JANUS, a spliceosome-associated protein, promotes miRNA biogenesis in Arabidopsis

Abstract MicroRNAs (miRNAs) are important regulators of genes expression. Their levels are precisely controlled through modulating the activity of the microprocesser complex (MC). Here, we report that JANUS, a homology of the conserved U2 snRNP assembly factor in yeast and human, is required for miRNA accumulation. JANUS associates with MC components Dicer-like 1 (DCL1) and SERRATE (SE) and directly binds the stem-loop of pri-miRNAs. In a hypomorphic janus mutant, the activity of DCL1, the numbers of MC, and the interaction of primary miRNA transcript (pri-miRNAs) with MC are reduced. These data suggest that JANUS promotes the assembly and activity of MC through its interaction with MC and/or pri-miRNAs. In addition, JANUS modulates the transcription of some pri-miRNAs as it binds the promoter of pri-miRNAs and facilitates Pol II occupancy of at their promoters. Moreover, global splicing defects are detected in janus. Taken together, our study reveals a novel role of a conserved splicing factor in miRNA biogenesis.

Here we report that JANUS plays important roles in miRNA biogenesis.JANUS interacts with DCL1 and SE, and directly binds the stem-loop of pri-miRNAs.The hypomorphic janus mutation reduces the activity of the DCL1 complex, causes reduced accumulation of miRNAs, and impairs the interaction of HYL1 with pri-miRNAs and the localization of HYL1 in the D-body, suggesting that JANUS may facilitate miRNA biogenesis by promoting the assembly of the DCL1 complex and the loading of pri-miRNAs into the DCL1 complex.Moreover, JANUS facilitate pri-miRNA transcription since the occupancy of Pol II at MIR promoter and MIR promoter activity are reduced in janus .Based on these observations, we propose that JANUS is a DCL1-associated protein that coordinates pri-miRNA transcription and processing.

Plant materials and growth condition
The CS16053 ( janus-1 ) line is in the Columbia ( Col-0 ) background and was obtained from the Arabidopsis Biological Resources Center ( ABRC ) .All plants were grown at 22 • C with 16-h light and 8-h dark cycles.The primers used for genotyping are listed in Supplementary Table S1 .

Complementation assay
The pJ ANUS::J ANUS-MYC plasmid was transformed into janus-1 and janus-2 ,and transgenic plants were identified through screening for Basta resistance.

RNA extraction, RT-PCR, and quantitative RT-PCR
Total RNAs from inflorescences were extracted with TRI reagent (Molecular Research Center).To perform RT-PCR, total RNAs were treated with DNase I (Thermo Fisher Scientific) followed by reverse transcription using M-MLV reverse transcriptase (Promega) with oligo-d(T) primers according to manufacturer's instructions.RT-qPCR was performed using iCycler apparatus (Bio-Rad) with Accuris Instruments PR2000-N-100 Qmax Green Real Time PCR Mix.The primers are listed in Supplementary Table S1 .

Small RNA sequencing
Small RNA libraries were prepared using total RNAs extracted from inflorescences.Two biological replicates were performed.After sequencing, miRNAs are analyzed as described ( 12 ).After removing reads aligned to t / r / sn / snoRNA, the total numbers of perfectly aligned reads were used for normalization (Nobuta et al., 2010).miRNAabundance was compared by using EdgeR with trimmed mean of M values normalization method ( 64 ).The dataset was deposited into National Center for Biotechnology Information Gene Expression Omnibus (Col-0 accession numbers: GSM7164784 and GSM7164785; janus accession numbers: GSM7164786 and GSM7164787).GFP-DCL1 or GFP-FLAG were co-expressed with JANUS-FLAG in N. benthamiana .IPs were performed using anti-GFP antibodies.JANUS-FLAG, GFP and GFP-DCL1 were detected by western blot.(E) Co-IP between truncated JANUS proteins and MYC-SE.JANUS protein contains two RNA recognition motifs (R) and a glycine-rich region (GR).Truncated JANUS proteins fused a GFP-tag at N-terminus.IPs were performed using anti-GFP antibodies.Truncated JANUS proteins and MYC-SE were detected by western blot.

