both constructs in planta has shown that phased processing of the syn-tasiRNAs is construct-specific. While in syn-tasiR-CP construct the processing was as predicted 21-nt phased in register with miR173-guided cleavage, the processing of syn-tasiR-3NCR is far from what was expected. A 22-nt species from the miR173-guided cleavage was a guide of two series of phased small RNAs, one of them in an exact 21-nt register, and the other one in a mixed of 21-/22-nt frame. In addition, both constructs produced abundant PPV-derived small RNAs in the absence of miR173 as a consequence of a strong sense post-transcriptional gene silencing induction. The antiviral effect of both constructs was also evaluated in the presence or absence of miR173 and showed that the impairment of PPV infection was not significantly higher when miR173 was present. The results show that syn-tasiRNAs processing depends on construct-specific factors that should be further studied before the so-called MIGS (miRNA-induced gene silencing) technology can be used reliably.

No MeSH data available.


Related in: MedlinePlus