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. 2016 Mar 10;171(1):359–368. doi: 10.1104/pp.16.00148

A Short Open Reading Frame Encompassing the MicroRNA173 Target Site Plays a Role in trans-Acting Small Interfering RNA Biogenesis1

Manabu Yoshikawa 1,2,3,*, Taichiro Iki 1,2,3,2, Hisataka Numa 1,2,3, Kyoko Miyashita 1,2,3, Tetsuo Meshi 1,2,3, Masayuki Ishikawa 1,2,3
PMCID: PMC4854708  PMID: 26966170

For efficient production of trans-acting siRNA, the third open reading frame on the primary precursor transcript that encompasses the microRNA173 target site needs to be translated.

Abstract

trans-Acting small interfering RNAs (tasiRNAs) participate in the regulation of organ morphogenesis and determination of developmental timing in plants by down-regulating target genes through mRNA cleavage. The production of tasiRNAs is triggered by microRNA173 (miR173) and other specific microRNA-mediated cleavage of 5′-capped and 3′-polyadenylated primary TAS transcripts (pri-TASs). Although pri-TASs are not thought to encode functional proteins, they contain multiple short open reading frames (ORFs). For example, the primary TAS2 transcript (pri-TAS2) contains 11 short ORFs, and the third ORF from the 5′ terminus (ORF3) encompasses the miR173 target site. Here, we show that nonsense mutations in ORF3 of pri-TAS2 upstream of the miR173 recognition site suppress tasiRNA accumulation and that ORF3 is translated in vitro. Glycerol gradient centrifugation analysis of Arabidopsis (Arabidopsis thaliana) plant extracts revealed that pri-TAS2 and its miR173-cleaved 5′ and 3′ fragments are fractionated together in the polysome fractions. These and previous results suggest that the 3′ fragment of pri-TAS2, which is a source of tasiRNAs, forms a huge complex containing SGS3, miR173-programmed AGO1 RNA-induced silencing complex, the 5′ fragment, and ribosomes. This complex overaccumulated, moderately accumulated, and did not accumulate in rdr6, sde5, and sgs3 mutants, respectively. The sgs3 sde5 and rdr6 sde5 double mutants showed phenotypes similar to those of sgs3 and sde5 single mutants, respectively, with regard to the TAS2-related RNA accumulation, suggesting that the complex is formed in an SGS3-dependent manner, somehow modified and stabilized by SDE5, and becomes competent for RDR6 action. Ribosomes in this complex likely play an important role in this process.

In plants, two groups of small RNAs (sRNAs), namely, microRNAs (miRNAs) and small interfering RNAs (siRNAs), play a role in RNA silencing (Axtell, 2013; Bologna and Voinnet, 2014). MiRNAs are excised from single-stranded primary MIRNA (MIR) transcripts by DICER-LIKE1 (DCL1) with the assistance of SERRETA, HYPONASTIC LEAVES1, and other proteins (Dong et al., 2008; Fang and Spector, 2007; Kurihara and Watanabe, 2004; Rogers and Chen, 2013). In contrast, siRNAs are produced from long double-stranded RNAs (dsRNAs) through endonucleolytic cleavage by DCL2, DCL3, or DCL4 (Xie et al., 2004, 2005; Yoshikawa et al., 2005). These sRNAs are bound by ARGONAUTE (AGO) proteins to form effector complexes called RNA-induced silencing complexes (RISCs) or silencing effector complexes. RISCs recognize RNAs with sequences complementary to sRNAs and down-regulate expression of the corresponding genes by mRNA cleavage or translational repression. Silencing effector complexes transcriptionally repress target genes via DNA methylation of target genomic loci (Chen, 2009, 2010).

trans-Acting siRNAs (tasiRNAs) are plant-specific endogenous siRNAs that posttranscriptionally repress the expression of corresponding target genes through mRNA cleavage (Yoshikawa, 2013). The initial step of tasiRNA biogenesis is the synthesis of primary TAS transcripts (pri-TASs) from the genomic DNA by RNA polymerase II (Allen et al., 2005; Yoshikawa et al., 2005). Like protein-coding mRNAs, pri-TASs are 5′-capped and 3′-polyadenylated. These modifications allow the export of transcripts from the nucleus to the cytoplasm where pri-TASs are cleaved by AGO1-RISCs containing 22-nucleotide (nt) miRNAs (e.g. microRNA173 [miR173] in Arabidopsis [Arabidopsis thaliana]) or AGO7-RISCs containing microRNA390 (miR390; Axtell et al., 2006; Chen et al., 2010; Cuperus et al., 2010; Felippes and Weigel, 2009; Montgomery et al., 2008a, 2008b). The cleavage results in poly(A)-deficient 5′ fragments and uncapped 3′ fragments, which are associated with the RISC and SUPPRESSOR OF GENE SILENCING3 (SGS3) and protected from degradation (Yoshikawa et al., 2005, 2013). Subsequently, either cleavage fragment is converted into dsRNAs by RNA-DEPENDENT RNA POLYMERASE6 (RDR6), and processed by DCL4 into 21-nt tasiRNAs that are incorporated by AGO1, AGO2, or AGO5 (Mi et al., 2008; Takeda et al., 2008; Xie et al., 2005; Yoshikawa et al., 2005). SILENCING DEFECTIVE5 (SDE5) was also identified as an essential factor for tasiRNA production (Hernandez-Pinzon et al., 2007; Jauvion et al., 2010). SDE5 is partially homologous to human TAP/NXF1 that participates in mRNA export from the nucleus to the cytoplasm (Hernandez-Pinzon et al., 2007). In addition, mutations in the genes encoding TEX1, HPR1/THO1, or THO6 reduce tasiRNA accumulation (Jauvion et al., 2010; Yelina et al., 2010). The homologs of these proteins in yeast and animals are contained in the transcription-export (THO/TREX) complex involved in transcription-coupled mRNA export (Köhler and Hurt, 2007; Masuda et al., 2005; Reed and Cheng, 2005; Rehwinkel et al., 2004; Strässer et al., 2002). However, the roles of SDE5 and a THO/TREX complex in tasiRNA production have remained unclear.