Differential gene expression and differential splicing analyses
RNA libraries were prepared using total RNAs extracted from inflorescences following standard protocol.Two biological replicates were performed.After sequencing, mRNAs are are analyzed as described ( 4 ).Differential gene expression analysis was conducted using R package Ballgown ( 58 ).Stattest function in Ballgown was used to test differential gene expression between Col-0 and janus .Only the genes with standard deviation of expression larger than 1 among all samples were used in the analysis and the genes with statistic Q -value ≤0.05 were considered as differentially expressed genes.Differential mRNA splicing analysis was conducted using R package VaSP ( 59 ).Only the introns supported with at least 5 junction reads in at least 1 sample were considered, and only the genes with the average read coverage larger than 1 in all samples were used in the analysis.Student's t-test was employed to test the difference of 3S scores between Col-0 and janus .Genes with any intron at P-value ≤ 0.05 and fold change of 3S scores ≥2 were considered as differential splicing genes.The dataset was deposited into National Center for Biotechnology Information Gene Expression Omnibus (Col-0 accession numbers: GSM7164780 and GSM7164781; janus accession numbers: GSM7164782 and GSM7164783).

RIP analyses
RIP was performed as described ( 12 ).A total of ∼4 g inflorescences of Col or transgenic plants harboring a p35S::MYC-JANUS transgene were cross-linked with 1% formaldehyde for 10 min.Then, glycine was added to quench the reaction for 10 min.Following this step, nuclei were extracted and lysed in 400 uL nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) by sonication five times.After debris was removed by centrifuge at 16000 × g for 10 min, equal amounts of proteins from various samples were diluted with RIP dilution buffer (16.7 mM Tris-HCl, 1.1% Triton X-100, 1.2 mM EDTA, pH 8.0, 167mM NaCl) and incubated with anti-MYC antibodies conjugated to protein G agarose beads or protein A / G agarose beads (for no-Ab controls).The immunoprecipitates were washed five times and then eluted with elution buffer (100 mM NaHCO3, 1% SDS) at 65 • C. Following reversing cross-linking with proteinase K (Invitrogen) and 200 mM NaCl at 65 • C, RNAs were extracted and used as templates for RT-PCR analyses.The primers are listed in Supplementary Table S1 .

ChIP assay
ChIP was performed using 14-day-old seedlings from Col-0 and amiR JANUS as described (Kim et al., 2011).Anti-CTD an-tibody (ab817, Abcam) was used for immunoprecipitation. qPCR was performed on DNAs copurified with CTD antibody.The primers are listed in Supplementary Table S1 .

Identification of JANUS as a candidate acting in the miRNA biogenesis
We performed network analyses to identify candidate proteins involved in miRNA biogenesis in Arabidopsis using MAC5 as a bait, based on the fact that functionally related genes usually occur in the same gene network ( 65 ).The MAC5 network constructed with the STRING program ( https://string-db.org ) was consisted of known protein factors involved in miRNA biogenesis such as MAC3, PRL1, MAC7, CDC5 and others, and proteins acting in RNA metabolisms ( Supplementary Figure S1 A & B).From this network, we prioritized potential splicing-related proteins as our candidates since miRNA biogenesis is interconnected with RNA splicing processes.Among these candidates, we focused on JANUS (AT2G18510) because it plays essential roles in plant development ( 61 ) and ( Supplementary Figure S2 A-E), which is consistent with the role of miRNAs in controlling development.
In order to determine if JANUS could function in miRNA biogenesis, we analyzed JANUS interactome.We immunoprecipitated (IP) MYC-JANUS from transgenic plants harboring a p35S::MYC-JANUS transgene with anti-MYC antibodies, and performed mass spectrometry analyses.IP was also performed using the same anti-MYC antibodies with Col-0 as a negative control.JANUS pulled down several known protein factors involved in miRNA biogenesis including MAC3, MA C5, MA C7, CDC5, PRL1,STV1 and SE (Figure 1 A), consistent with the result obtained from the functional network analysis of MAC5.

