RBV, an evolutionarily conserved WD40 repeat protein, promotes microRNA biogenesis and loading into ARGONAUTE1 in Arabidopsis

MicroRNAs (miRNAs) play crucial roles in gene expression regulation through RNA cleavage or translation repression. Although miRNA biogenesis has been extensively studied, new factors involved in miRNA biogenesis are still being found from various genetic screens, suggesting that knowledge of miRNA biogenesis or its regulation is still incomplete. Here, we report the identication of a previously uncharacterized and evolutionarily conserved WD40 domain protein as a player in miRNA biogenesis in Arabidopsis thaliana. A mutation in the REDUCTION IN BLEACHED VEIN AREA (RBV) gene encoding a WD40 domain protein led to the suppression of leaf bleaching caused by an articial miRNA; the mutation also led to a global reduction in the accumulation of endogenous miRNAs. RBV may act at multiple steps in miRNA biogenesis. RBV promotes the transcription of MIR genes into pri-miRNAs by enhancing the occupancy of RNA polymerase II (Pol II) at MIR gene promoters. The nuclear protein RBV may also participate in pri-miRNA processing, as the mutation of RBV leads to the reduction of dicing body number. Moreover, mutation of RBV leads to a defect of miRNA loading into AGO1.RBV function is required in messenger RNA splicing, as RNA-seq uncovered a global intron retention defect in the mutant. Thus, this previously uncharacterized, evolutionarily conserved, nuclear WD40 domain protein acts in miRNA biogenesis and RNA splicing.


Introduction
MicroRNAs (miRNAs), 20 to 24 nucleotides (nt) in length, are one class of endogenous noncoding small RNAs in eukaryotes. miRNAs are players in gene regulatory networks involved in many biological processes such as development, metabolism and immunity in plants and animals 1 . Target gene expression is regulated by miRNAs post-transcriptionally through RNA cleavage or translation repression 2 .
Biogenesis of plant miRNAs entails a series of steps. Genes encoding miRNAs (MIR) are transcribed into pri-miRNAs that form imperfect stem-loop structures by DNA-dependent RNA polymerase II (Pol II) 3,4 . MIR transcription is facilitated by Mediator 5 , NEGATIVE ON TATA LESS2 (NOT2) 6 , CELL DIVISION CYCLE5 (CDC5) 7 , Elongator 8 , and the TREX-2 component THP1 9 . Pri-miRNAs are processed into pre-miRNAs by DICER-LIKE1 (DCL1) 1,10,11,12 . In humans, pri-miRNAs are thought to be co-transcriptionally processed, as they are associated with chromatin during transcription and processed at sites of transcription before splicing 13,14 . It is proposed that the retention of pri-miRNAs at transcription sites may enhance processing 14 . In Arabidopsis, NOT2, CDC5 and Elongator interact with both Pol II and DCL1 6, 7, 8 , suggesting that pri-miRNA transcription and processing may be also coordinated in plants.
The e cient processing of pri-miRNAs requires the double-stranded RNA binding protein HYPONASTIC LEAVES1 (HYL1) 15,16 and the zinc nger protein SERRATE (SE) 17,18 , which form the microprocessor complex with DCL1 19,20 . The three proteins as well as pri-miRNAs are also found in nuclear foci called Dicing-bodies (D-bodies) 21 . Pre-miRNAs are processed to miRNA/miRNA* duplexes, which undergo 3' methylation by the methyltransferase HEN1 to maintain miRNA stability 22 . Finally, the mature miRNA strands are loaded into ARGONAUTE1 (AGO1) to form RNA Induced Silencing Complexes (RISCs), which are active forms of miRNAs 23,24,25 . In Arabidopsis, size exclusion chromatography showed that the molecular weight of miRISCs is similar to that of AGO1, suggesting that miRISCs are bi-molecular AGO1-miRNA complexes, although larger complexes containing AGO1 can also be detected 23,26,27 .
Here, we isolated a mutant with global defects in miRNA biogenesis in Arabidopsis. The mutation is in a previously uncharacterized gene encoding a protein containing seven WD40 repeats, which we designate as RBV. RBV promotes the transcription of MIR genes, as loss of function of RBV reduces MIR promoter activity and the occupancy of Pol II at MIR promoters. In addition, RBV promotes the localization of HYL1 in D-bodies. On the basis of these results, we propose that RBV may act to coordinate MIR transcription and pri-miRNA processing in plant miRNA biogenesis. Moreover, the association of miRNAs with AGO1 was drastically decreased and AGO1 resided in complexes larger than miRISCs in the rbv-1 mutant, suggesting that RBV1 promotes the loading of miRNAs into AGO1. RBV1 also has a global role in pre-mRNA splicing, affecting a set of short introns.