Previously, we demonstrated that miR173-cleaved TAS RNA fragments form huge complexes containing SGS3 and RISC (Yoshikawa et al., 2013). MiR173 triggers tasiRNA production from the primary TAS1a-c and TAS2 transcripts (pri-TAS1a-c and pri-TAS2, respectively) in Arabidopsis (Allen et al., 2005; Chen et al., 2010; Cuperus et al., 2010; Felippes and Weigel, 2009; Yoshikawa et al., 2005). Because the complex has large sedimentation coefficient (>80S), it was thought to contain additional unidentified components. On the other hand, although pri-TASs are not thought to encode functional proteins, multiple potential open reading frames (ORFs) > 12 nt (we designate coding regions between the initiating AUG codons and corresponding termination codons as ORFs in this study) have been identified. In pri-TAS1a-c and pri-TAS2, ORFs that encompass the miR173 cleavage sites are found (for pri-TAS1a and pri-TAS2, they are referred to as ORF3 because they correspond to the third ORFs from the 5′ termini; Fig. 1A, red bars). Zhang et al. demonstrated positive effects of translation on the production of synthetic tasiRNAs (syn-tasiRNAs) using an artificial tasiRNA production system based on a GFP-fused histone 2A-coding mRNA (Zhang et al., 2012). In this study, we investigated the function of ORF3 on pri-TAS2 and found that ORF3 is in fact translated and plays an important role in tasiRNA biogenesis.

Figure 1.

Figure 1.

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Effects of introduction of termination codons in ORF3 on the accumulation of TAS2-derived tasiRNAs in N. benthamiana. A, Structure of pri-TAS1a and pri-TAS2. The red bars and the red arrows show ORF3s and the miR173 cleavage sites, respectively. The numbers indicate the nucleotide positions on each transcript that correspond to the initiation codons, the miR173 cleavage sites, and the termination codons, respectively. The blue bars represent putative upstream ORFs. Downstream ORFs are omitted from this diagram. B, Structure of the plasmids used for transient expression analysis in N. benthamiana. Black and white arrows show the MIR and TAS2-related genes, respectively. The mutated nucleotides are shown by red characters. The termination codons generated in the mutated constructs are shown by boxed characters. C, Accumulation of TAS2-derived tasiRNAs in Agrobacterium-infiltrated N. benthamiana leaves. Cultures of Agrobacterium tumefaciens derived from two independent colonies for each construct were infiltrated into two independent plants, and leaves were separately harvested 48 h after infiltration. After RNA extraction, total RNA (10 μg) from each plant was applied to each lane of denaturing 15% polyacrylamide gel, separated by electrophoresis, and transferred to a nylon membrane. The RNA blot was hybridized with a 32P-labeled probe complementary to TAS2 RNA. The probe was stripped, and the same blot was reprobed with probes complementary to miR171a and miR173, respectively. D, Effect of the position of termination codons on tasiRNA production from pri-TAS2. sRNA (10 μg) was used for the analysis. The experiment was carried out as described in C. The intensity of each band was quantified and the ratio of TAS2-3′D3(+) to miR173 was normalized with the leftmost lane.