JANUS is associated with the DCL1 complex
Interestingly, SE was also identified as an interactor of JANUS, suggesting that JANUS may associate with the DCL1 complex (Figure 1 A).To test this possibility, we performed Bimolecular fluorescence complementation (BIFC) assay to test the potential interaction of JANUS with DCL1, HYL1, SE, CDC5 and AGO1.JANUS was fused with the C-terminal fragment of cyan fluorescent protein (cCFP), and DCL1, HYL1, SE, AGO1 and CDC5 were fused with the N-terminal fragment of Venus (nVenus).Co-expression of cCFP-JANUS with nVENUS-DCL1, -SE or -CDC5 produced strong YFP signals which were localized at the discrete bodies (Figure 1 B), while co-expression of cCFP-JANUS with nVenus-HYL1 or -AGO1 pairs produced weak and diffused YFP signals.These results indicated that JANUS might associate with these proteins.Next, we performed co-IP assay to confirm the association of JANUS with the DCL1 complex.We transiently co-expressed JANUS-FLAG with either MYC-SE or GFP-DCL1 in N .benthamiana and performed IP with anti-FLAG or anti-GFP antibodies.After IP, SE was detected in the JANUS-FLAG precipitates and JANUS-FLAG was detected in the GFP-DCL1 precipitates (Figure 1 C and D).Taken together, interactome and co-IP analysis show that JANUS interacts with DCL1 and associates with SE in Arabidopsis.

JANUS is required for miRNA accumulation
To evaluate if JANUS functions in miRNA biogenesis, we used a CRISPR / CAS9 system ( 62 ) to generate a weak allele of JANUS .In T1 generation, we obtained a hypomorphic janus mutant line, which contains a four-bp deletion in the seventh exon of JNAUS, and named it as janus-2 (Figure 2 A and B).The heterozygous janus-2 / + line was backcrossed to Col-0 for three times to remove the CRISPR / CAS9 transgene and other potential mutations.The resulting janus-2 mutant displayed pleiotropic development defects such as smaller plant size, serrated leaves, and entirely abolished fertility (Figure 2 C, Supplementary Figure S2 D).Moreover, the pavement cells in janus-2 were smaller than those in Col-0 as observed by scanning electronic microscope (SEM) (Figure 2 D).Expression of a pJ ANUS::J ANUS-MYC transgene fully recovered the developmental defects of janus-2 ( Supplementary Figure S2 D and E).In addition, we found that down regulation of JANUS transcripts with an artificial miRNA ( amiR JANUS ) ( Supplementary Figure S3 A) also caused delayed growth of plants ( Supplementary Figure S3 B and C).These results demonstrated that JANUS is required for plant development.
We next tested miRNA accumulation in inflorescences of janus -2.Indeed, reverse transcription quantitative PCR (RT-qPCR) analyses showed that several examined miR-NAs were reduced in janus-2 relative to Col-0 (Figure 2 E).The miRNA levels were restored in janus-2 harboring the pJ ANUS::J ANUS-MYC transgene (Figure 2 E).In addition, miRNA levels were also reduced in abundance in amiR JANUS relative to Col-0 ( Supplementary Figure S3 D).Illumina deep sequencing further confirmed that the accumulation of miR-NAs was globally reduced in janus-2 relative to Col-0 (Figure 2 F and Supplementary dataset 1 ).These results demonstrate that JANUS is required for miRNA accumulation.
We next performed reverse transcription quantitative PCR (qRT-qPCR) to examine the transcript levels of several miRNA target transcripts including ARF6 , AP2 , TOE1 , TAS2 and PHO2 , which are targets of miR167, miR172, miR173 and miR399, respectively.The levels of these targets were moderately increased in janus-2 compared with Col-0 and the complementation lines (Figure 2 G).This result is consistent with the decreased levels of miRNAs in janus-2 .

