Tejas functions as a core component in nuage assembly and precursor processing in Drosophila piRNA biogenesis.

PIWI-interacting RNAs (piRNAs), which protect genome from the attack by transposons, are produced and amplified in membraneless granules called nuage. In Drosophila, PIWI family proteins, Tudor-domain-containing (Tdrd) proteins, and RNA helicases are assembled and form nuage to ensure piRNA production. However, the molecular functions of the Tdrd protein Tejas (Tej) in piRNA biogenesis remain unknown. Here, we conduct a detailed analysis of the subcellular localization of fluorescently tagged nuage proteins and behavior of piRNA precursors. Our results demonstrate that Tej functions as a core component that recruits Vasa (Vas) and Spindle-E (Spn-E) into nuage granules through distinct motifs, thereby assembling nuage and engaging precursors for further processing. Our study also reveals that the low-complexity region of Tej regulates the mobility of Vas. Based on these results, we propose that Tej plays a pivotal role in piRNA precursor processing by assembling Vas and Spn-E into nuage and modulating the mobility of nuage components.

miRNA/siRNA-directed pathway to produce noncoding piRNAs from endogenous protein-coding regions ensures Drosophila spermatogenesis.

PIWI-interacting RNA (piRNA) pathways control transposable elements (TEs) and endogenous genes, playing important roles in animal gamete formation. However, the underlying piRNA biogenesis mechanisms remain elusive. Here, we show that endogenous protein coding sequences (CDSs), which are normally used for translation, serve as origins of noncoding piRNA biogenesis in Drosophila melanogaster testes. The product, namely, CDS-piRNAs, formed silencing complexes with Aubergine (Aub) in germ cells. Proximity proteome and functional analyses show that CDS-piRNAs and cluster/TE-piRNAs are distinct species occupying Aub, the former loading selectively relies on chaperone Cyclophilin 40. Moreover, Argonaute 2 (Ago2) and Dicer-2 activities were found critical for CDS-piRNA production. We provide evidence that Ago2-bound short interfering RNAs (siRNAs) and microRNAs (miRNAs) specify precursors to be processed into piRNAs. We further demonstrate that Aub is crucial in spermatid differentiation, regulating chromatins through mRNA cleavage. Collectively, our data illustrate a unique strategy used by male germ line, expanding piRNA repertoire for silencing of endogenous genes during spermatogenesis.

The Tudor Domain-Containing Protein, Kotsubu (CG9925), Localizes to the Nuage and Functions in piRNA Biogenesis in D. melanogaster.

Silencing of transposable elements (TEs) by Piwi-interacting RNAs (piRNAs) is crucial for maintaining germline genome integrity and fertility in animals. To repress TEs, PIWI clade Argonaute proteins cooperate with several Tudor domain-containing (Tdrd) proteins at membraneless perinuclear organelles, called nuage, to produce piRNAs to repress transposons. Here, we identify and characterize Kotsubu (Kots), one of the Drosophila Tudor domain-containing protein-1 (Tdrd1) orthologs, encoded by the CG9925 gene, that localizes to the nuage in gonads. We further show the dynamic localization of Kots in the male germline, where it shows perinuclear signals in spermatogonia but forms large cytoplasmic condensates in the spermatocytes that overlap with components of piNG-body, a nuage-associated organelle. The loss of kots results in a notable upregulation of stellate and a corresponding reduction in the suppressor of stellate piRNAs in the mutants. Furthermore, a moderate yet significant reduction of other piRNAs was observed in kots mutant testes. Taken together, we propose that Kots functions in the piRNA pathway, predominantly in the male germline by forming discrete cytoplasmic granules.

Modulation of Ago2 Loading by Cyclophilin 40 Endows a Unique Repertoire of Functional miRNAs during Sperm Maturation in Drosophila.