RESULTS

ORF3 of Pri-TAS2 Plays a Role in TasiRNA Accumulation

To examine whether ORF3 plays a role in tasiRNA production, we constructed wild-type and mutated TAS2 genes under control of the cauliflower mosaic virus 35S RNA promoter (P35S; Fig. 1B). TAS2-171 contained the miR171 target site in place of the miR173 target site (Yoshikawa et al., 2013). TAS2-MUT1 contained a single-nucleotide deletion in the four consecutive thymine residues immediately upstream of the miR173 target site. This frame shift produced a stop codon within the miR173 target site (Fig. 1B). In addition, the TTG codon present 15 nt upstream of the miR173 target site was replaced with the TGA termination codon in TAS2-MUT2 (Fig. 1B). Importantly, the latter two mutations did not modify the miR173 target site. These P35S::TAS2 gene cassettes were inserted into the binary vectors containing MIR171a or MIR173 (Fig. 1B) and were introduced into Nicotiana benthamiana leaves using the Agrobacterium-infiltration method (Allen et al., 2005). A plasmid harboring the GFP gene was used as a control. After RNA extraction from infiltrated leaves, the accumulation of tasiRNAs generated from each TAS2 transcript was analyzed (Fig. 1C). In leaves where wild-type TAS2 was coexpressed with the MIR173 transcript (pri-miR173), we observed an accumulation of tasiRNAs derived from the miR173-cleaved 3′ fragments of the TAS2 transcripts. Coexpression of MIR171a transcript (pri-miR171a) and wild-type TAS2 transcript resulted in a minor accumulation of sRNAs from the TAS2 transcript, whereas coexpression of pri-miR173 and TAS2-171 transcript did not (Fig. 1C). The level of accumulation of tasiRNAs in leaves expressing TAS2-MUT1 transcript and pri-miR173 was similar to that in leaves expressing the wild-type TAS2 transcript and pri-miR173. When pri-miR173 and TAS2-MUT2 transcript were coexpressed, the accumulation of TAS2-derived tasiRNAs was lower than that in leaves coexpressing pri-miR173 and wild-type TAS2 transcript.

To further investigate the effects of the location of the termination codons on TAS2-derived tasiRNA accumulation, we constructed three additional mutants of pri-TAS2 (Fig. 1B). TAS2-MUT3 has a single base substitution 21 nt upstream of the miR173 target site generating the TAG stop codon. TAS2-MUT4 contains two base substitutions 4 and 6 nt upstream of the miR173 target site, which create a termination codon at the mutation site. In TAS2-6×His, the 18-nt sequence just upstream of the miR173 target site was replaced with a sequence encoding six consecutive histidines, expecting changes in the secondary structure around the miR173 target site. Accumulation level of tasiRNAs from TAS2-MUT3 was lower than that from wild-type TAS2 and similar to that for TAS2-MUT2. TasiRNAs from TAS2-MUT4 and TAS2-6×His accumulated to similar levels to that from wild-type TAS2 (Fig. 1D). In the absence of miR173 overexpression, the full-length transcripts of the wild-type and the mutated TAS2 genes accumulated at similar levels (Supplemental Fig. S1). These results are consistent with the previous observation that introduction of a stop codon > 13 nt upstream of the miR173 target site reduces syn-tasiRNA accumulation (Zhang et al., 2012).

ORF3s of Pri-TAS2 and Pri-TAS1a Are Translated in Vitro

The above results suggest that translation of ORF3 is important for tasiRNA production from pri-TAS2. Next, we examined whether ORF3s of pri-TAS2 and pri-TAS1a, which encompass miR173 cleavage sites (Fig. 1A, red bars), are translated in an in vitro translation system using evacuolated tobacco (Nicotiana tabacum) protoplast extracts (mdBYL; Komoda et al., 2004). We modified TAS2 and TAS1a transcripts whose C-terminal eight amino acid residues in each ORF3 were replaced with those of the FLAG epitope (TAS2-FLAG and TAS1a-FLAG, respectively) so that their translation products could be detected with an anti-FLAG antibody (Fig. 2A). These transcripts were translated in mdBYL with an internal control mRNA encoding myc-tagged luciferase (LUC-myc). After translation, the products were analyzed by immunoblotting with anti-FLAG and anti-myc antibodies (Fig. 2B). The LUC-myc protein was detected in all reactions, except for the reaction in which LUC-myc mRNA was not added. When TAS2-FLAG and TAS1a-FLAG RNAs were added into the reaction mixture, FLAG-tagged products were detected, indicating that the ORF3s are translated.

Figure 2.

Figure 2.

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Detection of in vitro translation products from ORF3s of pri-TAS1a and pri-TAS2. A, Structure of pri-TAS1a and pri-TAS2 derivatives whose ORF3 sequences were partially replaced with FLAG-coding sequences. The changed nucleotides are shown by red characters. Thin and thick underlines represent the miR173 target sites and the FLAG epitope, respectively. B, Detection of FLAG-tagged translation products. After in vitro translation using mdBYL, products were separated using 4% to 12% Bis-Tris NuPAGE gel with MES running buffer. FLAG-tagged products and LUC-myc were detected with anti-FLAG antibody and anti-myc antibody, respectively.

TAS2-Derived RNAs Are Detected in Polysome Fractions

Previously, the accumulation patterns of TAS1a- and TAS2-derived RNAs in Arabidopsis mutants defective in tasiRNA production were determined (Yoshikawa et al., 2005). Each mutation showed unique effects on the accumulation pattern of tasiRNA and the precursors, which revealed the function and order of action of corresponding genes in tasiRNA biogenesis (Supplemental Fig. S2). The proposed outline of tasiRNA production is as follows: (1) miR173 is produced by DCL1 and loaded onto AGO1; (2) miR173-programmed AGO1 cleaves pri-TAS2; (3) SGS3 binds to the cleaved pri-TAS2-AGO1-miR173 complex and stabilizes the 5′ and 3′ cleavage fragments; (4) RDR6 is recruited to the 3′ cleavage fragment of pri-TAS2 and synthesizes complementary-strand RNA; and (5) the dsRNA is cleaved by DCL4 to produce tasiRNAs.