Isolation of a mutant with defects in miRNA biogenesis
We performed an ethylmethane sulfonate mutagenesis screen for Arabidopsis mutants in miRNA biogenesis, utilizing the vein-centered leaf beaching phenotype caused by the phloem-speci c expression of an arti cial miRNA (amiR-SUL) targeting SULFUR (SUL) as a visible marker for miRNA activity 28 . A series of mutants with reduced leaf bleaching was isolated and found to be in genes with known roles in miRNA biogenesis/activity, such as hyl1-11, dcl1-30, hen1-11 and ago1-25, suggesting that the genetic screen was effective (Supplementary Fig. 1a and b). The ago1-25 allele isolated in our study was thus named because it harbored the same mutation as the one previously reported 29 . In addition, we isolated a new suppressor mutant with reduced leaf bleaching ( Fig. 1a and b); the mutation was designated rbv-1 as it was later shown to be in a previously uncharacterized gene that we named REDUCTION IN BLEACHED VEIN AREA (RBV). This mutant exhibited pleiotropic developmental phenotypes, such as reduced root length, smaller plant size, narrow leaves, short stature, increased branching and reduced fertility ( Fig. 1c and Supplementary Fig. 2). Northern blot analyses showed that both amiR-SUL and endogenous miRNAs (miR156, miR159, miR164, miR165, miR167, miR319 and miR390) were moderately reduced in abundance in 14-day-old amiR-SUL rbv-1 seedlings as compared to amiR-SUL seedings (Fig.  1d). We also preformed small RNA sequencing with 14-day-old amiR-SUL and amiRSUL rbv-1 seedlings. Clustering analysis showed that the three biological replicates for each genotype were highly reproducible ( Supplementary Fig. 3a). Reads corresponding to miRNAs were normalized against total mapped reads and miRNAs with RPM (reads per million mapped reads)>10 in either genotype (average of three replicates) were included in our analyses (Supplementary Data 1). An overall reduction in miRNA abundance in the mutant relative to the amiR-SUL line was observed ( Fig. 1e and Supplementary Fig. 3b), while there were some miRNAs such as miR845a and miR845b showing increased accumulation in the amiRSUL rbv-1 mutant. We also performed RT-qPCR to examine the expression of some miRNAs target genes. The transcript levels of SPL3 and SPL10 (targets of miR156), PHB, REV and PHV (targets of miR165/6), MYB33 and MYB65 (targets of miR159), ARF8 (a target of miR167), and CUC2 (a target of miR164) were de-repressed in the amiR-SUL rbv-1 mutant (Fig. 1f).

RBV encodes an evolutionarily conserved WD40 domain protein
The amiR-SUL rbv-1 mutant was backcrossed with the parental amiR-SUL line. In a total of 614 F2 plants, 141 (23%) exhibited the mutant phenotypes, which is consistent with the phenotype being caused by a single, nuclear, and recessive mutation (c 2 =1.257; P =0.244; (Supplementary Table 1). In order to identify the causal mutation in amiR-SUL rbv-1, pooled DNA from mutant plants in the F2 of the amiR-SUL rbv-1 x amiR-SUL cross was used for whole-genome re-sequencing. The results revealed that the amiR-SUL rbv-1 phenotype was linked to a single nucleotide change (G-to-A) in the rst exon of AT5G64730, causing the change of the encoded amino acid from glycine to glutamic acid (see Methods; Fig. 2a).
In order to con rm that RBV is indeed AT5G64730, a construct of RBV (AT5G64730)-eYFP driven by its native promoter was generated and introduced into amiR-SUL rbv-1. Phenotypes of the transgenic plants showed that pRBV:RBV-eYFP fully rescued the morphological defects of amiR-SUL rbv-1 (Fig. 2c). Furthermore, the transgene restored amiR-SUL accumulation as well as leaf bleaching in amiR-SUL rbv-1 (Fig. 2d). While the expression of the amiR-SUL target gene, SUL, was increased in the mutant at the RNA and protein levels, the transgene also restored SUL RNA (Fig. 2e) and SUL protein (Fig. 2f) in amiR-SUL rbv-1 to wild-type levels. Moreover, the transgene rescued the defects in miR159 and miR319 accumulation in the amiR-SUL rbv-1 mutant (Fig. 2d). Therefore, the miRNA biogenesis and morphological defects of amiR-SUL rbv-1 were attributable to the mutation in RBV (AT5G64730).
