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. 2012 Jul;40(13):6241-54.
doi: 10.1093/nar/gks242. Epub 2012 Mar 19.

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

Rajendran Rajeswaran  1 , Michael Aregger, Anna S Zvereva, Basanta K Borah, Ekaterina G Gubaeva, Mikhail M Pooggin
Affiliations

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

Rajendran Rajeswaran et al. Nucleic Acids Res. 2012 Jul.

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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Figures

Figure 1.
Figure 1.
Blot hybridization analysis of dsRNA precursors of TAS1a/b/c and TAS2 tasiRNAs. (A) The panel shows blot hybridization analysis (5% PAGE) of total RNA from CaMV-infected (+) or mock-inoculated (−) wild-type (Col-0 and La-er), double (dcl2/3 and dcl3/4) and triple (dcl2/3/4) DCL-deficient mutants. The scans represent the same blot successively hybridized with probes specific for TAS1a/b/c-, TAS1a/b-, TAS1b-, TAS1c- and TAS2-derived 21-nt siRNAs and U6 RNA as a loading control. Single-stranded RNA markers are shown on the left. The scheme shows location and polarity of the probed siRNAs (colored boxes with their names indicated) with respect to the miR173 cleavage site (indicated by arrow) on hypothetical dsRNAs from the four TAS genes. Identical siRNAs/probes, i.e. siR255/siR255* duplex encoded by TAS 1a, 1b and 1c and 3′D1(−) siRNA of TAS 1a and 1b, are indicated in pink and purple, respectively. Cyan, blue and orange indicate siRNAs/probes specific for TAS1b, TAS1c and TAS2, respectively. (B) Interpretation of the analysis shown in panel A. The lanes corresponding to Col-0 and dcl2/3/4 samples were cropped from the above blots of sense (S) polarity. Positions of pri-RNAs and dsRNAs are indicated by arrows for each TAS gene (1a, 1b, 1c and 2).
Figure 2.
Figure 2.
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.
Figure 3.
Figure 3.
Two-cleavage mechanism for biogenesis of TAS1a-, TAS1b-, TAS1c- and TAS2-derived dsRNAs. Diagrammatic representation of TAS genes transcribed by Pol II into pri-RNAs which are then cleaved at the two sites by miR173- and TAS1c 3′D6(−) siRNA-AGO1 complexes, respectively. The excised fragment between the two-cleavage sites is converted by RDR6 to dsRNA. The cleavage sites for each pri-RNA and the siRNA/target pri-RNA duplexes are shown in the expanded regions. Additional complementary sites for TAS1c 3′D6(−) siRNA in the intervening regions of TAS2 and TAS1a pri-RNAs as well as the origin site of TAS1c 3′D6(−) are also shown. Colored arrowheads indicate positions of the 5′-terminus (miR173 site; in blue) and the 3′-terminus [TAS1c 3′D6(−) site; in red] of cloned cRT-PCR products representing the sense strands of TAS dsRNAs. The 5′- and 3′-terminal sequences of cRT-PCR clones representing the corresponding antisense strands of TAS dsRNAs are shown below the duplexes and highlighted in cyan. The number of specific cRT-PCR products/total products sequenced for each strand is indicated. Positions of potential cleavage sites for 3′D6(−) siRNA are indicated by open arrowheads. Positions of the cRT-PCR primers used for the short dsRNA mapping are indicated above and below of the TAS gene body.
Figure 4.
Figure 4.