Given that the pri-TAS2 RNA is translated, we performed polysome analysis with wild-type and tasiRNA-defective Arabidopsis plants to examine whether ribosomes are associated with the TAS2-related RNAs (Fig. 3; Supplemental Fig. S3). In this analysis, extracts from flower bud tissues of Arabidopsis plants were loaded onto 10% to 40% glycerol gradients, subjected to centrifugation, and fractionated into 12 fractions. Under our conditions, monosomes and polysomes were fractionated in the fourth and fifth to 12th fractions, respectively (profiles of A254 and rRNA distributions are shown in Fig. 3A and Supplemental Fig. S3).

Figure 3.

Figure 3.

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Polysome analysis of TAS2-derived RNAs in wild-type and tasiRNA-defective mutants. A, Patterns of A254 and distribution of ribosomal RNAs. An extract of floral tissues from wild-type (WT) plants was subjected to fractionation by 10% to 40% glycerol density gradient centrifugation. Absorbance was measured continuously. RNA was isolated from each fraction (1 mL), run on a 1.2% agarose gel, and stained with ethidium bromide. Fractions 1 and 12 represent the top and bottom fractions. 80S ribosomes were mainly fractionated in the fourth fraction. B, Polysome analyses of TAS2-derived RNAs in tasiRNA-defective mutants. Fractionation was performed as described in A. RNA blots were hybridized with the TAS2 probe. Total RNA (10 µg) from rdr6 mutant plants was used as a marker for the TAS2-related RNAs (the leftmost lane on each gel). Continuous absorbance measurement at 254 nm and RNA gels stained with ethidium bromide are shown in Supplemental Figure S3.

In wild-type plants, the 5′ TAS2 fragment (5′-TAS2) and more weakly the 3′ TAS2 fragment (3′-TAS2) were detected in the polysome fractions (eighth and ninth fractions) in which mRNAs associated with four to five ribosomes were observed (Fig. 3B). In plants with a hypomorphic allele of ago1 (ago1-25), the accumulation of tasiRNAs was lower than in wild-type plants (Supplemental Fig. S2A). However, the distribution pattern of TAS2-derived RNAs was not significantly different from that for the wild-type plants, probably due to the residual slicer activity (Fig. 3B). In dcl1-7 mutant plants, in which the accumulation of miR173 was strongly reduced (Supplemental Fig. S2A), pri-TAS2 overaccumulated in the fifth to seventh fractions. However, neither 5′-TAS2 nor 3′-TAS2 RNA fragments accumulated, perhaps due to a reduced level of miR173-programmed RISCs (Fig. 3B). This result suggests that a few ribosomes bound to pri-TAS2. In sgs3-11 mutant plants, pri-TAS2 accumulated at wild-type levels, whereas 5′-TAS2 and 3′-TAS2 were undetectable (Fig. 3B), supporting the above-mentioned model that SGS3 plays an important role in the stabilization of the pri-TAS2 cleavage fragments (Yoshikawa et al., 2013). In rdr6-11 mutant plants, large amounts of 5′-TAS2 and 3′-TAS2 were detected in the polysome fractions, peaking at the eighth and ninth fractions where the 5′-TAS2 RNA fragment was detected in wild-type plants (Fig. 3B). It is noteworthy that 5′- and 3′-TAS2 showed more rapid sedimentation than pri-TAS2 RNA in the dcl1 mutant. In the dcl4-10 mutant, the short TAS2-derived RNAs, including unprocessed RNAs complementary to 3′-TAS2 (Yoshikawa et al., 2005), were detected in the upper fractions (Fig. 3B). This result suggests that the dsRNAs synthesized from 3′-TAS2 by RDR6 are free from ribosomes.

In the sde5-5 mutant plants that are deficient in tasiRNA accumulation (Supplemental Fig. S2), 5′-TAS2 was detected in the polysome fractions, peaking at the eighth and ninth fractions like in wild-type plants (Fig. 3B). This result suggests that the protection of 5′-TAS2 by SGS3 is not significantly affected by a mutation in SDE5.

It has been shown that mutations in TEX1 and other genes that encode subunits of a THO/TREX complex affect the accumulation of pri-TASs and tasiRNAs (Jauvion et al., 2010; Yelina et al., 2010). Of these mutants, the tex1-4 mutant shows the most pronounced tasiRNA deficiency (Jauvion et al., 2010; Yelina et al., 2010). In the tex1-4 mutant, accumulation of 5′-TAS2 and 3′-TAS2 fragments and TAS2-derived tasiRNAs was not detected, while pri-TAS2 overaccumulated (Supplemental Fig. S2B). Unlike the dcl1-7 mutant, the accumulation level of miR173 in the tex1-4 mutant was similar to wild-type plants (Supplemental Fig. S2A; Jauvion et al., 2010; Yelina et al., 2010). In a tex1 mutant plant extract, pri-TAS2 was found mainly in the fifth to seventh fractions (Fig. 3B). In contrast to the TAS2-derived RNAs, miR173 and miR171 that can and cannot trigger tasiRNA production, respectively, showed similar sedimentation profiles in ago1, sgs3, rdr6, dcl4, and sde5 mutants to those in wild-type plants (Supplemental Fig. S4).

