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Research ArticleResearch Article
MAC3A and MAC3B, Two Core Subunits of the MOS4-Associated Complex, Positively Influence miRNA Biogenesis
Shengjun Li, Kan Liu, Bangjun Zhou, Mu Li, Shuxin Zhang, Lirong Zeng, Chi Zhang, Bin Yu
Shengjun Li
Qingdao Engineering Research Center of Biomass Resources and Environment, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao 266101, ChinaCenter for Plant Science Innovation University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0666School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0118
ORCID record for Shengjun Li
Kan Liu
Center for Plant Science Innovation University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0666School of Biological Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0118
Bangjun Zhou
Center for Plant Science Innovation University of Nebraska-Lincoln, Lincoln, Nebraska 68588-0666Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68583-0722
ORCID record for Bangjun Zhou
Mu Li
Shuxin Zhang
State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Taian 271018, China
Lirong Zeng
ORCID record for Lirong Zeng
Bin Yu
ORCID record for Bin Yu
For correspondence: byu3@unl.edu
Published February 2018. DOI: https://doi.org/10.1105/tpc.17.00953
© 2018 American Society of Plant Biologists. All rights reserved.
MAC3A and MAC3B are conserved U-box-containing proteins in eukaryotes. They are subunits of the MOS4-associated complex (MAC) that plays essential roles in plant immunity and development in Arabidopsis thaliana. However, their functional mechanisms remain elusive. Here, we show that Arabidopsis MAC3A and MAC3B act redundantly in microRNA (miRNA) biogenesis. Lack of both MAC3A and MAC3B in the mac3b mac3b double mutant reduces the accumulation of miRNAs, causing elevated transcript levels of miRNA targets. mac3a mac3b also decreases the levels of primary miRNA transcripts (pri-miRNAs). However, MAC3A and MAC3B do not affect the promoter activity of genes encoding miRNAs (MIR genes), suggesting that they may not affect MIR transcription. This result, together with the fact that MAC3A associates with pri-miRNAs in vivo, indicates that MAC3A and MAC3B may stabilize pri-miRNAs. Furthermore, we find that MAC3A and MAC3B interact with the DCL1 complex that catalyzes miRNA maturation, promote DCL1 activity, and are required for the localization of HYL1, a component of the DCL1 complex. Besides MAC3A and MAC3B, two other MAC subunits, CDC5 and PRL1, also function in miRNA biogenesis. Based on these results, we propose that MAC functions as a complex to control miRNA levels through modulating pri-miRNA transcription, processing, and stability.
MicroRNAs (miRNAs), ∼21 nucleotides in size, are endogenous noncoding RNAs that mainly repress gene expression at posttranscriptional levels (Baulcombe, 2004; Axtell, 2013). They are generated from the imperfect stem-loop residing in the primary miRNA transcripts (pri-miRNAs) (Voinnet, 2009), most of which are produced by DNA-dependent RNA polymerase II (Xie et al., 2005). In plants, the RNase III enzyme DICER-LIKE1 (DCL1) slices pri-miRNAs at least two times in the nucleus to release a miRNA-containing duplex (Baulcombe, 2004; Axtell, 2013; Zhang et al., 2015). Then, the small RNA methyltransferase HUA ENHANCER1 (HEN1) methylates the miRNA duplexes to protect them from degradation and untemplated uridine addition (Zhai et al., 2013; Ren et al., 2014). Following methylation, the miRNA strand is incorporated into the effector called ARGONAUTE1 (AGO1) with the assistance from HEAT SHOCK PROTEIN90 and CYCLOPHILIN40 and recognizes target transcripts through sequence complementarity (Baumberger and Baulcombe, 2005; Vaucheret, 2008; Smith et al., 2009; Earley and Poethig, 2011). AGO1 cleaves target mRNAs or inhibits their translation and therefore represses gene expression.