RBV encodes a previously uncharacterized protein with seven WD40 repeats (Fig. 2a). In plants, WD40 repeat proteins are numerous, interact with diverse proteins, and act in a variety of biological processes, such as plant development and immunity 30,31 . To investigate whether RBV is an evolutionarily conserved protein, a phylogenetic tree including homologs of RBV in various plants was generated (Supplementary Data 2); Supplementary Fig. 4a). According to the phylogenetic analysis, RBV orthologs can be found in plants ranging from single-cell green algae to core eudicots and grasses, and there is no close paralog of RBV in the latest common ancestor of land plants. In most eudicots that have undergone gamma whole genome duplication (WGD) and Brassicaceae that has also undergone beta and alpha WGDs, RBV remained as a single copy. Only in species that have undergone recent, speci c WGDs, such as apple, soybean, and maize, there are multiple copies of RBV ( Fig. 2b and Supplementary Fig. 4). This result indicated that RBV was functionally conserved and extra copies might be deleterious.
We also obtained two lines with T-DNA insertions in or near RBV (Salk_126634 and Salk_075672) (Supplementary Fig. 5a and 5c). Interestingly, the phenotypes of the T-DNA mutants were the same as Col ( Supplementary Fig. 5b). Next, we examined RBV transcripts in the two T-DNA insertion lines. RT-PCR showed that transcripts corresponding to the full-length coding region of RBV were present in the two mutants ( Supplementary Fig. 5d). In addition, real-time RT-PCR showed that the levels of RBV transcripts were only mildly reduced in the two mutants ( Supplementary Fig. 5e), consistent with the lack of morphological phenotypes.
In order to remove the amiR-SUL transgene background, we crossed the amiR-SUL rbv-1 mutant with wildtype (Col) plants. In the F2 population, we identi ed homozygous rbv-1 plants without the amiR-SUL transgene through genotyping; these plants showed the same pleiotropic phenotypes as amiR-SUL rbv-1 (Fig. 3a). The morphological phenotypes of rbv-1 were completely rescued by the pRBV:RBV-eYFP transgene (Fig. 3a). We further con rmed that the rbv-1 mutant without the amiR-SUL transgene had defects in miRNA accumulation. RNA gel blots showed that the levels of several endogenous miRNAs, such as miR156, miR159, miR164 and miR167, were mildly reduced in rbv-1 as compared to wild-type Col plants. Other miRNAs, such as miR319 and miR398, were unaffected ( Supplementary Fig. 6a). To rule out the possibility that RBV indirectly regulated miRNA accumulation through affecting the expression of the miRNA biogenesis machinery, we examined the expression of the known genes involved in miRNA biogenesis, and no signi cant changes were observed in rbv-1 (Supplementary Fig. 6b and 6c).

RBV promotes the transcription of MIR genes
We next investigated how RBV promotes miRNA biogenesis. MIR gene transcription to produce pri-miRNAs is the rst step in miRNA biogenesis. RT-qPCR showed that the levels of pri-miRNAs were reduced by ~30-50% in the rbv-1 mutant (Fig. 3b). For the pri-miRNAs, 298 pri-miRNAs annotated in Araport11 were examined by RNA-seq. In total, only 41 pri-miRNAs gave reads in any sample ( Supplementary Fig.  7). In the RNA-seq data, we only detected three (pri-miR159b, pri-miR166a and pri-miR319b) of the seven pri-miRNAs that we examined by qRT-PCR in Fig. 3b. All three pri-miRNAs showed reduced abundance in the rbv-1 mutant, which is consistent with results from qRT-PCR. The reduction in pri-miRNA abundance could be attributed to impaired MIR genes transcription, reduced stability of pri-miRNAs or enhanced pri-miRNA processing. To determine whether transcription was affected in the rbv-1 mutant, we crossed rbv-1 with a GUS reporter line (pMIR167a:GUS) under the control of the MIR167a promoter and obtained pMIR167a:GUS rbv-1 (with both the transgene and the mutation being homozygous). GUS activity was visibly lower in pMIR167a:GUS rbv-1 than in pMIR167a:GUS as revealed by GUS staining (Fig. 3c). RT-qPCR analysis con rmed that the rbv-1 mutant had lower GUS transcript levels (Fig. 3d). Thus, reduced transcription of MIR genes could be one of the problems in miRNA biogenesis in rbv-1.