JANUS regulates the transcription of MIR
Next we asked how JANUS acts in miRNA biogenesis.Because JANUS has been shown to regulate the transcription of several genes ( 66 ), we tested if janus-2 affects the transcript levels of several genes involved in miRNA biogenesis including CBP20 , CBP80 , DCL1 , DDL , HEN1 , HYL1 and SE, and pri-miRNA levels through RT-qPCR.While the transcript levels of miRNA biogenesis related genes did not show obvious change ( Supplementary Figure S4 ), pri-miRNA levels were reduced in both janus-2 and amiR JANUS relative to Col (Figure 3 A and Supplementary Figure S3 E), suggesting that JANUS may promote pri-miRNA transcription.We tested this pos-sibility using a GUS reporter gene driven by the MIR167a promoter ( pMIR167a::GUS ), which has been used to examine the function of several transcription factors in modulating pri-miRNA transcription ( 31 ,36 ).We crossed janus-2 / + with the transgenic line harboring the pMIR167a::GUS transgene and identified JANUS + (JANUS / JANUS or JANUS / janus-2 ) and janus-2 harboring the pMIR167a::GUS transgene.GUS staining on these plants revealed that the GUS activity was lower in janus-2 than that in JANUS + (Figure 3 B).qRT-PCR analysis confirmed that the GUS mRNA levels in janus-2 were reduced significantly relative to those in JANUS + (Figure 3 C).Then we used chromatin immunoprecipitation (ChIP) assays to examine the occupancy of Pol II at five MIR promoters in Col-0 and amiR JANUS with anti-RPB2 antibody.qPCR analysis showed that the occupancy of Pol II at three MIR promoters were reduced in amiR JANUS relative to Col-0 (Figure 3 D).Taken together, these results show that JANUS affects MIR transcription.

JANUS interacts with Pri-miRNAs in vitro and in vivo
Because JANUS is a putative RNA-binding protein, we investigated if JANUS binds pri-miRNAs in vivo .We performed an RNA immunoprecipitation assay (RIP) on the inflorescences of the transgenic plants harboring the p35S::MYC-JANUS transgene.Following cross-linking, nuclear isolation, and IP, RT-PCR was performed, and we can detect the enrichment of pri-miR156a , pri-miR159a , pri-miR167a and pri-miR172b in the MYC-JANUS IPs, but not in the no-antibody control (Figure 4 A).Moreover, the negative control EIF4A was not detected in the MYC-JANUS IPs (Figure 4 A).These results demonstrate that JANUS binds pri-miRNAs in vivo .Next, we performed an in vitro pull-down assay to examine whether JANUS could directly bind pri-miR162b .We expressed MBP and MBP-JANUS in E. coli and purified them with amylose resin ( Supplementary Figure S5 ).Then we incubated MBP and MBP-JANUS with radioactive labeled pri-miR162b , which was transcribed in vitro .MBP-JANUS, but not MBP, was able to pull down pri-miR162b.MBP-JANUS can also bind the precusor-miR162b (pre-miR162b) and precusor-miR172b (pre-miRN172b) (Figure 4 B).We also examined if JANUS binds dsRNAs and ssRNAs.However, we did not detect the interaction of JANUS with dsRNAs or ssRNAs (Figure 4 B).
To examine which region(s) within pri-miRNAs can be recognized by JANUS, we used an in vivo assay developed from our previous studies (Figure 4 C) ( 37 ,44 ).We first examined the interaction of MYC-JANUS with pri-miR172b in N. benthamiana transiently expressing p35S::MYC-JANUS and p35S::MIR172b which contains a full length pri-miR172b transcript ( MIR172bFL ).Consistent with our RIP assay, MYC-JANUS binds MIR172bFL , but not the endogenous control NtEF1A RNA from N. benthamiana (Figure 4 D).We further investigated the interaction of MYC-JANUS with the MIR172bF1 (a 287-nt 5 arm), MIR172bF2 (a 287-nt 5 arm + a stem loop region + a 6-nt 3 arm), MIR172bF3 (a 417-nt 3 arm), MIR172F4 (a 39-nt 5 arm + a stem loop region + a 6-nt 3 arm) (Figure 4 D).MYC-JANUS was able to bind MIR172bF2 , MIR172bF4 , but not MIR172bF1 and MIR172bF3 (Figure 4 E-H).These results together with the in vitro pull-down assay suggest that JANUS may bind the stemloop region of pri-miRNAs and require imperfect base-pair or the junction between dsRNA and single-stranded in the stemloop.