pri-miRNAs may be cotranscriptionally processed since DCL1 associates with MIR loci (Fang et al., 2015a). In the past decades, protein factors that regulate miRNA biogenesis through influencing pri-miRNA transcription, processing, and stability have been identified in plants. The transcriptional coactivator MEDIATOR (Kim et al., 2011), the CYCLIN-DEPENDENT KINASEs (CDKs) (Hajheidari et al., 2012), the transcription factor NEGATIVE ON TATA LESS2 (NOT2) (Wang et al., 2013), the DNA binding protein CELL DIVISION CYCLE5 (CDC5) (Zhang et al., 2013), and ELONGATOR (Fang et al., 2015a) are required for optimized Pol II activity at the MIR promoters. Following transcription, the forkhead domain-containing protein DAWDLE (DDL) (Yu et al., 2008) and the WD-40 protein PLEIOTROPIC REGULATORY LOCUS1 (PRL1) (Zhang et al., 2014) bind pri-miRNAs to prevent their degradation.
To efficiently and accurately process pri-miRNAs, DCL1 forms a complex with the double-stranded RNA (dsRNA) binding protein HYPONASTIC LEAVES1 (HYL1), the zinc-finger protein SERRATE (SE), and the RNA binding protein TOUGH (TGH) (Fang and Spector, 2007; Fujioka et al., 2007; Song et al., 2007; Dong et al., 2008; Ren et al., 2012). The formation of the DCL1 complex requires NOT2 (Wang et al., 2013), ELONGATOR (Fang et al., 2015a), MODIFIER OF SNC1, 2 (MOS2; an RNA binding protein) (Wu et al., 2013), and the DEAH-box helicase PINP1 (Qiao et al., 2015). How MOS2 and PINP1 participate in the assembly of the DCL1 complex remains unclear, since they do not interact with the DCL1 complex (Wu et al., 2013; Qiao et al., 2015). Efficient loading of pri-miRNAs to the DCL1 complex requires TGH (Ren et al., 2012), the THO/TREX complex that is involved in nuclear RNA transport (Francisco-Mangilet et al., 2015), and the ribosome protein STV1 (Li et al., 2017). Notably, several additional proteins, including the CAP BINDING PROTEINs (CBPs) (Gregory et al., 2008; Laubinger et al., 2008), NOT2, ELONGATOR, DDL, CDC5, and PRL1 also associate with the DCL1 complex to enhance pri-miRNA processing. In addition, SICKLE (SIC; a proline-rich protein) (Zhan et al., 2012), RECEPTOR FOR ACTIVATED C KINASE1 (RACK1) (Speth et al., 2013), STABILIZED1 (STA1; a pre-mRNA processing factor 6 homolog) (Ben Chaabane et al., 2013), REGULATOR OF CBF GENE EXPRESSION3 (RCF3; also known as HOS5 and SHI1) (Chen et al., 2015; Karlsson et al., 2015), and GRP7 (a glycine-rich RNA binding protein) (Köster et al., 2014) also regulate miRNA biogenesis. However, they do not associate with DCL1. Moreover, phosphorylation and dephosphorylation of HYL1 are crucial for pri-miRNA processing (Manavella et al., 2012). In addition, protein factors that act in miRNA biogenesis are also transcriptionally and posttranscriptionally regulated. For instance, DCL1 transcription is modulated by the histone acetyltransferase GCN5 (Kim et al., 2009), STA1 (Ben Chaabane et al., 2013), and the transcription factor XAP5 CIRCADIAN TIMEKEEPER (XCT) (Fang et al., 2015b). Notably, HYL1 protein levels are maintained by the SNF1-RELATED PROTEIN KINASE2 (Yan et al., 2017) and the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) (Cho et al., 2014) through unknown mechanisms. Recently, KETCH1 (KARYOPHERIN ENABLING THE TRANSPORT OF THE CYTOPLASMIC HYL1)-mediated transportation of HYL1 from the cytoplasm to the nucleus was shown to be crucial for miRNA biogenesis (Zhang et al., 2017). Interestingly, pri-miRNA structures also influence the DCL1 activity (Mateos et al., 2010; Song et al., 2010; Werner et al., 2010; Bologna et al., 2013; Zhu et al., 2013). For instance, the internal loop below the miRNA/miRNA* within the stem-loop is important for the processing of some pri-miRNAs.