To further con rm a positive role of RBV in MIR gene transcription, the occupancy of Pol II at MIR loci was determined by chromatin immunoprecipitation (ChIP) with an antibody against the Pol II C-terminal repeats. ChIP without an antibody served as the negative control. MIR166a, MIR167a and MIR171a promoter regions, as well as C1 (a region between the genes AT2G17460 and AT2G17470 known to not engage Pol II 7 , were examined by RT-qPCR after ChIP. The MIR166a and MIR167a promoter regions were enriched in the immunoprecipitates in both rbv-1 and Col relative to C1 (Fig. 3e). Pol II occupancy at MIR166a and MIR167a promoter regions was reduced in the rbv-1 mutant relative to Col, while the signal on MIR171a was too low to be calculated (Fig. 3e). Thus, RBV facilitates the recruitment of Pol II to MIR loci.

RBV is localized in the nucleoplasm and required for the proper localization of HYL1 in D-bodies
We studied the expression of RBV in various tissues and the subcellular localization of the protein. RT-PCR analyses of RNAs from seedlings, roots, cauline leaves, rosette leaves, stems and in orescences showed ubiquitous RBV expression ( Supplementary Fig.8). RBV:RBV-eYFP transgenic plants exhibited YFP uorescence in the nucleoplasm but not the nucleolus. SE is a component of the microprocessor in pri-miRNA processing. An mRuby3-tagged SE protein driven by the SE promoter 9 co-localized with RBV-eYFP in the nucleoplasm (Fig. 4a).
HYL1 and DCL1 form nuclear foci known as Dicing bodies (D-bodies), which are sites of pri-miRNA processing 21,32 . To determine the status of D-bodies in the rbv-1 mutant, we crossed a HYL1-YFP transgene 21 into the mutant and obtained plants homozygous for both the transgene and the rbv-1 mutation. D-body numbers were determined in 420 and 537 root nuclei of wild-type and rbv-1 plants, respectively. The number of HYL1-YFP D-bodies was signi cantly decreased in rbv-1 ( Fig. 4b and 4c), suggesting that mutation of RBV leads to a defect in D-body formation or the localization of HYL1 to Dbodies.
Mutation of RBV leads to a defect of miRNA loading into AGO1 miRNAs are loaded into AGO1 to form miRISCs that execute the silencing of target RNAs 23,25,33 . To determine whether RBV affects the formation of RISCs, we performed AGO1 IP followed by sRNA-seq with both input and IP samples from wild-type and rbv-1 seedlings. Three replicates were performed and showed high reproducibility ( Supplementary Fig. 9). From input samples, many miRNAs showed small but statistically signi cant reduction in abundance in the rbv-1 mutant (Supplementary Data 3). A few miRNAs, such as miR845a, miR845b and miR843, were elevated in abundance in the mutant ( Fig. 5a; Supplementary Data 3). The loading status of each miRNA was expressed as the ratio of miRNA abundance between AGO1 IP and input and differences between wild type and rbv-1 were evaluated by student's t-test. A global reduction in the AGO1 loading of miRNAs was observed in the rbv-1 mutant ( Fig.  5b; Supplementary Data 4). Notably, miR845a and miR845b, which exhibited elevated levels, were less associated with AGO1 in the mutant, indicating that RBV is crucial for miRISC formation. RNA gel blots were also performed to validate the sRNA-seq results. miR159 and miR166 levels were lower in input and further reduced in AGO1 IP in the mutant (Fig. 5c). miR845a levels were strongly increased in the rbv-1 input sample as compared with Col input but greatly reduced in the rbv-1 AGO1 IP sample as compared with Col AGO1 IP ( Fig. 5c; Supplementary Data 4). Thus, the rbv-1 mutant exhibits a global miRNA loading defect.
To determine how RBV might promote the loading of miRNAs into AGO1, we rst examined whether RBV interacts with AGO1. Co-IP was performed with pRBV:RBV-eYFP plants using anti-GFP and anti-AGO1 antibodies, but no interaction between RBV and AGO1 was detected. We next sought to determine whether the bi-molecular feature of miRISCs is affected in the mutant. Protein extracts from pRBV:RBV-eYFP rbv-1 and rbv-1 plants were subjected to gel ltration followed by western blotting to detect AGO1 and northern blotting to detect miR159. Among the 44 fractions, AGO1 was distributed in both high molecular weight (HMW) (fractions 9-11) and low molecular weight (LMW) (fractions 16-17) complexes in pRBV:RBV-eYFP rbv-1, consistent with previous ndings 27 . Notably, the LMW complexes, which corresponded to monomeric AGO1 in size, also showed peak levels of miR159, suggesting that they represent miRISCs with one AGO1 protein and one miRNA. However, such miRISCs were greatly reduced in rbv-1, with AGO1 being in HMW complexes instead (Fig. 5d). The distribution of miR159 also shifted towards HMW complexes in rbv-1 (Fig. 5d). Western blot analyses with anti-GFP antibody showed that RBV-eYFP was present in fractions 8-16 with estimated molecular weights much higher than that of an RBV-eYFP monomer, suggesting that RBV itself also resides in protein complexes ( Supplementary Fig.   10). Therefore, RBV promotes the formation of miRISCs that contain only AGO1 and miRNAs. The HMW AGO1 complexes may represent intermediates in RISC formation.