The above results prompted us to examine the sedimentation pattern of RNAs derived from other miR173-targeted TAS genes, namely, TAS1a and TAS1c, using flower tissue extracts from wild-type and rdr6 mutant plants (Fig. 4, A and B; Supplemental Fig. S5). In wild-type plants, the 5′ cleavage fragments of the TAS1a and TAS1c RNAs (5′-TAS1a and 5′-TAS1c) were weakly detected in the polysome fraction. In rdr6 mutant plants, the 3′ cleavage fragments of the TAS1a and TAS1c RNAs (3′-TAS1a and 3′-TAS1c), in addition to larger amounts of 5′-TAS1a and 5′-TAS1c, were detected in the polysome fraction. The fractionation patterns resembled that for TAS2-derived RNAs, suggesting that the cleavage products of pri-TAS1a and pri-TAS1c are also associated with ribosomes.

Figure 4.

Figure 4.

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Polysome analysis of TAS1a- and TAS1c-derived RNAs. Arabidopsis tissue extracts from wild-type (WT) and rdr6 mutant plants were fractionated. RNA was purified from each fraction, separated, and blotted on membranes as described in the legend to Figure 3. RNA blots were first hybridized with TAS1a probe (A). The probe was then stripped, and the same blots were reprobed for TAS1c (B). Total RNA (10 µg) from rdr6 mutant plants was used as a marker for the TAS1a- or TAS1c-related RNAs (the leftmost lane on each gel). Gels stained with ethidium bromide are shown in Supplemental Figure S3. Note that 80S ribosomes were mainly fractionated to the fourth fraction.

Ribosomes Associate with TAS2-Derived RNAs

The data presented here suggested that translation influences tasiRNA production from pri-TAS2. We evaluated whether ribosomes associate with TAS2-derived RNAs using the RNA-seq data deposited by Juntawong et al. (Juntawong et al., 2014; Supplemental Materials and Methods S1). This dataset was obtained by sequencing four types of RNA pools prepared from nonstressed wild-type Arabidopsis plants (GEO Accession GSE50597), i.e. polyA+ RNA purified from total RNA (GSM1224479 and GSM1224483 = total), polyA+ RNA purified from RNA that were coimmunopurified with FLAG-tagged ribosomes (GSM1224481 and GSM1224485 = polysomal), ribosome-associated RNA fragments that were obtained by RNase I digestion of Suc gradient-purified polysomes (GSM1224475 and GSM1224476 = RF), and ribosome-associated RNA fragments obtained by RNase I digestion of RNAs coimmunoprecipitated with FLAG-tagged ribosomes (GSM1224478 = TRAP). We calculated the number of reads per kilobase per million from reads that were mapped to the TAS1a-c, TAS2, and ACTIN2 (ACT2) gene loci (Supplemental Table S1). Like for the ACT2 transcript, reads for the TAS1a-c and TAS2 transcripts were detected in the polysomal, RF, and TRAP libraries as well as in the total fraction (Supplemental Table S1). Note that the TAS1a-c and TAS2 transcripts showed RF/total ratios similar to those for most protein-coding transcripts (Supplemental Fig. S6), supporting that these TAS transcripts are associated with ribosomes.

SDE5 Acts on the TAS2 Cleavage Fragments before DsRNA Synthesis by RDR6

In our polysome analysis, sedimentation patterns of 5′-TAS2 were similar between sde5 and wild-type plants (Fig. 3B). Although previous studies showed that SDE5 is an essential component in the tasiRNA production pathway and it was proposed that SDE5 functions to import TAS dsRNA from the cytosol to the nucleus or to forward single-stranded RNAs to RDR6 (Hernandez-Pinzon et al., 2007; Jauvion et al., 2010), the roles of SDE5 in the tasiRNA production pathway have remained unclear. Previous epistasis analysis between SGS3 and RDR6 showed that SGS3 plays a crucial role in stabilization of miR173-cleaved fragments (Yoshikawa et al., 2005). We took this approach to address the function of SDE5. We produced three double mutants of sgs3 sde5, rdr6 sde5, and dcl4 sde5, and accumulation of TAS2-related RNAs in these plants was analyzed (Fig. 5). In the sgs3 sde5 double mutant, the accumulation pattern of these RNAs was similar to that in sgs3 plants. i.e. the slight overaccumulation of 5′-TAS2 and 3′-TAS2 observed in sde5 plants was not observed in sgs3 sde5 plants, indicating that SGS3 acts on the miR173-cleaved fragments before the step facilitated by SDE5. In contrast, the accumulation pattern of TAS2-related RNAs in rdr6 sde5 and dcl4 sde5 double mutants was similar to that in the sde5 mutant. Notably, the accumulation of 5′-TAS2 and 3′-TAS2 in rdr6 sde5 plants was significantly lower than that in rdr6 plants. This observation is reminiscent of the previous results with regard to sgs3, rdr6, and sgs3 rdr6 mutants (Yoshikawa et al., 2005). However, in rdr6 sde5 plants, the cleaved fragments were clearly detectable, whereas the fragments were undetectable in sgs3 rdr6 plants like in sgs3 plants. These results strongly suggest that SDE5 acts on the cleavage fragments after SGS3 but before RDR6 and DCL4 act on the RNA and that SDE5 is needed for the accumulation of 5′-TAS2 and 3′-TAS2 in rdr6 plants.