Among proteins associated with the DCL1 complex, CDC5 and PRL1 are two core subunits of the MOS4-assoicated complex (MAC) (Monaghan et al., 2009). MAC is a conserved complex that associates with the spliceosome (Deng et al., 2016). Its homolog complexes in human and yeast are known as the CDC5-SNEVPrp19-Pso4 (PRP19) complex and the Nineteen complex (NTC), respectively (Palma et al., 2007). Both PRP19 and NTC function in splicing, DNA repair, the cell cycle, and genome stability (Chanarat and Straßer, 2013). MAC contains three additional core subunits, MAC3A, MAC3B, and MOS4, and at least 13 accessory proteins with diversified functions (Monaghan et al., 2009). Deficiency in MAC impairs plant immunity and development (Monaghan et al., 2009). However, related mechanisms still need investigation. We have previously shown that CDC5 and PRL1 have overlapping roles in regulating DCL1 activity, but distinct functions in pri-miRNA transcription and stability (Zhang et al., 2013, 2014). These results raise the possibility that other MAC components may also have diversified effects on miRNA biogenesis. Among core MAC components, MAC3A and MAC3B are two homologous U-box type E3 ubiquitin ligases (∼82% identity and 90% similarity) (Monaghan et al., 2009). E3 ligase activity of MAC3B has been demonstrated in vitro (Wiborg et al., 2008). We previously showed that a loss-of-function mutation in MAC3A does not affect miRNA accumulation (Zhang et al., 2014). However, this result may reflect the redundant function of MAC3B with MAC3A.
In this study, we found that lack of both MAC3A and MAC3B reduces the accumulation of miRNAs and impairs the localization of HYL1 in the D-body. MAC3A associates with the DCL1 complex and pri-miRNAs and promotes pri-miRNA processing. MAC3A and MAC3B are also required for accumulation of pri-miRNAs. However, unlike CDC5, MAC3A neither interacts with Pol II nor affects MIR transcription. These results suggest that MAC3A/3B may stabilize pri-miRNAs and act as a cofactor to promote D-body formation and pri-miRNA processing. In addition, we show that MAC3A is a phosphorylation-dependent E3 ligase and its E3 ligase activity is required for miRNA biogenesis. We propose that MAC may act as a complex to promote miRNA biogenesis and different MAC components may have distinct and cooperative effects on pri-miRNA transcription, stability, and processing.
MAC3A and MAC3B Are Required for miRNA Biogenesis
The fact that CDC5 and PRL1, two core components of MAC, are required for miRNA biogenesis suggests that other MAC components may also function in miRNA biogenesis. However, we previously showed that a single mac3a mutation does not affect miRNA accumulation in Arabidopsis thaliana (Zhang et al., 2014). To evaluate if this result might reflect redundancy between MAC3A and MAC3B in miRNA biogenesis, we generated a mac3a mac3b double mutant through crossing mac3a (Salk_089300) to mac3b (Salk_050811) (Monaghan et al., 2009). Compared with Col (wild-type plant), mac3a mac3b displayed pleiotropic development defects (Figure 1). For instance, the root length of mac3a mac3b is much shorter (Figures 1A and 1I). Moreover, the size of the mac3a mac3b was smaller (Figure 1B). Reduced cell number was likely responsible for the smaller size of mac3a mac3b, since the size of palisade cells from mac3a mac3b was comparable to that from Col (Figures 1C, 1D, and 1J). In addition, mac3a mac3b leaves had three to four branch points (four to five branches) on average, while most trichomes of Col had two branch points (three branches) (Figures 1E, 1F, and 1K). Furthermore, the silique length of mac3b mac3b was shorter than that of Col (Figures 1G and 1L). Moreover, the amounts of aborted seeds were higher in the siliques of mac3a mac3b than those of the wild type (Figures 1H and 1M), suggesting that MAC3A and MAC3B also affect fertility.
mac3a mac3b Displays Pleiotropic Developmental Defects.
(A) Seven-day-old seedlings of Col and mac3a mac3b. Bar = 5 mm.
(B) Three-week-old Col and mac3a mac3b. Bar = 1cm.
(C) and (D) Palisade cells of the fifth leaves from Col (C) and mac3a mac3b (D). Bar = 100 μm.