AGO1 is known to associate with trans-acting small interfering RNAs (ta-siRNAs) in addition to miRNAs 34,35 . Contrary to miRNAs, which are loaded into AGO1 in the nucleus, ta-siRNAs are loaded into AGO1 in the cytoplasm 24 . We investigated whether RBV affects the ta-siRNA-AGO1 association. We quanti ed 21-nt siRNAs that mapped to 100-bp windows that overlapped with TAS1A, TAS1B, TAS1C and TAS2 loci. The ta-siRNAs were not signi cantly altered in rbv-1 input as compared to Col input in each 100-bp window ( Supplementary Fig. 11 a, c, e and g). We then analyzed their levels in AGO-IP vs. input, and found that the loading of ta-siRNAs into AGO1 was largely unaffected. Only ta-siRNAs in window 5 of TAS1A, window 3 of TAS2B and window 3 of TAS1C were affected ( Supplementary Fig.11 b, d, f and h). The levels of miR173, the trigger of ta-siRNA biogenesis from TAS1 and TAS2 loci, were not signi cantly different between Col and the rbv-1 mutant ( Supplementary Fig. 11 i). The fact that ta-siRNA loading was largely unaffected in the mutant is consistent with RBV being a nuclear protein.
RBV is required for the splicing of short introns in certain pre-mRNAs In order to determine whether RBV affects the expression of protein-coding genes, we performed RNA-seq with 14-day-old seedlings of Col and rbv-1 in triplicates. The three biological replicates for each genotype were highly reproducible ( Supplementary Fig. 12).
Differentially expressed genes (DEGs) were identi ed between Col and mutant samples with FPKM >1, fold-change >2 and FDR<0.05 as the cutoff. In total, we identi ed 632 up-regulated (hyper-DEGs) and 363 down-regulated (hypo-DEGs) genes, respectively (Supplementary Data 5 and 6, Supplementary Fig. 13a). Gene Ontology (GO) analyses showed that both the hyper-DEGs and hypo-DEGs were enriched in genes with roles in responses to stimuli (Supplementary Fig. 13b and 13c). We also examined the global transcript levels of miRNA targets from the RNA-seq data. Although several examined miRNA targets were found to be derepressed in the mutant by RT-qPCR, a global trend of increased levels of miRNA target transcripts was not observed (Supplementary Fig. 13d, Supplementary Data 7). It is possible that the RBV mutation leads to a defect in the transcriptional regulation of these genes as well.
It has been reported that MOS4-Associated Complex (MAC) components MAC3A, MAC3B, MAC7, and two WD40 repeat proteins (PRL1 and PRL2) affect both miRNA biogenesis and the splicing of protein-coding transcripts 36,37,38 . This prompted us to examine whether the rbv-1 mutant had splicing defects using the RNA-seq data (see Methods). In total, 474 Genes were found to have intron retention defects in the rbv-1 mutant compared to Col, with a total of 511 intron retention events (Supplementary Data 8). Two examples (At4g15790 and At1g03280) are presented in Fig. 6a. PI (percent of intron reads) was increased in the rbv-1 mutant (Fig. 6b). No differential expression of the genes with intron retention was observed between the rbv-1 mutant and wild type (Supplementary Fig. 14a). Next, we examined whether the genes with intron retention or the retained introns in the rbv-1 mutant had any common features. Intriguingly, the retained introns in the rbv-1 mutant seemed to be shorter as compared to the overall length distribution of introns (P= 1.984083e-22) (Fig. 6c). Besides, genes affected in rbv-1 tended to have more introns than all genes (P=2.823e-05) (Fig. 6d). Intron retention defects are also found in prl1 prl2 and mac3a mac3b mutants 36 . We compared the retained introns in rbv-1, prl1 prl2 and mac3a mac3b and found little overlap between the introns affected in rbv-1 with those affected in either prl1 prl2 or mac3a mac3b (Fig. 6d), suggesting that RBV affects different introns from the MAC components.
