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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

In-phase processing of syn-tasiRNA precursors directed by miR173.Phased syn-tasiRNAs (21 nt) (D1-D7) in register with the miR173-guided cleavage site (indicated with an arrow) from syn-tasiR-CP (A) and syn-tasiR-3NCR (B) constructs. (C) Two series of phased syn-tasiRNAs (21 and 22 nt) in register with the miR173-guided cleavage site (indicated with an arrow). One series is marked as a-aa and the second one as a-ab. Numbers above and below the syn-tasiRNAs indicate reads from samples syn-tasiR+173/syn-tasiR-173. The mi173 target site (in purple) and the PPV sequences are shown in bold.
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pone.0132281.g004: In-phase processing of syn-tasiRNA precursors directed by miR173.Phased syn-tasiRNAs (21 nt) (D1-D7) in register with the miR173-guided cleavage site (indicated with an arrow) from syn-tasiR-CP (A) and syn-tasiR-3NCR (B) constructs. (C) Two series of phased syn-tasiRNAs (21 and 22 nt) in register with the miR173-guided cleavage site (indicated with an arrow). One series is marked as a-aa and the second one as a-ab. Numbers above and below the syn-tasiRNAs indicate reads from samples syn-tasiR+173/syn-tasiR-173. The mi173 target site (in purple) and the PPV sequences are shown in bold.

Mentions: Seven PPV CP-specific sRNAs induced by miR173-expression were 21-nt in length and were in register with the expected miR173-guided cleavage site of the syn-tasiR-CP transcript (Fig 4A). In agreement with its origin from double stranded products, some of these sRNAs mapped to the transcribed strand [D1(+), D2(+) and D3(+)] and some others did to the complementary strand [D2(-), D3(-), D4(-) and D6(-)]. Whereas similar accumulation of both strands was observed for some of the PPV CP-specific phased sRNAs (D2 and D3), in other cases, one strand, either the positive (D1), or the negative (D4 and D6), was strongly preferred (Fig 4A). In the case of the syn-tasiR-3NCR two contiguous 21-nt sRNAs [D1(+) and D2(-)] laying just downstream the miR173 cleavage site were shown to be induced in response to miR173 (Fig 4B) but the 21-nt register did not go beyond. However, eight phased miR173-induced 21-nt sRNAs that accumulated more than 50 reads in the +miR173 library could be coupled to the expected miR173-guided cleavage site by a 22-nt sRNA [D1a(+)], which was also induced when miR173 was expressed (Fig 4C, species a and aa). But this was not the only register of phased sRNAs. Two other 22-nt sRNAs species [D4ab(+) and D6ab(-)] allow to couple another series of phased sRNAs, which included nine species fulfilling the threshold conditions when miR173 is expressed (Fig 4C, species a and ab); one of them, D5ab(-), accumulates up to 10305 reads.


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)

In-phase processing of syn-tasiRNA precursors directed by miR173.Phased syn-tasiRNAs (21 nt) (D1-D7) in register with the miR173-guided cleavage site (indicated with an arrow) from syn-tasiR-CP (A) and syn-tasiR-3NCR (B) constructs. (C) Two series of phased syn-tasiRNAs (21 and 22 nt) in register with the miR173-guided cleavage site (indicated with an arrow). One series is marked as a-aa and the second one as a-ab. Numbers above and below the syn-tasiRNAs indicate reads from samples syn-tasiR+173/syn-tasiR-173. The mi173 target site (in purple) and the PPV sequences are shown in bold.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132281.g004: In-phase processing of syn-tasiRNA precursors directed by miR173.Phased syn-tasiRNAs (21 nt) (D1-D7) in register with the miR173-guided cleavage site (indicated with an arrow) from syn-tasiR-CP (A) and syn-tasiR-3NCR (B) constructs. (C) Two series of phased syn-tasiRNAs (21 and 22 nt) in register with the miR173-guided cleavage site (indicated with an arrow). One series is marked as a-aa and the second one as a-ab. Numbers above and below the syn-tasiRNAs indicate reads from samples syn-tasiR+173/syn-tasiR-173. The mi173 target site (in purple) and the PPV sequences are shown in bold.
Mentions: Seven PPV CP-specific sRNAs induced by miR173-expression were 21-nt in length and were in register with the expected miR173-guided cleavage site of the syn-tasiR-CP transcript (Fig 4A). In agreement with its origin from double stranded products, some of these sRNAs mapped to the transcribed strand [D1(+), D2(+) and D3(+)] and some others did to the complementary strand [D2(-), D3(-), D4(-) and D6(-)]. Whereas similar accumulation of both strands was observed for some of the PPV CP-specific phased sRNAs (D2 and D3), in other cases, one strand, either the positive (D1), or the negative (D4 and D6), was strongly preferred (Fig 4A). In the case of the syn-tasiR-3NCR two contiguous 21-nt sRNAs [D1(+) and D2(-)] laying just downstream the miR173 cleavage site were shown to be induced in response to miR173 (Fig 4B) but the 21-nt register did not go beyond. However, eight phased miR173-induced 21-nt sRNAs that accumulated more than 50 reads in the +miR173 library could be coupled to the expected miR173-guided cleavage site by a 22-nt sRNA [D1a(+)], which was also induced when miR173 was expressed (Fig 4C, species a and aa). But this was not the only register of phased sRNAs. Two other 22-nt sRNAs species [D4ab(+) and D6ab(-)] allow to couple another series of phased sRNAs, which included nine species fulfilling the threshold conditions when miR173 is expressed (Fig 4C, species a and ab); one of them, D5ab(-), accumulates up to 10305 reads.

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