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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: 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.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 for syn-tasiRNA accumulation in N. benthamiana transient assays.Mapping of small RNA sequences obtained by high-throughput sequencing are shown. Analyses of total small RNA (18–26 nt) reads in the presence (+miR173) or absence of miR173 (-miR173) is shown in percentages in pie charts. Those reads mapping across the syn-tasiRNA constructs (construct specific) are shown in detail. CP: reads mapping to CP region included in the syn-tasiR-CP. 3NCR: reads mapping to 3’NCR region included in the syn-tasiR-3NCR. Ath-MIR173: reads mapping to the MIR173 construct.
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pone.0132281.g002: Effect of miR173 expression for syn-tasiRNA accumulation in N. benthamiana transient assays.Mapping of small RNA sequences obtained by high-throughput sequencing are shown. Analyses of total small RNA (18–26 nt) reads in the presence (+miR173) or absence of miR173 (-miR173) is shown in percentages in pie charts. Those reads mapping across the syn-tasiRNA constructs (construct specific) are shown in detail. CP: reads mapping to CP region included in the syn-tasiR-CP. 3NCR: reads mapping to 3’NCR region included in the syn-tasiR-3NCR. Ath-MIR173: reads mapping to the MIR173 construct.

Mentions: The total numbers of reads after filtering to remove rRNA and tRNA were 9,433,876 and 8,893,736 for the pools of syn-tasiRNA constructs expressed alone (library-miR173) or together with miR173 (library +miR173), respectively. To determine how expression of miR173 affects the patterns of sRNA populations, we classified sRNAs in five groups: i) matching to N. benthamiana genome, ii) matching Agrobacterium genome, iii) matching common regions of agroinfiltrated pMDC32-derived plasmids, iv) matching specific regions of each pMDC32-derived plasmids, and v) unknown sequences (Fig 2). Approximately 32%, 1.5% and 60% of the reads of the-miR173 library corresponded to sRNAs of the N. benthamiana, Agrobacterium and pMDC-shared groups, respectively (Fig 2). These percentages were marginally lower, and that of the unknown sequences marginally higher, in the +miR173 library (Fig 2). As corresponds to the small size of the cloned PPV sequence, the percentages of both PPV CP- and PPV 3’NCR-specific reads were low in both libraries. Interestingly, the percentage of 3’NCR-specific reads was 83% higher in the library derived from leaves expressing miR173 (0.75% versus 0,41%) (Fig 2), suggesting that miR173 enhanced the production of siRNAs downstream of its target site. However, an equivalent increase of CP-specific siRNAs as consequence of miR173 expression was not observed (Fig 2).


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 for syn-tasiRNA accumulation in N. benthamiana transient assays.Mapping of small RNA sequences obtained by high-throughput sequencing are shown. Analyses of total small RNA (18–26 nt) reads in the presence (+miR173) or absence of miR173 (-miR173) is shown in percentages in pie charts. Those reads mapping across the syn-tasiRNA constructs (construct specific) are shown in detail. CP: reads mapping to CP region included in the syn-tasiR-CP. 3NCR: reads mapping to 3’NCR region included in the syn-tasiR-3NCR. Ath-MIR173: reads mapping to the MIR173 construct.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132281.g002: Effect of miR173 expression for syn-tasiRNA accumulation in N. benthamiana transient assays.Mapping of small RNA sequences obtained by high-throughput sequencing are shown. Analyses of total small RNA (18–26 nt) reads in the presence (+miR173) or absence of miR173 (-miR173) is shown in percentages in pie charts. Those reads mapping across the syn-tasiRNA constructs (construct specific) are shown in detail. CP: reads mapping to CP region included in the syn-tasiR-CP. 3NCR: reads mapping to 3’NCR region included in the syn-tasiR-3NCR. Ath-MIR173: reads mapping to the MIR173 construct.
Mentions: The total numbers of reads after filtering to remove rRNA and tRNA were 9,433,876 and 8,893,736 for the pools of syn-tasiRNA constructs expressed alone (library-miR173) or together with miR173 (library +miR173), respectively. To determine how expression of miR173 affects the patterns of sRNA populations, we classified sRNAs in five groups: i) matching to N. benthamiana genome, ii) matching Agrobacterium genome, iii) matching common regions of agroinfiltrated pMDC32-derived plasmids, iv) matching specific regions of each pMDC32-derived plasmids, and v) unknown sequences (Fig 2). Approximately 32%, 1.5% and 60% of the reads of the-miR173 library corresponded to sRNAs of the N. benthamiana, Agrobacterium and pMDC-shared groups, respectively (Fig 2). These percentages were marginally lower, and that of the unknown sequences marginally higher, in the +miR173 library (Fig 2). As corresponds to the small size of the cloned PPV sequence, the percentages of both PPV CP- and PPV 3’NCR-specific reads were low in both libraries. Interestingly, the percentage of 3’NCR-specific reads was 83% higher in the library derived from leaves expressing miR173 (0.75% versus 0,41%) (Fig 2), suggesting that miR173 enhanced the production of siRNAs downstream of its target site. However, an equivalent increase of CP-specific siRNAs as consequence of miR173 expression was not observed (Fig 2).

Bottom Line: 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.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