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Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis.

Rajeswaran R, Aregger M, Zvereva AS, Borah BK, Gubaeva EG, Pooggin MM - Nucleic Acids Res. (2012)

Bottom Line: TAS1c is also feedback regulated by a cis-acting siRNA.We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation.TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNAs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Botany, University of Basel, Schönbeinstrasse 6, 4056 Basel, Switzerland.

ABSTRACT
Biogenesis of trans-acting siRNAs (tasiRNAs) is initiated by miRNA-directed cleavage of TAS gene transcripts and requires RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Here, we show that following miR173 cleavage the entire polyadenylated parts of Arabidopsis TAS1a/b/c and TAS2 transcripts are converted by RDR6 to double-stranded (ds)RNAs. Additionally, shorter dsRNAs are produced following a second cleavage directed by a TAS1c-derived siRNA. This tasiRNA and miR173 guide Argonaute 1 complexes to excise the segments from TAS2 and three TAS1 transcripts including TAS1c itself to be converted to dsRNAs, which restricts siRNA production to a region between the two cleavage sites. TAS1c is also feedback regulated by a cis-acting siRNA. We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation. TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNAs.

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The entire, polyadenylated 3′-products of miR173-cleaved pri-RNAs of TAS1a, TAS1b, TAS1c and TAS2 genes are converted by RDR6 to dsRNAs. Diagrammatic representation of TAS genes transcribed by Pol II into 5′-capped and 3′-polyadenylated pri-RNAs which are then cleaved by miR173-AGO1 complexes. The 3′-products are converted by RDR6 to long dsRNAs. The regions of the miR173 cleavage site and the poly(A) sites of each TAS pri-RNA are enlarged. Arrows above the upper strand indicate positions of the 5′-terminus (miR173 site) and the 3′-terminus of at least one cloned cRT-PCR product representing the sense strands of TAS dsRNAs; the 3′-termini to which poly(A) is added are indicated in pink. Arrows below the complementary strand indicate the termini of one or more cloned cRT-PCR product representing the corresponding antisense strands of dsRNAs. Termini of the antisense RNA that begin with a poly(U) stretch are indicated in cyan. Thick arrows indicate the termini of the major sense and antisense RNAs; the number of specific cRT-PCR products/total products sequenced for each strand are indicated. Positions of cRT-PCR primers used for long dsRNA mapping are indicated above and below of the TAS gene body.
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gks242-F2: The entire, polyadenylated 3′-products of miR173-cleaved pri-RNAs of TAS1a, TAS1b, TAS1c and TAS2 genes are converted by RDR6 to dsRNAs. Diagrammatic representation of TAS genes transcribed by Pol II into 5′-capped and 3′-polyadenylated pri-RNAs which are then cleaved by miR173-AGO1 complexes. The 3′-products are converted by RDR6 to long dsRNAs. The regions of the miR173 cleavage site and the poly(A) sites of each TAS pri-RNA are enlarged. Arrows above the upper strand indicate positions of the 5′-terminus (miR173 site) and the 3′-terminus of at least one cloned cRT-PCR product representing the sense strands of TAS dsRNAs; the 3′-termini to which poly(A) is added are indicated in pink. Arrows below the complementary strand indicate the termini of one or more cloned cRT-PCR product representing the corresponding antisense strands of dsRNAs. Termini of the antisense RNA that begin with a poly(U) stretch are indicated in cyan. Thick arrows indicate the termini of the major sense and antisense RNAs; the number of specific cRT-PCR products/total products sequenced for each strand are indicated. Positions of cRT-PCR primers used for long dsRNA mapping are indicated above and below of the TAS gene body.

Mentions: To precisely map TAS dsRNAs we used cRT-PCR that allows simultaneous sequencing of 5′- and 3′-termini of a given RNA [(23), Supplementary Figure S2A]. Pyrophosphatase treatment of total RNA, prior to RNA ligation, allowed circularization of transcripts not only with a 5′-monophosphate present on 3′-fragments of miRNA-cleaved TAS pri-RNA, but also with a 5′-triphosphate likely present on an RDR6-synthesized complementary RNA. cRT-PCR primers proximal to the termini of a given sense or antisense RNA (Figures 2 and 3; Supplementary Figure S2) were designed based on the sizes of putative dsRNAs estimated by blot hybridization (Figure 1B). cRT-PCR products were gel-purified, cloned and sequenced (∼10–30 individual clones for each).Figure 2.


Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis.