Figure 5.

Figure 5.

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Epistasis analysis of the function of SGS3, RDR6, DCL4, and SDE5. A, Accumulation patterns of TAS2-related RNAs in wild type (WT), single mutants, and sde5 mutation-containing double mutants. Total RNA (20 µg) from each plant was used for northern analysis. RNA blots were hybridized with TAS2 probe. Gels were stained with ethidium bromide, and rRNA was shown as a loading control. B, Detection of tasiR255 derived from TAS1a-c in wild-type, sde5, dcl4, and dcl4 sde5 plants. sRNA (20 µg) was separated on a denaturing 15% polyacrylamide gel and blotted onto a sheet of Hybond-N+ membrane. The blot was probed with tasiR255-AS. The probe was then stripped, and the same blots were reprobed with miR173-AS.

To explore the order of action further, we examined the accumulation of 21-nt tasiR255 derived from TAS1a-c. In dcl4 plants, 21-nt tasiR255 was not detected, and instead 22-nt tasiR255 was detected, suggesting that dsRNAs synthesized by RDR6 are processed by DCL2 that produces 22-nt siRNA in the absence of DCL4 (Henderson et al., 2006; Xie et al., 2005). When sRNAs extracted from dcl4 sde5 plants were analyzed, neither 21-nt nor 22-nt tasiR255 was detected, indicating that dsRNA synthesis by RDR6 does not occur in this mutant. Taken together, these data support the idea that SDE5 acts on the miR173-cleaved fragments before RDR6 does.

DISCUSSION

Pri-TASs are synthesized by RNA polymerase II and have a 5′ cap and 3′ poly(A) that facilitate translation by recruiting ribosomes to the RNA. It had been unclear whether short ORFs on TAS RNAs are translated, and such translation events affect tasiRNA production. Here, we showed that some TAS2-, TAS1a-, and TAS1c-derived RNAs are fractionated in the polysome fraction and that the ORF3s of pri-TAS2 and pri-TAS1a are in fact translated. We also found that introduction of a termination codon in ORF3 of pri-TAS2 15 nt or more upstream of the miR173 recognition site reduces tasiRNA accumulation. Such reduction was not observed when a termination codon was introduced within or 6 nt upstream of the recognition site. Position of termination codons of major ORFs is critical for susceptibility of mRNAs to the nonsense-mediated mRNA decay system (Kertész et al., 2006). However, the pri-TAS2 mutant transcripts with altered position of ORF3 termination codon are unlikely targeted by the mRNA decay system because accumulation of the transcripts was not significantly affected by the mutations in the absence of miR173 overexpression (Supplemental Fig. S1). Thus, these results support an important role of ORF3 translation in tasiRNA production and are consistent with previous observations obtained by Zhang et al. using an artificial tasiRNA production system that the stop codons located > 13 nt upstream of the miR173 cleavage site reduce syn-tasiRNA accumulation (Zhang et al., 2012).

In contrast to pri-TAS2, the primary TAS4 transcript (pri-TAS4) whose tasiRNA production is triggered by miR828-mediated cleavage does not contain an ORF starting upstream of and encompassing the miR828 target site (Rajagopalan et al., 2006). The TAS3a-c transcripts (pri-TAS3a-c) that require two miR390 target sites for tasiRNA production have some short ORFs, but no > 30-nt ORFs that encompass either miR390 target site are found (Axtell et al., 2006; Howell et al., 2007; Montgomery et al., 2008a). The fact that tasiRNAs are produced even without ORFs that encompass miRNA recognition sites is consistent with our results of mutational analysis for TAS2 ORF3 and indicates that these ORFs are not absolutely required but facilitate tasiRNA production. Thus, the ORFs on pri-TASs serve as regulators that fine-tune the expression of tasiRNA target genes.

Our previous in vitro analysis showed that 5′-TAS2 and 3′-TAS2 associate with miR173-programmed AGO1-RISC and SGS3, and 3′-TAS2, which lacks the 5′ cap and should be unstable in the cell extract, escapes from degradation (Yoshikawa et al., 2013). In rdr6 mutant plants, the 5′ and 3′ fragments of TAS2 overaccumulated and were found together in the fraction that contained mRNAs associating with three to four ribosomes. Taken together, it is possible that, after cleavage of pri-TASs by miR173-programmed RISC, the RISC and SGS3 complex anchors the 5′ and 3′ cleavage fragments through base pairing with miR173, forming a single complex. Because ribosomes initially bind mRNA near the 5′ termini and move in the 5′-to-3′ direction on the mRNA, it is likely that the movement of ribosomes beyond the cleavage site is inhibited. Consequently, a complex that contains the 5′ cleavage fragment with stalled ribosomes and the 3′ cleavage fragment that are linked together by the RISC and SGS3 would be formed. The lengths between the initiation codon of ORF3 and the miR173 recognition site are 150 nt, 95 nt, and 138 nt for pri-TAS1a, pri-TAS1c, and pri-TAS2, respectively (Fig. 1A). Considering that a ribosome covers ≈ 30-nt region (Wolin and Walter, 1988), three to five ribosomes can bind to the 5′ cleavage fragments, which is consistent with the observations in our polysome analysis. Importantly, because the 3′ cleavage fragment should be free from ribosomes, RDR6 would be able to efficiently synthesize RNA complementary to the 3′ cleavage fragment without collision with translating ribosomes, which move on the same RNA in the opposite (5′-to-3′) direction.