(E) and (F) Trichomes of Col and mac3a mac3b. Bar = 0.3 mm.
(G) Mature siliques of Col and mac3a mac3b. Bar = 2mm.
(H) Dissected siliques of Col and mac3a mac3b. White arrow: aborted seeds.
(I) Quantification of root length in Col and mac3a mac3b. Thirty plants were measured to calculate average root length. **P < 0.01 by Student's t test.
(J) Quantification of cell size of the fifth leaves in Col and mac3a mac3b. The value of Col was set as 100. Eighty cells of the fifth leaves from each genotype were measured.
(K) Quantification of trichome branches in Col and mac3a mac3b. Sixty trichomes from each genotype were analyzed. Numbers (3, 4, or 5) indicate the number of branches.
(L) Quantification of silique length in Col and mac3a mac3b. Thirty siliques from the same position of each genotype were measured. **P < 0.01 by Student's t test.
(M) Quantification of aborted seeds in Col and mac3a mac3b. Twenty siliques from Col or mac3a mac3b were analyzed. **P < 0.01 by Student's t test, compared with Col-0 value. Error bars in (I) to (M) indicate sd.
The pleiotropic growth defects of mac3a mac3b are consistent with the effect of miRNAs on plant development; we therefore examined the accumulation of miRNAs in mac3a mac3b and Col through RNA gel blot. The abundance of all nine examined miRNAs was reduced in mac3a mac3b relative to Col (Figure 2A). RT-qPCR analyses further confirmed that miRNA levels were decreased in mac3a mac3b (Figure 2B). We also examined the effect of MAC3A and MAC3B on trans-acting siRNAs (ta-siRNAs), which is another class of sRNAs that represses gene expression at posttranscriptional levels (Peragine et al., 2004; Allen et al., 2005; Yoshikawa et al., 2005; Axtell et al., 2006). Similar to miRNAs, ta-siR255 was reduced in abundance in mac3a mac3b (Figure 2A). However, the effect MAC3A and 3B on ta-siR255 might be indirect, since the production of ta-siRNAs depending on miRNAs, whose abundance was reduced in mac3a mac3b.
mac3a mac3b Reduces the Accumulation of miRNAs.
(A) The levels of small RNAs in Col and mac3a mac3b detected by RNA gel blot. U6 RNA serves as the loading control. The numbers shown below the picture indicates the amount of small RNAs in mac3a mac3 relative to that of Col (set as 1) and represent mean of three replicates (P < 0.05). miR159/319: the upper band was miR159 and the lower band showed miR319.
(B) The levels of miRNAs detected by RT-qPCR. miRNA levels in mac3a mac3b were normalized to those of U6 RNA and compared with Col (value set as 1). Error bars indicate sd of three replicates (*P < 0.05 and **P < 0.01 by Student's t test, compared with Col-0 value).
(C) The transcript levels of miRNA targets in Col and mac3a mac3b detected by RT-qPCR. The transcript levels of miRNA targets were normalized to those of UBIQUITIN5 (UBQ5) and compared with Col (set as 1). Error bars indicate sd of three replicates (**P < 0.01 by Student's t test, compared with Col-0 value).
We further compared miRNA profile from inflorescences of mac3a mac3b with that of the wild type through deep sequencing. The abundance of many miRNAs was reduced in mac3a mac3b relative to the wild type (Supplemental Figure 1A and Supplemental Data Set 1), suggesting that MAC3A MAC3B may have a global effect on miRNA accumulation. We also compared the effect of mac3a mac3b on miRNA accumulation with that of dcl1-9 (a weak allele of dcl mutants) and cdc5. As expected, cdc5 and dcl1-9 reduced the abundance of most miRNAs (Supplemental Figures 1B and 1C and Supplemental Data Set 1). Among significantly downregulated miRNAs (P < 0.1), DCL1, CDC5, and MAC3A/MAC3B showed overlapping effects on many of them (Supplemental Figure 1D). However, some miRNAs were differentially affected by DCL1, CDC5, and MAC3A/MAC3B (Supplemental Figure 1D). These results suggest that these proteins may have overlapping and distinct roles in miRNA biogenesis.