In gene silencing, Hsp90 chaperone machinery assists Argonaute (Ago) binding and unwinding of silencing small RNA (sRNA) duplexes. This enables the formation of effector RNA-induced silencing complex (RISC) that often displays cargo preferences. Hence, in Drosophila, microRNAs (miRNAs) and small-interfering RNAs (siRNAs) are differentially sorted into Ago1-RISC and Ago2-RISC, respectively. Here, we identify fly Cyclophilin 40 (Cyp40) as a testis-specialized Hsp90 co-chaperone essential for spermatogenesis and for modulating Ago2-RISC formation. We show that testis-distinctive Ago-sorting and strand-selection mechanisms accumulate a unique set of miRNAs on Ago2. Cyp40 interacts with duplex-incorporating Ago2 through Hsp90 in vitro and selectively promotes the build-up of Ago2-bound miRNAs, but not endogenous siRNAs, in vivo. Moreover, one of Cyp40-dependent Ago2-sorted miRNAs is required for late spermatogenesis, unraveling the physiological relevance of the unconventional yet conserved Drosophila miRNA-Ago2 sorting pathway. Collectively, these results identify RISC-regulatory roles for Hsp90 machinery and, more generally, highlight the tissue-specific adaptation of sRNA pathways through chaperone diversification.

A Suppressor Screen for AGO1 Degradation by the Viral F-Box P0 Protein Uncovers a Role for AGO DUF1785 in sRNA Duplex Unwinding.

In Arabidopsis thaliana, ARGONAUTE1 (AGO1) plays a central role in microRNA (miRNA) and small interfering RNA (siRNA)-mediated silencing and is a key component in antiviral responses. The polerovirus F-box P0 protein triggers AGO1 degradation as a viral counterdefense. Here, we identified a motif in AGO1 that is required for its interaction with the S phase kinase-associated protein1-cullin 1-F-box protein (SCF) P0 (SCFP0) complex and subsequent degradation. The AGO1 P0 degron is conserved and confers P0-mediated degradation to other AGO proteins. Interestingly, the degron motif is localized in the DUF1785 domain of AGO1, in which a single point mutation (ago1-57, obtained by forward genetic screening) compromises recognition by SCFP0 Recapitulating formation of the RNA-induced silencing complex in a cell-free system revealed that this mutation impairs RNA unwinding, leading to stalled forms of AGO1 still bound to double-stranded RNAs. In vivo, the DUF1785 is required for unwinding perfectly matched siRNA duplexes, but is mostly dispensable for unwinding imperfectly matched miRNA duplexes. Consequently, its mutation nearly abolishes phased siRNA production and sense transgene posttranscriptional gene silencing. Overall, our work sheds new light on the mode of AGO1 recognition by P0 and the in vivo function of DUF1785 in RNA silencing.

Structural Flexibility Enables Alternative Maturation, ARGONAUTE Sorting and Activities of miR168, a Global Gene Silencing Regulator in Plants.

In eukaryotes, the RNase-III Dicer often produces length/sequence microRNA (miRNA) variants, called "isomiRs", owing to intrinsic structural/sequence determinants of the miRNA precursors (pre-miRNAs). In this study, we combined biophysics, genetics and biochemistry approaches to study Arabidopsis miR168, the key feedback regulator of central plant silencing effector protein ARGONAUTE1 (AGO1). We identified a motif conserved among plant pre-miR168 orthologs, which enables flexible internal base-pairing underlying at least three metastable structural configurations. These configurations promote alternative, accurate Dicer cleavage events generating length and structural isomiR168 variants with distinctive AGO sorting properties and modes of action. Among these isomiR168s, a duplex with a 22-nt guide strand exhibits strikingly preferential affinity for AGO10, the closest AGO1 paralog. The 22-nt miR168-AGO10 complex antagonizes AGO1 accumulation in part via "transitive RNAi", a silencing-amplification process, to maintain appropriate AGO1 cellular homeostasis. Furthermore, we found that the tombusviral P19 silencing-suppressor protein displays markedly weaker affinity for the 22-nt form among its isomiR168 cargoes, thereby promoting AGO10-directed suppression of AGO1-mediated antiviral silencing. Taken together, these findings indicate that structural flexibility, a previously overlooked property of pre-miRNAs, considerably increases the versatility and regulatory potential of individual MIRNA genes, and that some pathogens might have evolved the capacity or mechanisms to usurp this property.

In Vitro Formation of Plant RNA-Induced Silencing Complexes Using an Extract of Evacuolated Tobacco Protoplasts.