Many MIR genes have introns 39,40,41 . The RNA-seq did not detect intron retention events in pri-miRNAs, but the low abundance of pri-miRNAs could have precluded the detection of intron retention events. We performed RT-PCR to test the splicing of introns from three miRNA precursors (pri-miR163, pri-miR156 and pri-miR168) with intron-anking primers. Genomic DNA was ampli ed with the same primers to indicate the size of the intron-containing fragments. No defects in pri-miRNA splicing were observed in the rbv-1 mutant (Supplementary Fig. 14b).

Discussion
RBV is involved in MIR gene transcription, pri-miRNA processing and miRISC formation RBV, a WD40 protein, is an evolutionarily conserved protein in plants. However, it has not been studied from any species. In this study, we show that a recessive mutation in RBV reduces the levels of many endogenous miRNAs, indicating that mutation of RBV leads to a defect in miRNA biogenesis. Consistent with the known roles of plant miRNAs in various developmental processes, the rbv-1 mutant exhibits strong developmental defects. How does RBV promote miRNA biogenesis? Our ndings suggest a role of RBV in promoting Pol II transcription of MIR genes, which is supported by the reduced levels of pri-miRNAs, compromised MIR167a promoter activity, and decreased Pol II occupancy at MIR genes in the rbv-1 mutant. In Arabidopsis, a series of proteins have been found to promote MIR transcription and/or pri-miRNA processing, and can be grouped into two main classes 1 . One group contains CBP80 and CBP20 42 , STA1 43 , SICKLE 40 , TOUGH 44 , PINP1 45 , THO1 and THO2 46, 47 , and MOS2 48 . A common feature is that mutants in these genes show reduced levels of miRNAs and increased abundance of pri-miRNAs. A second group of proteins acts in a different manner. In loss-of-function mutants in the genes in this group, the abundances of both pri-miRNAs and mature miRNAs are reduced. Proteins in this group include DAWDLE 49 , CDC5 7 , NOT2 6 , Elongator 8 , PRL1 50 , MAC7 36 , PP4 38 and THP1 9 . Besides, mutants in PRL1, CDC5, MAC7, PP4 and THP1 show a reduced number of HYL1 D-bodies 7,9,36,38,50 . A number of proteins in this group interact with DCL1, HYL1, or SE and thus are thought to bridge MIR gene transcription and pri-miRNA processing 6,7,8,9,38,49 . Besides the two main classes, the third group, which includes CHR2, a partner of SE, promotes the transcription of MIR genes but represses miRNA accumulation by inhibiting pri-miRNA processing 51 . Our studies show that RBV belongs to the second group of proteins that promotes MIR gene transcription and possibly pri-miRNA processing.
RBV also differs from the second group of proteins in that it has a clear role in miRISC formation. In the rbv-1 mutant, both AGO1 and miR159 shift into complexes with higher molecular weights. RBV itself is also found in high molecular weight complexes. We suspect that the high molecular weight complexes containing AGO1 and miR159 represent intermediates in miRISC formation, and that RBV helps the dissociation of AGO1-miRNA from other proteins to form active miRISCs. Heat Shock Protein 90 (HSP90) is required for sRNA loading into AGO1 in tobacco lysates 52 and for RISC formation in Drosophila and humans 53 . HSP90 proteins are involved in RNA silencing in animals 54 and plants 55,56 . Consistently, HSP90.4 was found in GFP-AGO1 IP spectrometry experiments 24 . Another protein that has recently been shown to play a role in miRISC formation is CARP9, which interacts with both HYL1 and AGO1 in the nucleus 57 . The relationship between RBV, HSP90 and CARP9 is worth investigating in the future.
In summary, RBV, as a nuclear WD40 protein, is involved in multiple steps in miRNA biogenesis, including MIR transcription, pri-miRNA processing and AGO1 loading (Fig.7).
RBV is also required for the splicing of certain mRNAs Proteins such as MAC7, PP4, MAC3A/3B, and PRL1 act not only in miRNA biogenesis but also in pre-mRNA splicing in Arabidopsis 36, 38 . We found that RBV also plays a role in splicing. Similar to mutants in MAC7, PP4, MAC3A/3B, and PRL1, no signi cant correlations between intron retention and changes in gene expression were found in the rbv-1 mutant. Introns retained in the rbv-1 mutant had minimal overlap with those in the other mutants, suggesting the presence of different categories of introns, whose splicing requires distinct factors. RBV acts on genes with more introns and introns that are shorter in length. Furthermore, intron retention was not found for intron-containing pri-miRNAs in rbv-1, suggesting that mutation of RBV leads to a defect in miRNA biogenesis and pre-mRNA splicing independently. It is possible that RBV function is required for yet other aspects of nuclear RNA metabolism.