Rajeswaran R, Aregger M, Zvereva AS, Borah BK, Gubaeva EG, Pooggin MM - Nucleic Acids Res. (2012)

The entire, polyadenylated 3′-products of miR173-cleaved pri-RNAs of TAS1a, TAS1b, TAS1c and TAS2 genes are converted by RDR6 to dsRNAs. Diagrammatic representation of TAS genes transcribed by Pol II into 5′-capped and 3′-polyadenylated pri-RNAs which are then cleaved by miR173-AGO1 complexes. The 3′-products are converted by RDR6 to long dsRNAs. The regions of the miR173 cleavage site and the poly(A) sites of each TAS pri-RNA are enlarged. Arrows above the upper strand indicate positions of the 5′-terminus (miR173 site) and the 3′-terminus of at least one cloned cRT-PCR product representing the sense strands of TAS dsRNAs; the 3′-termini to which poly(A) is added are indicated in pink. Arrows below the complementary strand indicate the termini of one or more cloned cRT-PCR product representing the corresponding antisense strands of dsRNAs. Termini of the antisense RNA that begin with a poly(U) stretch are indicated in cyan. Thick arrows indicate the termini of the major sense and antisense RNAs; the number of specific cRT-PCR products/total products sequenced for each strand are indicated. Positions of cRT-PCR primers used for long dsRNA mapping are indicated above and below of the TAS gene body.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401431&req=5

gks242-F2: The entire, polyadenylated 3′-products of miR173-cleaved pri-RNAs of TAS1a, TAS1b, TAS1c and TAS2 genes are converted by RDR6 to dsRNAs. Diagrammatic representation of TAS genes transcribed by Pol II into 5′-capped and 3′-polyadenylated pri-RNAs which are then cleaved by miR173-AGO1 complexes. The 3′-products are converted by RDR6 to long dsRNAs. The regions of the miR173 cleavage site and the poly(A) sites of each TAS pri-RNA are enlarged. Arrows above the upper strand indicate positions of the 5′-terminus (miR173 site) and the 3′-terminus of at least one cloned cRT-PCR product representing the sense strands of TAS dsRNAs; the 3′-termini to which poly(A) is added are indicated in pink. Arrows below the complementary strand indicate the termini of one or more cloned cRT-PCR product representing the corresponding antisense strands of dsRNAs. Termini of the antisense RNA that begin with a poly(U) stretch are indicated in cyan. Thick arrows indicate the termini of the major sense and antisense RNAs; the number of specific cRT-PCR products/total products sequenced for each strand are indicated. Positions of cRT-PCR primers used for long dsRNA mapping are indicated above and below of the TAS gene body.
Mentions: To precisely map TAS dsRNAs we used cRT-PCR that allows simultaneous sequencing of 5′- and 3′-termini of a given RNA [(23), Supplementary Figure S2A]. Pyrophosphatase treatment of total RNA, prior to RNA ligation, allowed circularization of transcripts not only with a 5′-monophosphate present on 3′-fragments of miRNA-cleaved TAS pri-RNA, but also with a 5′-triphosphate likely present on an RDR6-synthesized complementary RNA. cRT-PCR primers proximal to the termini of a given sense or antisense RNA (Figures 2 and 3; Supplementary Figure S2) were designed based on the sizes of putative dsRNAs estimated by blot hybridization (Figure 1B). cRT-PCR products were gel-purified, cloned and sequenced (∼10–30 individual clones for each).Figure 2.

Bottom Line: TAS1c is also feedback regulated by a cis-acting siRNA.We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation.TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNAs.

View Article: PubMed Central - PubMed

Affiliation: Institute of Botany, University of Basel, Schönbeinstrasse 6, 4056 Basel, Switzerland.

ABSTRACT
Biogenesis of trans-acting siRNAs (tasiRNAs) is initiated by miRNA-directed cleavage of TAS gene transcripts and requires RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Here, we show that following miR173 cleavage the entire polyadenylated parts of Arabidopsis TAS1a/b/c and TAS2 transcripts are converted by RDR6 to double-stranded (ds)RNAs. Additionally, shorter dsRNAs are produced following a second cleavage directed by a TAS1c-derived siRNA. This tasiRNA and miR173 guide Argonaute 1 complexes to excise the segments from TAS2 and three TAS1 transcripts including TAS1c itself to be converted to dsRNAs, which restricts siRNA production to a region between the two cleavage sites. TAS1c is also feedback regulated by a cis-acting siRNA. We conclude that TAS1c generates a master siRNA that controls a complex network of TAS1/TAS2 siRNA biogenesis and gene regulation. TAS1/TAS2 short dsRNAs produced in this network are processed by DCL4 from both ends in distinct registers, which increases repertoires of tasiRNAs.

Show MeSH
Related in: MedlinePlus