JANUS is required for the formation of the D-Bodies
The interaction of JANUS with DCL1 and SE led us to test if JANUS could modulate pri-miRNA processing using an in vitro assay ( 12 ).In this assay, we incubated radioactive labeled pri-miR162b with the protein extracts from young flower buds of Col, or amiR JANUS .The production of miR162 was reduced in the protein extracts of amiR JANUS relative to Col (Figure 5 A and B), suggesting that JANUS may promote pri-miRNA processing.Next, we tested the effect of janus-2 on the formation of the D-body using HYL1-YFP as a reporter gene ( 25 ).We crossed a HYL1-YFP transgenic line into janus -2 / + and calculated the percentage of cells containing D-bodies in the root tips and elongation region.As previously reported ( 34 ,42 ), the HYL1-containing D-bodies existed in most cells ( ∼90%; Figure 5 C and D) in JANUS + (JANUS / JANUS or JANUS / janus-2 ), whereas Dbodies were observed in only ∼40% of cells in janus-2 .This result demonstrates that JANUS is required for correct HYL1 localization, revealing its potential role in facilitating D-body formation.
The interaction of HYL1 with pri-miRNAs is impaired in amiR JANUS SAP49 binds the pre-mRNA-U2 snRNA helix to promote the interaction between U2 snRNP and pre-mRNAs ( 56 ,57 ).By analogy, we sought that JANUS might also facilitate the interaction of pri-miRNA with the DCL1 complex.Therefore, we tested the interaction of HYL1 with pri-miRNAs using an RNA IP (RIP) assay, which is an indicator of pri-mRNA loading ( 12 ).HYL1 was IPed from protein extracts of amiR JANUS and Col-0 using antibodies recognizing HYL1 ( Supplementary Figure S6 ), and qRT-PCR was performed to examine the amounts of pri-miRNAs associated with HYL1.The result showed that three examined HYL1-bound pri-miRNAs were reduced in abundance in amiR JANUS relative to Col-0 ( Supplementary Figure S6 ).This result shows that JANUS may enhance pri-miRNA loading to the DCL1 complex.

JANUS affects gene expression at global levels
Next we examined the general role of JANUS in modulating gene expression.We compared mRNA profiling in the flowers from the janus-2 with that in WT by RNA-Seq analyses in two biological replicates.After sequencing, we identified DEGs between janus-2 and Col-0 with fold change of 1.5 or more.A total of 6149 down-regulated and 4147 up-regulated genes were identified, respectively, in janus-2 (Figure 6 A and Supplementary dataset 2 ).To better understand the function of JANUS, we performed gene ontology (GO) analyses on DEGs in janus-2 .Genes related to RNA splicing, embryo development, pollen development, ribonucleoprotein complex assembly, cell wall modification, DNA repair were enriched in down-regulated genes (Figure 6 B and Supplementary dataset 3 ), while genes involved in regulation of response to stimulus, glucosinolate metabolism, RNA processing, meiotic cell cy- cle and leaf development were detected in up-regulated genes (Figure 6 C and Supplementary dataset 3 ).