Next, we evaluated the influence of mac3a mac3b on the transcript levels of ARF4, ARF8, CKB3, CUC1, MYB33, PHO2, PHV, PPR, and SPL9/10/13, which are targets of tasiR-ARF, miR167, miR397, miR164, miR159, miR399, miR166, miR400, and miR156, respectively. The levels of these target transcripts were increased in mac3a mac3b compared with Col (Figure 2C), suggesting that MAC3A and 3B are required for optimal activity of miRNAs and ta-siRNAs.
To determine if the lack of MAC3A and MAC3B was responsible for the observed phenotypes, we expressed a genomic copy of MAC3A fused with a GUS gene at its 3ʹ end under the control of its native promoter (proMAC3A:MAC3A-GUS) in mac3a mac3b. The expression of this transgene rescued the developmental defects of mac3a mac3b (Supplemental Figure 2A). In addition, fusion constructs MAC3B-GFP (pro35S:MAC3B-GFP) or MYC-MAC3A (pro35S:MYC-MAC3A) under the control of the 35S promoter also complemented the developmental defects of mac3a mac3b (Supplemental Figures 2B and 2J). Consistent with this observation, miRNA and target transcript levels in the complementation lines were comparable to those in Col (Supplemental Figures 2K and 2L). We also examined the expression pattern of MAC3A in mac3a mac3b harboring proMAC3A:MAC3A-GUS through GUS histochemical staining. MAC3A was universally expressed and displayed high expression levels in primary root tip, lateral root, and young leaves (Supplemental Figures 2C to 2I). These results demonstrate that MAC3A and MAC3B act redundantly to control development and miRNA accumulation of Arabidopsis.
MAC3A and MAC3B Do Not Affect MIR Transcription
We have previously shown that CDC5 and PRL1 regulate pri-miRNA levels through modulating pri-miRNA transcription and stability, respectively (Zhang et al., 2013, 2014). This led us to test if pri-miRNA levels were also altered in mac3a mac3b. As expected, all examined pri-miRNAs were reduced in abundance in mac3a mac3b compared with Col (Figure 3A). We suspected that as in cdc5, this reduction could be caused by alteration in transcription. Thus, we evaluated the effect of mac3a mac3b on MIR promoter activity. The MIR promoter reporter construct, pMIR167a:GUS (Zhang et al., 2014), was crossed into mac3a mac3b. Histochemical staining and RT-qPCR analyses revealed that the expression levels of GUS in mac3a mac3b were similar to those in the wild type (Figures 3B and 3C), indicating that MAC3A and MAC3B may have no effect on MIR promoter activity. Furthermore, we tested the interaction between MAC3A and the second largest subunit of Pol II (RPB2) through coimmunoprecipitation assay (co-IP) in the mac3a mac3b expression pro35S:MAC3A-GFP. In MAC3A-GFP precipitates, we did not detect the presence of RPB2 (Figure 3D), suggesting that unlike CDC5 and PRL1, MAC3A does not associate with RPB2. We also examined the occupancy of Pol II at the MIR promoters through chromatin immunoprecipitation (ChIP) assays in mac3a mac3b and Col performed using anti-RPB2 antibody. qPCR analysis did not detect an obvious difference of Pol II occupancy at various MIR promoters between mac3a mac3b and Col (Figure 3E). Taken together, these results suggest that MAC3A and MAC3B do not affect MIR transcription.
mac3a mac3b Reduces the Accumulation of pri-miRNAs without Affecting Transcription.
(A) The levels of pri-miRNAs in Col and mac3a mac3b. RT-qPCR was used to analyze pri-miRNA levels. pri-miRNA levels in mac3a mac3b were normalized to those of UBQ5 and compared with Col (values were set as 1). Error bars indicate sd of three replicates (*P < 0.05 and **P < 0.01 by Student's t test, compared with Col-0 value).
(B) Histochemical staining of GUS in Col and mac3a mac3b harboring proMIR167a:GUS. Fifteen plants containing GUS were analyzed for each genotype. A representative image for each genotype is shown. Bar = 2 mm.