All seeds were sown on 1/2 Murashige and Skoog Basal Medium (Sigma-Aldrich, M5519) plus 1% Sucrose and 0.8% Agar. All plants were grown at 22°C under 16 h light and 8 h dark cycles.

Mutagenesis and mapping
EMS mutagenesis was performed as described 9 . A mutant, amiR-SUL rbv-1, with reduced leaf bleaching, was isolated and then backcrossed with the parental line pSUC2:amiR-SUL. In the F2 generation, ~100 plants with the amiR-SUL rbv-1 phenotype were identi ed. Genomic DNA was extracted by the CTAB method 58 from individual plants and equal amount of DNA was pooled for genomic DNA library construction. The library was paired-end (PE151bp) sequenced on the Illumina platform HiSeq4000 at 50x coverage at BGI-Shenzhen, China. Focusing only on G-to-A mutations in coding regions, a mutation in At5g64730 was identi ed in amiR-SUL rbv-1. A Derived Cleaved Ampli ed Polymorphic Sequences (dCAPS) marker was designed to genotype this mutation (Supplementary Table 2). The PCR products from wild type can be digested by NcoI, whereas those from amiR-SUL rbv-1 could not. Genotyping thẽ 100 individuals showed that this mutation was linked to the plant phenotype.

DNA constructs and complementation
The genomic region of RBV (At5g64730) including ~1 kb promoter was ampli ed with the primers proRBV-F and RBV-R (Supplementary Table 2) and cloned into the pTSK108 vector. The clone was sequenced to ensure the absence of mutations and was recombined with the pGWB640 gateway vector 59 to generate the pRBV:RBV-eYFP construct via LR reaction. The pRBV:RBV-eYFP plasmid was transformed into the rbv-1 mutant in both amiR-SUL and Col backgrounds through the Agrobacterium tumefaciensmediated oral dip method as described 60

Reverse transcription quantitative PCR (RT-qPCR)
RT-qPCR was performed to quantify mRNA and pri-miRNAs levels. 1μg total RNA was reverse transcribed with oligo (dT) using the PrimeScript™ 1st Strand cDNA Synthesis Kit (TAKARA, 6110A) according to the manufacturer's instructions. RT-qPCR was performed in the 96-well StepOnePlus real-time system (ABI) using the SYBR premix ExTaq II kit (TAKARA, RR820A). The following scheme was used: 95°C for 30s and 40 cycles of 95°C for 5s and 60°C for 30s. The levels of transcripts were normalized to the level of the internal standard UBQ5 and the 2 -△△ CT values of control samples were set to 1. The primers for pri-miRNAs are listed in Supplementary Table 2. Three independent biological replicates were carried out for each genotype. Student's t test was used for the evaluation of statistical signi cance.
Histochemical GUS staining 14-day-old seedlings of pMIR167a:GUS and pMIR167a:GUS rbv-1 (homozygous for both transgene and the rbv-1 mutation) were subjected to histochemical GUS staining according to the standard protocol 66 .
Chromatin Immunoprecipitation (ChIP) assay RNA Polymerase II ChIP was performed as described (Kim et al., 2011) with 14-day-old Col and rbv-1 seedlings using an antibody against RPB1 (Abcam, ab817). RT-qPCR was performed with co-immunoprecipitated DNA, using primers listed in Supplementary Table 2. Relative enrichment was calculated by normalizing the amount of ChIP-ed DNA to the corresponding amount in the input.

Western blot Analysis
Western blots were performed as described 65 . Proteins from 14-day-old seedlings were extracted, resolved in 12% (v/v) SDS-polyacrylamide gels, and transferred to Hybond C-Extra membranes (Amersham Biosciences). The membranes were blocked with 5% (w/v) non-fat milk in Tris buffered saline Tween (TBST) buffer and then probed with speci c antibodies. Antibodies used included anti-GAPDH were identi ed between Col and rbv-1 using cuffdiff 69 with FPKM >1, fold-change >2 and FDR<0.05 as lters. Expression levels of all genes and DEGs were plotted using the value of log2 (FPKM+1). GO enrichment analysis was performed with agriGO 70 using a webtool (http://bioinfo.cau.edu.cn/agriGO/).
Only the top 20 terms were presented in this paper. The analysis of splicing defects was carried out using Araport 11 intron annotation and a previously developed pipeline known as SQUID (https://github.com/Xinglab/SQUID). In brief, the level of retained introns was calculated using two methods: PI_Junction (intron -exon junction reads/[intronexon junction reads + exon-exon reads]) and PI_density (normalized intronic reads/normalized exonic reads). The differentially spliced introns were de ned using a stringent cutoff: combined_FDR < 0.1, Diff_PI_Junction > 0.05, Diff_PI_Density > 0.05.