Epistasis analysis showed that SDE5, which has been proposed as an RNA transporter, facilitates tasiRNA generation acting upstream of RDR6 and downstream of SGS3. Considering that cleaved TAS2 RNAs are overaccumulated in an rdr6 mutant and this overaccumulation is canceled in an sde5 rdr6 double mutant, we presume that SDE5 somehow changes the state of the cleaved TAS2 RNA-miR173 RISC-SGS3-ribosome complex to stabilize it in the absence of RDR6. Without a status change caused by SDE5, RDR6 could not be recruited to this complex to synthesize RNA that is complementary to the 3′ cleavage fragment. Although it remains unclear whether SDE5 is present within this complex, SDE5 may somehow facilitate the RDR6 recruitment (Fig. 6). To know more details, biochemical function of SDE5 should be uncovered.

Figure 6.

Figure 6.

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Possible involvement of ribosomes in tasiRNA biogenesis from pri-TAS2: a model. i, An 80S ribosome is formed at the initiation codons of ORF3 on pri-TAS2 and moves in the 3′ direction. In steady state, a few ribosomes bind to pri-TAS2. ii, A miR173-programmed RISC cleaves a pri-TAS2. iii, A ribosome that is translating the ORF3 encounters the complex containing the RISC and SGS3 near the cleavage site, and then stalls at this point. If translation initiation continues, multiple ribosomes become stacked on the 5′ cleavage fragment, followed by SDE5 action on the cleavage fragments. iv, RDR6 converts the ribosome-free 3′ cleavage fragment into a dsRNA. ORF3 and ORFs longer than 12 nt are shown as red and blue bars, respectively. Where two coding regions of different frames overlap, only upstream ORFs are shown.

Another potential RNA transporter, THO/TREX, is also related to tasiRNA production. Mutants defective in its subunits show overaccumulation of pri-TAS2 and loss of 5′-TAS2 compared with the wild type regardless of similar accumulation level of miR173 (Jauvion et al., 2010; Yelina et al., 2010). Our results in the tex1 mutants showed that pri-TAS2 was distributed in the polysome fractions and the cleavage fragments were not detected. These observations suggest that the THO/TREX complex and SDE5 function in the transport of TAS2-related RNAs at a distinct step in the tasiRNA production pathway. However, further studies are required to show that pri-TAS2 is exported to the cytoplasm and translated and to determine the function of the THO/TREX complex in tasiRNA biogenesis.

MATERIALS AND METHODS

Oligonucleotides

All oligonucleotides used in this study are listed in Supplemental Table S2 (primers) and Supplemental Table S3 (sRNA probes).

Transient Expression in Nicotiana benthamiana

Plasmids containing MIR171a or MIR173 were constructed as described previously (Montgomery et al., 2008b). TAS2 and its derivative genes were amplified using the TAS2-GWF1 and TAS2-R1 primers, and cloned into the pENTR/D-TOPO vector (Life Technologies). TAS2 mutant fragments were generated using the corresponding primers listed in Supplemental Table S2. After transferring the TAS2-related genes into pEarleyGate100 (Earley et al., 2006), fragments containing the TAS2-related genes with P35S and an octopine synthase terminator were amplified using the 35S-SbfI-F and OCS-HindIII-R primers, subcloned into the pCR4 Blunt-TOPO vector (Life Technologies), digested with SbfI and HindIII, and inserted between the SbfI and HindIII sites of the pMDC32 plasmids (Curtis and Grossniklaus, 2003) containing MIR171a or MIR173.

Agrobacterium tumefaciens EHA105 cells harboring each pMDC32-based plasmid were suspended in agroinfiltration buffer (10 mm MES, pH 5.6, 10 mm MgCl2, and 150 µm acetosyringone) to a concentration of optical density at 600 nm of 0.1, and were infiltrated into leaves of N. benthamiana plants (Kubota et al., 2003). Leaves were harvested 48 h after infiltration, and sRNA was extracted with ISOGENII (NIPPON GENE). To analyze sRNA by northern hybridization, sRNA (10 µg) was loaded onto a 15% polyacrylamide gel, transferred to a Hybond-N+ nylon membrane (GE Healthcare), and hybridized with probes in ULTRAhyb-oligo hybridization buffer (Ambion). Oligonucleotides labeled with 32P using T4 polynucleotide kinase (New England BioLabs) were used as probes. TAS2-3′D3(+)AS-LNA probe was prepared as described previously (Allen et al., 2005).