(C) GUS transcript levels in Col and mac3a mac3b harboring the proMIR167a:GUS transgene. GUS transcript levels detected by RT-qPCR were normalized to those of UBQ5 and compared with Col (value was set as 1). Error bars indicate sd of three replicates.
(D) MAC3A does not coimmunoprecipitate with RPB2. Anti-GFP antibody was used to immunoprecipitate MAC3A-GFP. MAC3A-GFP and RPB2 were detected with anti-GFP and anti-RPB2 antibodies, respectively. Input, total proteins before IP; NoAb, immunoprecipitates with agarose beads.
(E) The occupancy of Pol II at MIR promoters in Col and mac3a mac3b detected by ChIP followed by qPCR. The intergenic region between At2g17470 and At2g17460 (POL II C1) was used as a negative control.
MAC3A and MAC3B Associate with the DCL1 Complex
To further understand how MAC3A and MAC3B affect miRNA biogenesis, we examined the effect of mac3a mac3b on the expression of DCL1, DDL, SE, HYL1, CBP20/80, and HEN1, which are known to function in miRNA biogenesis. The transcript levels of HYL1 and CBP20/80 were slightly increased, while the abundance of DDL transcripts was marginally reduced (Supplemental Figure 3A). In addition, the levels of DCL1, HEN1, and SE did not show significant change. Immunoblot analyses further showed that the protein levels of SE and DCL1 were not changed in mac3a mac3b, whereas the HYL1 protein was slightly increased in abundance (Supplemental Figure 3B). Moreover, we also examined the effect of mac3a mac3b on the splicing of DCL1, DDL, HEN1, HYL1, and SE using RT-PCR with primers targeting a subset of introns (Supplemental Figure 3C). MAC3A and MAC3B did not have an obvious effect on the splicing of these introns (Supplemental Figures 3C and 3D). However, it is not clear if MAC3A and MAC3B affect the splicing of other introns in these examined genes.
Since MAC3A and MAC3B are components of the MAC, we suspected that like CDC5 and PRL1, MAC3A, and MAC3B might also interact with the DCL1 complex. We performed a bimolecular fluorescence complementation (BiFC) assay to test this possibility. In the leaves of Nicotiana benthamiana transiently coexpressing MAC3A or MAC3B fused with the C-terminal fragment of cyan fluorescent protein (cCFP) with CDC5, PRL1, DCL1, or SE fused with the N-terminal fragment of Venus (nVenus), yellow fluorescence signals were observed (shown in green color; Figure 4A; Supplemental Figure 4). BiFC signals of MAC3A or MAC3B with PRL1, DCL1, and SE were localized at the discrete bodies (Figure 4A; Supplemental Figure 4). Interestingly, the interaction between MAC3A/3B and CDC5 produced not only discrete signals but also diffused ones, agreeing with the role of MAC in mRNA splicing (Figure 4A; Supplemental Figure 4). Coexpression cCFP-MAC3A or cCFP-3B with nVenus-HYL1 resulted in weak and diffused YFP signals (Figure 4A; Supplemental Figure 4), consistent with the observation that CDC5 and PRL1 do not coimmunoprecipitate with HYL1 (Zhang et al., 2014).
MAC3A Associates with the DCL1 Complex.
(A) The interaction of MAC3A with CDC5, PRL1, DCL1, HYL1, SE, and AGO1 detected by BiFC analysis. Paired cCFP- and nVenus-fusion proteins were coexpressed into N. benthamiana leaves. Green color indicates the BiFC signal (originally yellow fluorescence) detected by a confocal microscopy at 48 h after infiltration. One hundred nuclei were examined for each pair and a representative image is shown. Red: autofluorescence of chlorophyll.
(B) Co-IP between MAC3A and CDC5.
(C) Co-IP between MAC3A and PRL1. CDC5-YFP, PRL1-YFP or YFP were coexpressed with MYC-MAC3A in N. benthamiana. IP was performed using anti-YFP antibodies. MYC-MYC3A, CDC5-YFP, PRL1-YFP, and YFP were detected by immunoblot.
(D) Co-IP between MAC3A and DCL1.