JANUS promotes pre-mRNA splicing
Next, we asked if JANUS also plays a role in splicing by examining the effect of janus-2 on intron retention of pre-mRNAs.The ratio between RNA-Seq reads mapped to introns and those mapped to exons was used to calculate the intron retention.The intron retention at global levels was determined using all annotated transcripts that passed the abun-dance filter.The result showed that the intron retention rate in janus-2 was higher than in WT.A total of 675 genes were found to have higher intron retention rate in janus-2 relative to WT (Figure 6 D and Supplementary dataset 4 ). Figure 6 E showed two examples of impaired mRNA intron retention in janus-2 relative to WT.We randomly selected three genes with increased intron retention in janus-2 for validation using RT-PCR analysis.The intron retention was increased in all these three genes in janus-2 , agreeing with the RNA-Seq result ( Supplementary Figure S7 A and B).We next asked if splicing defects caused altered expression levels in janus-2 .The co-occurrence of genes with intron retention defects with both upregulated and downregulated DEGs were examined.Only a small portion of DEGs had splicing defects (Figure 6 A).

Discussion
SF3B4 / HSH49 is a conserved U2-snRNP assembly factor from yeast to human (53)(54)(55).Its role in regulating transcription and translation of specific genes have also been reported (58)(59)(60).However, the function of JANUS, a homolog gene of SF3B4 / HSH49 from plant remains largely unknown.In this study, network analysis suggests that JANUS is functionally correlated with MAC5.Moreover, JANUS is physically associated with the MAC complex and the DCL1 complex, suggesting that it may function in miRNA biogenesis.Indeed, both the weak janus-2 mutation and knockdown of JANUS by amiR JANUS cause pleiotropic development defects and reduce the accumulation of miRNA.
JANUS appear to affect pri-miRNA processing, based on the facts that miRNA levels are reduced in janus and that the production of miR162 from both pri-miR162b and pre-miR162b is reduced in janus protein extract relative to WT.How does JANUS promote pri-miRNA processing?It may promote DCL1 activity through its interaction with the DCL1 complex.In human and yeast, JANUS homologs are considered as a platform for the U2 snRNP assembly through their interactions with other proteins ( 67 ).We suspect that JNAUS may play a similar role in miRNA biogenesis.Supporting this notion, the number of D-bodies is reduced in janus-2 .In addition, it may directly modulate DCL1 activity through its R2-PG domain.In human, SF3B4 modulates the activity of Bone morphogenetic protein (BMP)-2 through R2-PG-mediated SF3B4-BMP-2 interaction ( 68 ).We propose that JANUS may have a similar effect on SE.Interestingly, the R2 domain of JANUS also interact with Pol II ( 61 ), suggesting that JANUS may coordinate co-transcriptional processing of pri-miRNAs.JANUS may also promote pri-miRNA processing through its interaction with pri-miRNAs.The assembly of RNA into protein complex or efficient RNA processing often needs conformation changes.Our results show that JANUS binds the stem-loop of pri-miRNAs and promotes the interaction of HYL1 with pri-miRNAs.Thus, it is possible that JANUS may alter the structure of pri-miRNAs to facilitate their assembly into the DCL1 complex.Indeed, SF3B4 / HSH49 binds the U2 snRNA and pre-mRNA to alter their structure, and thereby promotes their interaction with U2snRNP ( 69 ).
JANUS may also affect the accumulation of some miR-NAs through its effect on pri-miRNAs.The levels of some pri-miRNAs are reduced in janus , which could be caused by reduced stability or transcription.The interaction of JANUS with Pol II together with the fact that Pol II occupancy at some MIR promoters and MIR167a promoter activity are reduced in janus demonstrate that JNAUS likely promotes the transcription of some pri-miRNAs.It has been shown that JANUS] promotes the transcription of W O X2 and PIN7 during early embryogenesis.These results suggest that like SF3B4 / HSH49, JANUS modulates the transcription of a subset of genes including some pri-miRNAs.Indeed, our RNA seq-analysis suggest that the transcript levels of many genes are reduced in janus .
The association of JANUS with MAC suggest that like SF3B4 / HSH49m, JANUS may play a role in splicing.Indeed, RNA-seq analysis shows that intron retention of many pre-mRNAs is altered in janus .Interestingly, only few pri-miRNAs have intron retention defects, indicating that JANUS may act in splicing independent of its function in miRNA biogenesis.Notably, the transcript levels of genes with altered intron retention can be up-regulated, down-regulated or unchanged, showing that the correlation between intron defection and changes in transcript levels is not significant.This result suggests that JANUS a broad role in RNA metabolism.
To summarize, our work uncovers a role of JANUS in miRNA biogenesis.It may promote miRNA accumulation through facilitating the assembly of the DCL1 complex and the interaction of pri-miRNAs with the DCL1 complex, and / or functioning as accessory protein factor to enhance DCL1 activity.Beside this function, JANUS also plays essential role in splicing and modulates the transcription of a subset of genes.Given the function of SF3B4 / HSH49m in regulating translation of specific genes ( 53 ), it is possible JANUS has additional functions in plants.Through these combined functions, JANUS ensures the proper development of plants.

