A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis

Abstract

The Arabidopsis genes, TAS2 and TAS1a, produce structurally similar noncoding transcripts that are transformed into short (21-nucleotide [nt]) and long (24-nt) siRNAs by RNA silencing pathways. Some of these short siRNAs direct the cleavage of protein-coding transcripts, and thus function as trans-acting siRNAs (ta-siRNAs). Using genetic analysis, we defined the pathway by which ta-siRNAs and other short siRNAs are generated from these loci. This process is initiated by the miR173-directed cleavage of a primary poly(A) transcript. The 3' fragment is then transformed into short siRNAs by the sequential activity of SGS3, RDR6, and DCL4: SGS3 stabilizes the fragment, RDR6 produces a complementary strand, and DCL4 cleaves the resulting double-stranded molecule into short siRNAs, starting at the end with the miR173 cleavage site and proceeding in 21-nt increments from this point. The 5' cleavage fragment is also processed by this pathway, but less efficiently. The DCL3-dependent pathway that generates long siRNAs does not require miRNA-directed cleavage and plays a minor role in the silencing of these loci. Our results define the core components of a post-transcriptional gene silencing pathway in Arabidopsis and reveal some of the features that direct transcripts to this pathway.

Figure 1.

At2g39680 (TAS2) produces ta-siRNAs that target PPR genes. (A) Sequences in TAS2 that have reverse complementarity to mRNAs encoding PPR proteins. Fractions indicate the number of sequenced RLM-5′RACE clones that terminated at the indicated position. (B) sgs3-11 and rdr6-11 prevent the formation of RLM-5′RACE products from PPR transcripts targeted by siRNAs from TAS2. Amplification of the miR171 target, SCL6-IV, occurs normally in these mutants. (C) Blots of LMW RNA hybridized with oligo nucleotide probes complementary to the TAS2 sequences in A and to the cloned TAS2 siRNA, siR1511. U6 was used as loading control. (D) Quantitative real-time RT-PCR analysis of the abundance of PPR transcripts in total RNA from wild type (WT), sgs3-11, rdr6-11, and plants homozygous for the SALK_014168 T-DNA insertion. PCR was performed with primers that flank the cleavage site in these transcripts. Three to six biological replicates were performed for each sample. Values were normalized to EIF4A and are expressed as the ratio of the value relative to wild type. (E) Blot of LMW RNA from wild type (WT), two families of plants homozygous for the SALK_014168 T-DNA insertion, and rdr6-11 hybridized with probes to the indicated siRNAs.

Figure 2.

TAS2(+) and TAS1(+) are cleaved by miR173. (A) Schematic illustration of TAS2 cDNAs and transcripts identified by 5′ and 3′ RACE, RLM-5′RACE, and RT-PCR of self-ligated RNA. (Black box) Exon; (open box) intron. (B) Structure of the unspliced TAS1a(+) transcript (Vazquez et al. 2004b). (C) DNA probes used in this study. Similarly positioned probes were used for TAS2 and TAS1a. (D) Blots of total and poly(A)-selected RNA hybridized with the indicated TAS2 probes. The abundance of the ≈1-kb polyadenylated (+) transcript is not affected by rdr6-11; however, the 0.44-kb nonadenylated 5′ (+) fragment and the 0.66-kb polyadenylated 3′ (+) fragment accumulate to high levels in this mutant. (E) TAS2(+) and TAS1(+) transcripts are cleaved at the miR173 target site. Fractions indicate the number of RLM-5′ RACE clones that terminated at the indicated position.

Figure 3.

The position and predicted nucleotide sequence of TAS2 and TAS1 siRNAs. (A) siRNAs arising from the 3′ fragment of TAS2, TAS1a, TAS1c, and TAS1b are positioned in 21-nt increments relative to the miR173 cleavage site (arrow). The sequence of these siRNAs is available from http://asrp.cgrb.oregonstate.edu. siRNAs in boxes are within 2 nt of the site predicted by a 21-nt spacing pattern. (B) The sequence of hypothetical ta-siRNAs from TAS2 and TAS1a, and the PPR genes targeted by these ta-siRNAs. Transcripts that are cleaved 10 nt from the 5′ end of the ta-siRNA are in red. Sequences that are similar in TAS2 and TAS1a are indicated by a bracket. (C) At1g12770 has two closely spaced cleavage sites potentially targeted by ta-siRNAs from TAS2 and TAS1a.

Figure 4.

Production of ta-siRNAs from TAS2(+) and TAS1a(+) requires miR173-directed cleavage. Blots of LMW RNA (A,C) or total RNA (B,D) hybridized with the indicated probes. (A) Mutations that interfere with the biogenesis of miR173 have correlated effects on the accumulation of siR1511 and siR255. (B) Cleavage of TAS2(+) in dcl1-7, hyl1-2, and hst-6 is inversely correlated with the abundance of miR173 in these mutants. (C) A T-DNA-inserted 3′ of the miR173 cleavage site destabilizes TAS2(+) and reduces the accumulation of the 5′ cleavage fragment. (D) The 5′ and 3′ portions of TAS2 and TAS1a produce different size classes of siRNAs. Production of 24-nt siRNAs requires DCL3 but not DCL1. (E) dcl2-1 and dcl3-1 have no effect on the accumulation of TAS2(+) and TAS1a(+) or their cleavage products. See Figure 2B for the location of the probes used in these experiments.

Figure 5.

Epistasis analysis of the function of SGS3, RDR6, and XRN4. Blots of total RNA (A-C) and LMW RNA (C) hybridized with the indicated probes. (A) dcl1-7 rdr6-11 double mutants are indistinguishable from dcl1-7. (B) sgs3-11 reduces the accumulation of the TAS2(+) and TAS1a(+) cleavage fragments and is epistatic to rdr6-11.(C) xrn4-1 does not affect the accumulation of TAS2(+) and its cleavage fragments (top) and also has no effect on siRNAs derived from TAS2 and TAS1a (bottom).

Figure 6.

DCL4 is a component of the SGS3/RDR6 pathway and acts downstream of RDR6. (A) Two-week-old wild-type and mutant plants. The vegetative phenotype of dcl4-2 is identical to that of sgs3-11 and rdr6-11. (B) The domain structure of the DCL4 protein (Schauer et al. 2002) and the location of the dcl4-2 mutation. dcl4-2 affects a glutamic acid that is completely conserved in Dicers from Arabidopsis (At), Homo sapiens (Hs), C. elegans (Ce), Drosophila melanogaster (Dm), and S. pombe (Sp). (C) dcl4-2 interferes with the biogenesis of siR1511 and siR255 but does not affect miR173. (D) dcl4-2 accumulates an array of small transcripts, some of which hybridize to an oligonucleotide probe specific for the (-) strand of TAS2(3′). These small transcripts are not produced in rdr6-11 dcl4-2 double mutants.

Figure 7.

Model for the biogenesis of short siRNAs from TAS2(+) and TAS1a(+). The biogenesis of short siRNAs is initiated by cleavage of these transcripts by miR173. This event produces fragments that are protected from degradation by SGS3 and are subsequently transcribed by RDR6. The resulting double-stranded fragments are then cleaved into small siRNAs by DCL4 and possibly DCL1, starting at the end of the fragment. Some of these small siRNAs function as ta-siRNAs. The 3′ fragment is particularly susceptible to DCL4 because transcription by RDR6 frequently terminates at the miR173 cleavage site, yielding a duplex end. The 5′ fragment is less susceptible to DCL4 because transcription frequently begins internal to the 3′ end of this fragment, leaving a single-stranded end. Cleavage from the 5′ end of this fragment is inhibited by the 5′ cap.