Small RNA-mediated gene silencing is involved in a variety of biological processes among many eukaryotic organisms. The silencing effector, generally referred to as RNA-induced silencing complex (RISC), comprises an ARGONAUTE (AGO) protein and a small single-stranded guide RNA in its core. RISCs recognize target genes containing sequences complementary to the guide RNA and repress their expression transcriptionally or posttranscriptionally. In vitro systems that recapitulate RISC assembly are useful not only to decipher the molecular mechanisms underlying the assembly process itself but also to dissect the downstream silencing pathways mediated by RISCs. Here, we describe a method for in vitro plant RISC assembly, which relies on an extract of evacuolated protoplasts derived from Nicotiana tabacum BY-2 suspension-cultured cells. In this extract, synthetic duplexes of small RNAs are incorporated into AGO proteins that are synthesized by in vitro translation, and then duplex unwinding and selective strand elimination result in formation of mature RISCs.

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing.

Phytoviruses encode viral suppressors of RNA silencing (VSRs) to counteract the plant antiviral silencing response, which relies on virus-derived small interfering (si)RNAs processed by Dicer RNaseIII enzymes and subsequently loaded into ARGONAUTE (AGO) effector proteins. Here, a tobacco cell-free system was engineered to recapitulate the key steps of antiviral RNA silencing and, in particular, the most upstream double-stranded (ds)RNA processing reaction, not kinetically investigated thus far in the context of plant VSR studies. Comparative biochemical analyses of distinct VSRs in the reconstituted assay showed that in all cases tested, VSR interactions with siRNA duplexes inhibited the loading, but not the activity, of antiviral AGO1 and AGO2. Turnip crinkle virus P38 displayed the additional and unique property to bind both synthetic and RNA-dependent-RNA-polymerase-generated long dsRNAs, and inhibited the processing into siRNAs. Single amino acid substitutions in P38 could dissociate dsRNA-processing from AGO-loading inhibition in vitro and in vivo, illustrating dual-inhibitory strategies discriminatively deployed within a single viral protein, which, we further show, are bona fide suppressor functions that evolved independently of the conserved coat protein function of P38.

A complex of Arabidopsis DRB proteins can impair dsRNA processing.

Small RNAs play an important role in regulating gene expression through transcriptional and post-transcriptional gene silencing. Biogenesis of small RNAs from longer double-stranded (ds) RNA requires the activity of dicer-like ribonucleases (DCLs), which in plants are aided by dsRNA binding proteins (DRBs). To gain insight into this pathway in the model plant Arabidopsis, we searched for interactors of DRB4 by immunoprecipitation followed by mass spectrometry-based fingerprinting and discovered DRB7.1. This interaction, verified by reciprocal coimmunoprecipitation and bimolecular fluorescence complementation, colocalizes with markers of cytoplasmic siRNA bodies and nuclear dicing bodies. In vitro experiments using tobacco BY-2 cell lysate (BYL) revealed that the complex of DRB7.1/DRB4 impairs cleavage of diverse dsRNA substrates into 24-nucleotide (nt) small interfering (si) RNAs, an action performed by DCL3. DRB7.1 also negates the action of DRB4 in enhancing accumulation of 21-nt siRNAs produced by DCL4. Overexpression of DRB7.1 in Arabidopsis altered accumulation of siRNAs in a manner reminiscent of drb4 mutant plants, suggesting that DRB7.1 can antagonize the function of DRB4 in siRNA accumulation in vivo as well as in vitro. Specifically, enhanced accumulation of siRNAs from an endogenous inverted repeat correlated with enhanced DNA methylation, suggesting a biological impact for DRB7.1 in regulating epigenetic marks. We further demonstrate that RNase three-like (RTL) proteins RTL1 and RTL2 cleave dsRNA when expressed in BYL, and that this activity is impaired by DRB7.1/DRB4. Investigating the DRB7.1-DRB4 interaction thus revealed that a complex of DRB proteins can antagonize, rather than promote, RNase III activity and production of siRNAs in plants.

Messages on small RNA duplexes in plants.

Small RNA-mediated gene silencing encompasses diverse developmental events, stress responses, defense against pathogens, and maintenance of genome integrity. Extensive studies in model organisms have unveiled the molecular mechanisms underpinning the RNA silencing phenomena, and the accumulating knowledge have characterized the intricate pathways and the repertoire of proteins responsible for the actions of small RNAs characterized as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Although the single-stranded, matured guide small RNAs direct the effector ribonucleoprotein complexes to induce gene silencing in sequence-specific manner, the double-stranded intermediate, the small RNA duplexes, which are processed as nascent products of the RNase III enzyme activities, act as key to determine the downstream molecular pathways and the fate of small RNAs. Based at the small RNA duplex-centered view, this review describes the recent advances in understanding the small RNA pathways in plants.