Figure 1 .
Figure 1.JANUS associates with DCL1 complex.(A) Protein factors identified from MYC-JANUS-associated proteins identified by mass spectrometry analysis.(B) Bimolecular fluorescence complementation (BiFC) analysis of JANUS with DCL1, HYL1, SE, AGO1 and CDC5.Paired cCFP-and nVENUS fusion proteins were co-expressed in N. benthamiana .Green color indicates the BiFC signal detected by a confocal microscopy at 48 h after infiltration.(C) Co-IP between JANUS-FLAG and MYC-SE.JANUS-FLAG or GFP-FLAG were co-expressed with MYC-SE in tobacco leaves.IPs were performed using anti-FLAG antibodies.JANUS-FLAG, FLAG and MYC-SE were detected by western blot.(D) Co-IP between JANUS-FLAG and GFP-DCL1.GFP-DCL1 or GFP-FLAG were co-expressed with JANUS-FLAG in N. benthamiana .IPs were performed using anti-GFP antibodies.JANUS-FLAG, GFP and GFP-DCL1 were detected by western blot.(E) Co-IP between truncated JANUS proteins and MYC-SE.JANUS protein contains two RNA recognition motifs (R) and a glycine-rich region (GR).Truncated JANUS proteins fused a GFP-tag at N-terminus.IPs were performed using anti-GFP antibodies.Truncated JANUS proteins and MYC-SE were detected by western blot.

Figure 2 .
Figure 2. Hypomorphic janus-2 mutation reduces miRNA accumulation.(A) SgRNA target site at JANUS gene.(B) Alignment of nucleotide sequences and sanger sequencing result at the target site in janus-2.The letters highlighted in red indicate four-bp deletion.(C) Five-week-old plants of Col-0 and junus-2 .(D) Scanning electron microscopy of Col-0 and junus-2 leaf surfaces.(E) The relative levels of miRNAs detected by RT-qPCR.miRNA levels in Col-0, junus-2 and junus-2 harboring the pJ ANUS::J ANUS-MYC transgene.The levels of miRNAs were normalized to those of U6 RNAs and compared with Col-0 (set as 1).Different letters indicate significant difference determined by ANO V A ( P < 0.05).(F) Small RNA sequencing analysis in Col-0 and junus-2 .The miRNA abundance was calculated as reads per million, and a log2-transformed ratio of junus-2 / Col-0 was plotted.(G) The levels of miRNA target transcripts in Col-0 junus-2 and junus-2 harboring the pJ ANUS::J ANUS-MYC transgene detected by RT-qPCR.The levels of miRNA target transcripts were normalized to those of UBQ5 and compared with Col-0 (set as 1).Different letters indicate significant difference determined by ANO V A ( P < 0.05).