TAS1c is a master tasiRNA-generating unit regulated by feedback loops. (A) Genetic requirements for the biogenesis of TAS1/2-derived siRNAs. The panel shows blot hybridization analysis (15% PAGE) of total RNA from CaMV-infected (+) or mock-inoculated (−) wild-type Arabidopsis (Col-0), single (dcl2-5, dcl3-1 and dcl4-2) and double (dcl2/3, dcl2/4 and dcl3/4) DCL null mutants, AGO mutants (ago1-25 and ago2-1) and TAS1c siRNA-deficient mutant (tas1c-1) plants. The cropped scans represent blots successively hybridized with probes specific for TAS1c-, TAS1b-, TAS1a/b-, TAS1a/b/c-, TAS2- and TAS3a-derived siRNAs and miR173. EtBr-stained rRNA is shown as a loading control. The relative levels of TAS siRNA accumulation in a mutant line versus Col-0 were calculated using the miR173 level as an internal control for each sample; for each blot, siRNA levels in Col-0 were set to 1. (B) Blot hybridization analysis of TAS1c-derived dsRNA-D6 and dsRNA-D10. The cropped scans are from the blot shown in panel (A) (the second lane from left). (C) Blot hybridization analysis of TAS1c-, TAS1b-, TAS1a-, TAS2-derived dsRNAs in CaMV-infected Col-0 and tas1c-1 plants using 5% PAGE. The total RNA samples analyzed are the same as in panel A (the two from right). EtBr-stained RNA is shown as a loading control. Positions of dsRNAs derived from TAS1c, TAS1b, TAS1a and TAS2 are indicated by arrows (same as in Figure 1B). TAS1c 3′D6(−) tasiRNA-dependent dsRNAs are highlighted in pink, TAS1c 3′D10(−)-dependent dsRNA is in green and tasiRNA-independent longest dsRNAs are in blue. (D) Model for TAS1c tasiRNA biogenesis and feedback regulation.
Figure 5.
Figure 5.
Loading of TAS1c siRNAs into AGO1 and AGO2 complexes that mediate in trans and in cis cleavages. (A) The upper panel shows blot hybridization analysis of total sRNAs (input) and sRNAs associated with AGO1 protein in mock-inoculated (−) or CaMV-infected (+) plants following IP with AGO1-antibodies (IP:AGO1). The scans represent the same blot successively hybridized with probes specific for TAS1c siRNAs, miR173 and, as a negative control, the transposon SIMPLEHAT2-derived 24-nt siRNA. The lower panel shows Western blot analysis of AGO1 protein accumulation in the input and the IP:AGO1 fractions using AGO1-antibody (@AGO1). Underneath is the same blot stained with antibodies specific for CaMV TAV protein (@TAV); (B) The in cis cleavage site for TAS1c-derived siRNA 3′D10(−) at TAS1c pri-RNA (the numbering is from the 5′ cap-site). Thick and thin arrows indicate the major and minor cleavage products, respectively, determined by cRT-PCR; the number of clones is given when more than one clone had the same 3′-terminus. The sequence of 3′D10(−) siRNA guiding the cleavage is shown under the pri-RNA sequence. The 5′-termini of predominant antisense RNAs of 1c dsRNA-D10 are shown, with corresponding numbers of specific cRT-PCR products/total products indicated; (C) Blot hybridization analysis of TAS1c-derived dsRNAs in CaMV-infected Col-0 and ago2-1 plants using 5% PAGE. The blot was hybridized with TAS1c 3′D6(−) siRNA-specific probe. EtBr-stained RNA is shown as a loading control. Positions of TAS1c-derived dsRNAs and dsRNA-D10 are indicated by arrows; (D) sRNA sequencing reads for TAS1c-derived siRNAs and miR173 in sRNA libraries prepared from AGO:IP complexes [(15); GEO accession: GSE10036].
Figure 6.
Figure 6.
TAS1c 3′D6(−) tasiRNA-directed cleavage regulates TAS1c 3′D10(−) siRNA biogenesis and precludes TAS2 siRNA production downstream of the target site. (A) The wild-type and mutant targets sites (TS) of the wild-type TAS1c pri-RNA and its TS mutant version, respectively. Mutagenized positions are in lower case and highlighted in cyan. Position of the cleavage directed by the wild-type TAS1c 3′D6(−) tasiRNA or its seed-mutant version is indicated by red arrow. (B) Reconstruction of TAS1c and TAS2 tasiRNA biogenesis in a transient expression assay using N. benthamiana. 35S:TAS1c constructs with mutagenized 3′D6(−), 3′D10(−) siRNAs, the 3′D6(−) target site sequences, or both 3′D6(−) and its target site sequences were expressed or coexpressed with 35S:miR173 and also with 35S:TAS2 constructs as indicated above the blot panels. The siRNAs detected by successive hybridization of the blots are shown to the right of each panel.
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