PI_Junction was used to represent the levels of retained introns.
AGO1 IP assay 1g of 14-day-old seedlings was ground in liquid nitrogen, and IP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% Glycerol, 0.1% NP-40, 1mM PMSF) and EDTA-free protease inhibitor mixture (Roche) were added to the powder, which was followed by 20 min incubation with gentle shaking at 4°C. The supernatant was incubated with 100μL of dynabeads (Invitrogen, 10002D) for 2h at 4°C. After centrifugation, the supernatant was used for IP. 50 ul was saved as input, and the rest was incubated with anti-AGO1 antibody (Agrisera, AS09527, dilution, 1:2000) for 2 h at 4 ℃. The beads were washed with wash buffer (IP buffer with 0.5% NP-40), and 1/10 (v/v) was added to 2x SDS-loading buffer for western blot analysis and 9/10 (v/v) was used for RNA isolation. Small RNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, E7300), and sequenced on an Illumina HiSeq2500 platform at BerryGenomics, China. The proteins were separated by SDS-PAGE and protein gel blot analysis was performed using anti-AGO1 antibody (Agrisera, AS09527, dilution, 1:2000).
Gel ltration assay 1g of 14-day-old T3 pRBV: RBV-eYFP rbv-1 seedlings and wild type was collected and ground in liquid nitrogen. Then 1.5 ml phosphate buffer supplemented with 1mM PMSF, 1% EDTA-free protease inhibitor mixture, RNase Inhibitor (TAKARA) and 0.4% CA630 was added to the powder. The homogenized crude extracts were kept on ice for 20 min. After two rounds of centrifugation for 20 min (12000 g) each at 4℃, 1.5ml of the cleared crude extract was immediately used for sample injection. Gel-ltration chromatography was carried out as described 27   Isolation of a silencing suppressor mutant rbv-1 from an amiR-SUL line. a Phenotypes of 1-month-old amiR-SUL rbv-1 and amiR-SUL plants. Bar=1cm. b Images of rosette leaves from 1-month-old plants grown under long-day conditions. c 14-day-old seedlings showing reduced root length in amiR-SUL rbv-1.   b Levels of seven pri-miRNAs in 14-day-old seedlings of Col, rbv-1 and the complementation line pRBV:RBV-eYFP rbv-1 as determined by RT-qPCR. UBQ5 was used as the internal control. The expression values were relative to those in Col, which were set to 1. Error bars represent standard deviation calculated from three independent replicates. (*Student's t test, P < 0.05). c Representative images of GUS staining of pMIR167a:GUS and pMIR167a:GUS rbv-1 in orescences. Bars = 2 mm. d The transcript levels of GUS from the two genotypes as determined by RT-qPCR. The expression values were relative to pMIR167a:GUS. Error bars represent standard deviation calculated from three independent replicates.
(*Student's t test, P < 0.05). e RBV is required for the recruitment of Pol II to MIR166a and MIR167a promoters. The occupancy of Pol II at various regions was determined by ChIP with rbv-1 and Col using an antibody that recognizes the C-terminal repeat (YSPTSPS) of the largest subunit of Pol II. ChIP performed without the antibody served as a negative control. A genomic region between the genes AT2G17460 and AT2G17470 named Pol II C1 was also used as a negative control. Means and standard deviation from three independent replicates are presented. (*Student's t test, P < 0.05) miRNAs with RPM value >10 in either genotype in the input samples (as in a) are shown here. The red dots indicate miRNAs with increased AGO1 association in rbv-1, and the blue dots indicate miRNAs with reduced AGO1 association in rbv-1. c RNA gel blot analysis of three miRNAs before (input) and after AGO1 IP. U6 was used as an internal control for the input samples. For the IP samples, a portion was used for protein gel blot analysis to quantify AGO1 protein levels. The levels of miRNAs in the IP samples were normalized against AGO1 protein levels. No matter whether the assayed miRNAs were increased or reduced in abundance in input samples, they all showed reduced AGO1 association. d Size exclusion chromatography with pRBV:RBV-eYFP rbv-1 and rbv-1 samples followed by western blotting to detect AGO1 and northern blotting to detect miR159. The upper panel indicates the distribution of AGO1 while the lower panel represents the distribution of miR159 among the fractions. The numbers above the AGO1 blots indicate those of the fractions. Note that no AGO1 or miR159 was detected in fractions 1-7 (not shown). The positions of the molecular weight standards are shown above the AGO1 blots.

Figure 7
A model for the role of RBV in MIR gene transcription, pri-mRNA processing and AGO1 loading.

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