(E) Co-IP between MAC3A and SE. GFP or MAC3A-GFP was coexpressed in N. benthamiana with MYC-DCL1 or MYC-SE, respectively. IP was performed using an anti-MYC antibody. After IP, proteins were detected by immunoblot. Inputs in (B) to (E) show the total protein before IP. RNase A was used to digest RNA stands.
Next, we used co-IP to confirm the interaction of MAC3A with CDC5, PRL1, DCL1, and SE. We first coexpressed MYC-MAC3A with CDC5-YFP, PRL1-YFP, or YFP and performed IP with anti-YFP antibodies. MYC3A was detected in CDC5-YFP and PRL1-YFP precipitates, but not in YFP precipitates (Figures 4B and 4C), confirming the interaction of MAC3A with CDC5 and PRL1. We next coexpressed MAC3A-GFP or GFP with MYC-DCL1 or MYC-SE and tested the interaction of coexpressed proteins. MAC3A-YFP, but not YFP, coimmunoprecipitated with MYC-DCL1 and MYC-SE (Figures 4D and 4E). Furthermore, RNase A treatment did not disrupt the interaction of MAC3A with DCL1 and SE (Figures 4B to 4D). These results suggest that MAC3A and MAC3B associate with the DCL1 complex in an RNA-independent manner.
mac3a mac3b Reduces pri-miRNA Processing in Vitro
The association of MAC3A and MAC3B suggests that they may modulate DCL1 activity. We used an in vitro pri-miRNA processing assay to test this possibility. As previously described, we first generated a radiolabeled pri-miR162b (MIR162b) composed of the stem-loop of miR162b with 6-nucleotide arms at each end using in vitro transcription (Figure 5A). Processing of MIR162b was then tested in the protein extracts from young flowers of mac3a mac3b or Col. The production of miR162b from MIR162b was reduced in the protein extracts of mac3a mac3b relative to Col (Figure 5B). At 50- and 100-min time points, the levels of miR162 generated in mac3a mac3b were ∼20% of those produced in Col (Figure 5C). These results suggest that MAC3A/3B may be required for the optimal activity of the DCL1 complex.
MAC3A Associates with pri-miRNAs and Promotes pri-miRNA Processing.
(A) Diagram of MIR162b used in the in vitro processing assay.
(B) MIR162b processing in protein extracts from mac3a mac3b and Col.
(C) Quantification of miR162 production in mac3a mac3b extracts relative to Col. The processing reaction was performed for 50 or 100 min. The radioactive signal of miR162 was normalized to input in mac3a mac3b and compared with that in Col (value set as 1). The value is the mean of three repeats. **P < 0.01 by Student's t test, compared with Col-0 value.
(D) MAC3A interacts with pri-miRNAs in vivo. RIP assay was performed on transgenic plants harboring pro35S:MYC-MYC3A using an anti-MYC antibody. Five percent RNAs were used as the input. NoAb: No antibody control.
(E) MAC3A does not bind RNAs in vitro. pri-miR162b, pre-miR162b, ssRNA, and dsRNA were generated through in vitro transcription.
MAC3A Binds pri-miRNAs in Vivo
The WD domain of MAC3A and MAC3B is known to mediate protein-protein interaction. However, it can also interact with RNAs (Lau et al., 2009). Thus, it is possible that MAC3A and MAC3B could bind pri-miRNAs. To test this hypothesis, we performed an RNA immunoprecipitation assay (RIP) on seedlings of the mac3a mac3b complementation line harboring the MYC-MAC3A transgene (Ren et al., 2012). Following cross-linking, nuclear isolation, and immunoprecipitation, we examined the presence of pri-miRNAs in MAC3A IPs using RT-PCR. All examined pri-miRNAs, but not the control EIF4A RNAs, were enriched in the MAC3A IPs (Figure 5D). By contrast, pri-miRNAs were not detected in the no-antibody controls (Figure 5D). These results suggest that MAC3A/3B associates with pri-miRNAs in vivo.