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

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.

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.

Cytoplasmic assembly and selective nuclear import of Arabidopsis Argonaute4/siRNA complexes.

In plants, DNA methylation can be mediated by a class of Argonaute4 (AGO4)-associated heterochromatic siRNAs (hc-siRNAs), through a pathway termed RNA-directed DNA methylation (RdDM). It has been thought that RdDM is solely a nuclear process, as both the biogenesis and functioning of hc-siRNAs take place in the nucleus. In this study, we unexpectedly found that hc-siRNAs are predominantly present in the cytoplasm. We demonstrated that AGO4 is loaded with hc-siRNAs in the cytoplasm and the formation of mature AGO4/siRNA complexes requires HSP90 and the cleavage activity of AGO4. Intriguingly, siRNA binding facilitates the redistribution of AGO4 into the nucleus, likely through inducing conformational change that leads to the exposure of the nuclear localization signal (NLS). Our findings reveal an unsuspected cytoplasmic step in the RdDM pathway. We propose that selective nuclear import of mature AGO4/siRNA complexes is a key regulatory point prior to the effector stage of RdDM.

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.

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.

An outer membrane autotransporter, AoaA, of Azorhizobium caulinodans is required for sustaining high N2-fixing activity of stem nodules.

In this study, we investigated the function of a putative high-molecular-weight outer membrane protein, azorhizobial outer membrane autotransporter A (AoaA), of Azorhizobium caulinodans ORS571. Sequence analysis revealed that AoaA was an autotransporter protein belonging to the type V protein secretion system. Azorhizobium caulinodans forms N(2)-fixing nodules on the stems and roots of Sesbania rostrata. The sizes of stem nodules formed by an aoaA mutant having transposon insertion within this ORF were as large as those in the wild-type strain, but the N(2)-fixing activity of the nodules by the aoaA mutant was lower than that of wild-type nodules. cDNA-amplified fragment length polymorphism and reverse transcriptase-PCR analysis revealed that the expressions of several pathogen-related genes of host plants were induced in the aoaA mutant nodules. Furthermore, exopolysaccharide production was defective in the aoaA mutant under free-living conditions. These results indicate that AoaA may have an important role in sustaining the symbiosis by suppressing plant defense responses. The exopolysaccharide production controlled by AoaA might mediate this suppression mechanism.

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis.

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.

Evidence for functional differentiation of duplicated nifH genes in Azorhizobium caulinodans.

Azorhizobium caulinodans is a symbiotic diazotroph that contains duplicated nifH genes. This study focused on the biological sense behind the duplication. In-frame deletion mutants of nifH1 and nifH2 were constructed in order to analyze nitrogen fixation activity, both in symbiosis and in free-living conditions. Symbiotic nitrogen fixation activity was not affected by deletion of nifH1 or nifH2, while free-living nitrogen fixation activity was significantly decreased. Deletion of nifH1 had a significant effect in semi-aerobic condition, while deletion of nifH2 was significant in microaerobic condition, suggesting functional differences between nifH1 and nifH2. Transcriptional activity of nifH1 was higher than nifH2, both in microaerobic and semi-aerobic conditions.

Isolation and differential expression of ß-1,3-glucanase messenger RNAs, SrGLU3 and SrGLU4, following inoculation of Sesbania rostrata.

We report here the isolation and characterisation of two new ß-1,3-glucanase cDNAs, SrGLU3 and SrGLU4, from a tropical legume Sesbania rostrata Bremek. & Oberm., which form N2-fixing nodules on the stem after infection by Azorhizobium caulinodans. SrGLU3 was characterised as being grouped in a branch with tobacco class I ß-1,3-glucanases, where the isoforms were reported to be induced by either pathogen infection or ethylene treatment. SrGLU4 was characterised as separate from other classes, and we propose this new branch as a new class (Class VI). The SrGLU3 gene was constitutively expressed in normal stem nodules induced by the wild type strain of A. caulinodans (ORS571), and also even in immature stem nodules induced by a mutant (ORS571-C1), which could not form mature stem-nodules. In contrast, the transcript accumulation of SrGLU4 was hardly detectable in immature nodules inoculated by the ORS571-C1 mutant. We suggest that S. rostrata makes use of SrGLU4 to discriminate between symbionts and non-symbionts (mutants) in developing nodules. We propose the SrGLU4 gene as a new nodulin during nodulation.