Figure 3 .
Figure 3. JANUS is required for the transcription of pri-miRNAs.(A) The accumulation of pri-miRNAs in Col-0 and janus-2 detected by RT-qPCR.P ri-miRNA le v els in janus-2 w ere normaliz ed to those of UBQ5 and compared with Col-0 (set as 1).Error bars: standard deviations (SD) of three replicates.** P < 0.01, * P < 0.05 (Student's t test).(B) The levels of GUS in JANUS + and janus-2 harboring pMIR167a::GUS .JANUS+ : J ANUS / J ANUS , or JANUS / janus-2.(C) The transcript levels of GUS driv en b y MIR167a promoter in JANUS + and janus-2.GUS transcript le v els w ere determined b y qR T-PCR.T he GUS mRNA le v els in janus-2 w ere normaliz ed to UBQ5 and compared with those in JANUS+ .* P < 0.05, ** P < 0.01 (Student's t test).(D) The occupancy of Pol II at MIR promoters in Col-0 and amiR JANUS was detected by chromatin immunoprecipitation (ChIP) f ollo w ed b y qPCR.IP w as perf ormed using antibodies against CTD on protein extracts from Col-0 and janus-2 .The intergenic region between At2g17470 and At2g17460 (POL II C1) was amplified as a negative control.* P < 0.05, ** P < 0.01 (Student's t test).

Figure 4 .
Figure 4. JANUS binds the stem-loop region of pri-miRNAs.(A) JANUS binds pri-miRNAs in vivo .RIP assay was performed on the transgenic plants harboring p35S::MYC-JANUS using anti-MYC antibodies.After RIP, RNA was extracted and detected by RT-PCR.No Ab means no antibody control.EIF4A was used as negative control.(B) JANUS binds pri-miR1 62b, pre-miR1 62b and pre-miR172b in vitro .ssRNA: single stranded-RNA; dsRNA: double stranded-RNA.(C) Diagrams of various MIR172b constructs used for the JANUS-binding assay.miR172 is shown in red; miR172b* is shown in blue.(D-H) The interaction of JANUS with full-length and truncated MIR172b RNAs.MYC-JANUS and MIR172b were transiently co-expressed in tobacco lea v es.IP was performed with anti-MYC antibodies.NtEF1A was used as negative control.No Ab: IP without antibody.

Figure 5 .
Figure 5. JANUS is required for the maturation of miRNA, the localization of HYL1 and the interactions between HYL1 and pri-miRNAs.(A) The amount of miR162b produced from MIR162b was reduced in amiR JANUS .P roteins w ere isolated from seedlings of Col-0 and amiR JANUS and incubated with MIR162b .The reactions were stopped at various time points as indicated in the picture.(B) Quantification of miR162b production in amiR JANUS compared to that in Col-0.The radioactive signal of miR162 were normalized to input and compared with that of Col-0.The amount of miR162 produced in Col-0 was set as 1.The value represents mean of two repeats.* P < 0.05, ** P < 0.01 (Student's t test).(C) Image of HYL1 localization in the cells of root elongation region of Col-0 and janus-2 .Ten-da y -old plants w ere e xamined.(D) Quantification of root cells harboring HYL1-localized D-bodies in Col-0 and janus-2 .Over 100 cells for each genotype were examined.Error bar indicates SD. ** P < 0.01 (Student's t test).

Figure 6 .
Figure 6.JANUS affects gene expression and splicing at global levels.(A) Venn diagram showing the degree of overlap of splicing defective genes with up-regulated or down-regulated in janus-2 relative to Col-0.(B) GO enrichment of down-regulated genes in janus-2 relative to Col-0.(C) GO enrichment of up-regulated genes in janus-2 relative to Col-0.(D) Genome − wide intron Single Splicing Strength (3S) scores in Col-0 and janus-2 .The red dots indicate introns with significant differential splicing between Col-0 and janus-2 .and the gray dots indicate no significant difference.(E and F) Two examples of differentially spliced transcripts between Col-0 and janus-2 .For each transcript, the x-axis is the genomic position, the y-axes are the RNA-seq read numbers, the arcs indicate e x on-e x on junctions (introns) and the numbers are Single Splicing Strength (3S, see Methods) scores.Transcripts from Col-0 are in grey.Transcripts from janus-2 are in pink and gene str uct ures are in blue.The significant differentially spliced introns are highlighted in vertical gray box.