In Vitro Translation Using Evacuolated Tobacco Protoplast Extracts

Full-length DNA fragments of TAS1a and TAS2 were amplified by PCR using TAS1a-F1 and TAS1a-R1 primers for TAS1a and TAS2-F2 and TAS2-R1 primers for TAS2, and then were cloned into the pCR4 Blunt-TOPO vector. To generate TAS1a-FLAG and TAS2-FLAG fragments, we used TAS1a-FLAG-F1 and TAS1a-FLAG-R1 primers for TAS1a-FLAG and TAS2-FLAG-F1 and TAS2-FLAG-R1 primers for TAS2-FLAG. These fragments were cloned into the pCR4 Blunt-TOPO vector. Template DNA fragments for in vitro transcription were prepared using the SP6-TAS1a-F and TAS1a-polyA-R primers for TAS1a and TAS1a-FLAG, and the SP6-TAS2-F and TAS2-polyA-R primers for TAS2 and TAS2-FLAG. LUC-myc mRNA was prepared as described previously (Yoshikawa et al., 2013). The AmpliCap SP6 High Yield Message Maker Kit (CELLSCRIPT) was used for in vitro transcription.

In vitro translation was performed using mdBYL as described previously (Iki et al., 2010; Komoda et al., 2004). Translation products were separated on 4% to 12% Bis-Tris NuPAGE gels (Life Technologies) using MES running buffer. FLAG-tagged products and LUC-myc were detected on western blots with anti-FLAG (DYKDDDDK) antibody (Wako) and anti-myc antibody (Applied Biological Materials), respectively, using the ECL plus detection system (GE Healthcare).

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) mutants dcl1-7, ago1-25, sgs3-11, rdr6-11, dcl4-10, sde5-5, and tex1-4 have been described previously (Havecker et al., 2012; Jauvion et al., 2010; Mlotshwa et al., 2008; Morel et al., 2002; Peragine et al., 2004; Yelina et al., 2010). Plants were grown as described previously (Yoshikawa et al., 2013).

Glycerol Density Gradient Ultracentrifugation Analysis

Flower buds of Arabidopsis plants were harvested in liquid nitrogen and stored at −80°C. Frozen flower buds were ground to a powder in liquid nitrogen, and 400 µL of packed powder was combined with 850 µL of extraction buffer (20 mm HEPES, pH 7.5, 500 mm KCl, 10 mm MgCl2, 10 mm Ribonucleoside Vanadyl Complex [New England BioLabs], 5 mm DTT, and 250 µg/mL cycloheximide). After clarification by centrifugation at 12,000g for 5 min at 4°C, 750 µL of the supernatant was recovered. After second-round clarification by centrifugation at 12,000g for 2 min at 4°C, 650 µL of the supernatant was recovered. The supernatant (600 µL) was loaded onto a 12-mL glycerol gradient (10%–40% [w/v] glycerol in 20 mm HEPES, pH 7.5, 100 mm KCl, and 10 mm MgCl2) and centrifuged at 39,000 rpm for 90 min at 4°C using a SW40Ti rotor (Beckman). Gradients were fractionated to 12 fractions (1 mL each) using the Density Gradient Fractionation System (ISCO), continuously measuring A254.

To analyze TAS2-derived RNAs and miRNAs, RNA was isolated from each glycerol gradient fraction using the High Pure miRNA Isolation Kit (Roche). Each fraction (1 mL) was mixed with 2.08 mL of the binding buffer and 1.33 mL of the binding enhancer buffer included in the kit. After elution with 100 µL of elution buffer, aliquots of 60 µL or 20 µL were used for TAS2-derived RNA analysis or miRNA analysis, respectively. To analyze TAS1a- and TAS1c-derived RNAs, RNA was isolated from each glycerol gradient fraction (1 mL) using the RNeasy Mini Kit (QIAGEN) by adding 3.5 mL of RLT buffer containing 1% β-mercaptoethanol followed by elution with 100 µL of elution buffer. Aliquots (85 µL) were used for northern analysis.

For northern analysis, RNA was loaded onto 1.2% agarose gels and transferred to Hybond-N+. Hybridization was performed using PerfectHyb plus hybridization buffer (Sigma-Aldrich) and 32P-labeled probes with the Prime-It II Random Primer Labeling Kit (Agilent). For miRNAs, hybridization was performed as described above.

Accession Number

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number GSE50597.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_171_1_359__index.html (1.4KB, html)

Acknowledgments

We thank Dr. R. Scott Poethig for valuable comments on the manuscript. We also thank Yoriko Fujibayashi for technical assistance and the members of “RNA and Biofunctions” in PRESTO for useful advice.

Glossary

tasiRNA

trans-acting small interfering RNA

pri-TAS

primary TAS transcript

ORF

open reading frame

sRNA

small RNA

miRNA

microRNA

dsRNA

double-stranded RNA

syn-tasiRNA

synthetic tasiRNA

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Associated Data

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Supplementary Materials

Supplemental Data
supp_171_1_359__index.html (1.4KB, html)
supp_pp.16.00148_PP2016-00148DR1_Supplemental_Materialfigs.pdf (869.5KB, pdf)
supp_pp.16.00148_PP2016-00148DR1_Supplemental_Materialtbls.pdf (90.8KB, pdf)
supp_pp.16.00148_PP2016-00148DR1_Supplemental_Materialmethods.pdf (65.5KB, pdf)

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