Manabu Yoshikawa

In vitro assembly of plant RNA-induced silencing complexes facilitated by molecular chaperone HSP90.

RNA-induced silencing complexes (RISCs) play central roles in posttranscriptional gene silencing. In plants, the mechanism of RISC assembly has remained elusive due to the lack of cell-free systems that recapitulate the process. In this report, we demonstrate that plant AGO1 protein synthesized by in vitro translation using an extract of evacuolated tobacco protoplasts incorporates synthetic small interfering RNA (siRNA) and microRNA (miRNA) duplexes to form RISCs that sequester the single-stranded siRNA guide strand and miRNA strand, respectively. The formed RISCs were able to recognize and cleave the complementary target RNAs. In this system, the siRNA duplex was incorporated into HSP90-bound AGO1, and subsequent removal of the passenger strand was triggered by ATP hydrolysis by HSP90. Removal of the siRNA passenger strand required the ribonuclease activity of AGO1, while that of the miRNA star strand did not. Based on these results, the mechanism of plant RISC formation is discussed.

Cyclophilin 40 facilitates HSP90-mediated RISC assembly in plants

Posttranscriptional gene silencing is mediated by RNA‐induced silencing complexes (RISCs) that contain AGO proteins and single‐stranded small RNAs. The assembly of plant AGO1‐containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90‐dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex‐bound AGO1. Moreover, CYP40 that was synthesized by in‐vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin‐binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90–AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

3′ fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3

trans-acting small interfering RNAs (tasiRNAs) are plant-specific endogenous siRNAs produced via a unique pathway whose first step is the microRNA (miRNA)-programmed RNA-induced silencing complex (RISC)–mediated cleavage of tasiRNA gene (TAS) transcripts. One of the products is subsequently transformed into tasiRNAs by a pathway that requires several factors including SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6. Here, using in vitro assembled ARGONAUTE (AGO)1–RISCs, we show that SGS3 is recruited onto RISCs only when they bind target RNA. Following cleavage by miRNA173 (miR173)-programmed RISC, SGS3 was found in complexes containing cleaved TAS2 RNA and RISC. The 3′ cleavage fragment (the source of tasiRNAs) was protected from degradation in this complex. Depletion of SGS3 did not affect TAS2 RNA cleavage by miR173-programmed RISC, but did affect the stability of the 3′ cleavage fragment. When the 3′ nucleotide of 22-nt miR173 was deleted or the corresponding nucleotide in TAS2 RNA was mutated, the complex was not observed and the 3′ cleavage fragment was degraded. Importantly, these changes in miR173 or TAS2 RNA are known to lead to a loss of tasiRNA production in vivo. These results suggest that (i) SGS3 associates with AGO1–RISC via the double-stranded RNA formed by the 3′-terminal nucleotides of 22-nt miR173 and corresponding target RNA, which probably protrudes from the AGO1–RISC molecular surface, (ii) SGS3 protects the 3′ cleavage fragment of TAS2 RNA from degradation, and (iii) the observed SGS3-dependent stabilization of the 3′ fragment of TAS2 RNA is key to tasiRNA production.

Tandemly arranged chalcone synthase A genes contribute to the spatially regulated expression of siRNA and the natural bicolor floral phenotype in Petunia hybrida

The natural bicolor floral traits of the horticultural petunia (Petunia hybrida) cultivars Picotee and Star are caused by the spatial repression of the chalcone synthase A (CHS-A) gene, which encodes an anthocyanin biosynthetic enzyme. Here we show that Picotee and Star petunias carry the same short interfering RNA (siRNA)-producing locus, consisting of two intact CHS-A copies, PhCHS-A1 and PhCHS-A2, in a tandem head-to-tail orientation. The precursor CHS mRNAs are transcribed from the two CHS-A copies throughout the bicolored petals, but the mature CHS mRNAs are not found in the white tissues. An analysis of small RNAs revealed the accumulation of siRNAs of 21 nucleotides that originated from the exon 2 region of both CHS-A copies. This accumulation is closely correlated with the disappearance of the CHS mRNAs, indicating that the bicolor floral phenotype is caused by the spatially regulated post-transcriptional silencing of both CHS-A genes. Linkage between the tandemly arranged CHS-A allele and the bicolor floral trait indicates that the CHS-A allele is a necessary factor to confer the trait. We suppose that the spatially regulated production of siRNAs in Picotee and Star flowers is triggered by another putative regulatory locus, and that the silencing mechanism in this case may be different from other known mechanisms of post-transcriptional gene silencing in plants. A sequence analysis of wild Petunia species indicated that these tandem CHS-A genes originated from Petunia integrifolia and/or Petunia inflata, the parental species of P. hybrida, as a result of a chromosomal rearrangement rather than a gene duplication event.

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

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. 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.

Locus-Specific Requirements of DDR Complexes for Gene-Body Methylation of TAS Genes in Arabidopsis thaliana

Posttranscriptional gene silencing via 21-nucleotide (nt) trans-acting small interfering RNAs (tasiRNAs) is a plant-specific RNA silencing mechanism. tasiRNAs are generated by RNA-DEPENDENT RNA POLYMERASE 6, SUPPRESSOR OF GENE SILENCING 3, and DICER-LIKE 4 using TAS transcripts as templates and degrade their target mRNA in a manner similar to micro-RNA. TAS gene-derived small RNAs also trigger cytosine methylation in cis together with known components of RNA-directed DNA methylation (RdDM). RdDM is known to be required for silencing of repetitive elements and transposons via a 24-nt small interfering RNA complementary to the target DNA. Plant-specific DNA-dependent RNA polymerase V (Pol V) is thought to be required for the recruitment of other RdDM components to the target region, together with the DDR complex, which contains DEFECTIVE IN RNA-DIRECTED DNA METHYLATION 1, DEFECTIVE IN MERISTEM SILENCING 3, and RNA-DIRECTED DNA METHYLATION 1. Although genome-wide studies indicate that Pol V and the DDR complex mostly share endogenous targets, the requirement for each component of RdDM at TAS genes remains obscure. Here, we investigated the involvement of components of the DDR complex in RdDM triggered by TAS gene-derived small RNAs. Our results show that methylation of the TAS3a gene was reduced in dms3 and drd1, whereas methylation of the TAS1c gene was decreased in dms3 but not in drd1. These findings indicate that Pol V and components of the DDR complex act separately in a locus-specific manner.