Next, we tested if MAC3A could directly bind pri-miRNA in vitro using the RNA pull-down assay (Ren et al., 2012). In this assay, MBP and recombinant MAC3A fused with maltose binding protein (MBP) at its N terminus (MBP-MAC3A) were expressed in Escherichia coli, purified with amylose resin, and then incubated with [32P]-labeled MIR162b (Supplemental Figure 5A; Figure 5E). After washing, neither MBP-MAC3A nor MBP retained MIR162b (Figure 5E). MBP-MAC3A also did not interact with an ∼100-nucleotide single-stranded RNA (ssRNA), which was generated through in vitro transcription using a N-terminal fragment of the UBIQUITIN5 (N-UBQ5), or a dsRNA generated through annealing of sense and antisense strands of N-UBQ5 (Figure 5E). Because MAC3A activity needs phosphorylation (see below), we treated the recombinant MAC3A protein with extracts from Col (see below) to modify the protein and then tested its interaction with MIR162b. The modified MAC3A also did not interact with RNAs (Supplemental Figure 5B). These results suggest that MAC3A is not an RNA binding protein.
MAC3A and MAC3B Are Required for the Localization of HYL1 in D-Bodies
The interaction of MAC3A/B with the DCL1 complex also prompted us to test the effect of mac3a mac3b on the formation of the D-body. We crossed a HYL1-YFP transgenic line, which has been used as a reporter for the D-body (Wang et al., 2013; Wu et al., 2013; Qiao et al., 2015), into mac3a mac3b and examined the percentage of cells containing D-bodies in the root tips and elongation region. As previously reported (Wu et al., 2013), the HYL1-containing D-bodies existed in most cells (∼84%; Figures 6A and 6B; Supplemental Figures 6A and 6B) in the wild type. By contrast, D-bodies were observed in only ∼26% of cells in mac3a mac3b. This result demonstrates that MAC3A and MAC3B are required for correct HYL1 localization, indicating their potential role in facilitating D-body formation.
mac3a mac3b Affects the Localization of HYL1 in the Nucleus.
(A) Image of HYL1 localization in the root cells of Col and mac3a mac3b. Seven-day-old plants were examined. A typical image is shown. Arrows indicate the D-bodies. Bar = 5 μm.
(B) Quantification of root cells harboring HYL1-localized D-bodies in Col and mac3a mac3b. More than 400 cells from 12 roots for each genotype were examined. Error bar indicates sd (n = 400). **P < 0.01 by Student's t test, compared with Col-0 value.
MAC3A Is a U-Box Ubiquitin E3 Ligase Whose Activity Depends on Phosphorylation
Both MAC3A and MAC3B contain an N-terminal ligase U-box domain that confers E3 ubiquitin ligase activity and recruits the E2 conjugating enzyme, a coiled-coil region that exists in all Prp19 homologs, mediates the tetramerization of Prp19 and interacts with CDC5L and SFP27 in metazoans, and a C-terminal WD domain composed of seven WD repeats that is required for substrate recruitment (Figure 7A). Homologous of MAC3A and MAC3B exist in all plants, while their copy numbers vary among different genomes (Supplemental Figure 7 and Supplemental Data Set 2).
MAC3A Is a Bona Fide U-Box Ubiquitin E3 Ligase.
(A) The protein domains of MAC3A and MAC3B.
(B) Ubiquitin ligase activity of MAC3A. The in vitro ubiquitin ligase activity assay was performed in the presence of FLAG-ubiquitin (FLAG-Ub) and recombinant E1 and E2. MBP and no E3 ligase protein serve as negative controls. Polyubiquitination of MAC3A demonstrates its ubiquitin ligase activity. Anti-FLAG antibody and anti-MBP antibody were used to detect FLAG-ubiquitin and MBP/MBP-MAC3A, respectively. mac3a mac3b extracts: protein extracts from inflorescences of mac3a mac3b.
Because MAC3A has considerable sequence difference from MAC3B, we tested if it is a ubiquitin E3 ligase using MBP-MAC3A (Supplemental Figure 5A). We examined the E3 ligase activity in the presence of ubiquitin, the ubiquitin-activating enzyme (E1) SlUBA, and the ubiquitin-conjugating enzyme (E2) UBC8 (Zhou et al., 2017). However, MBP-MAC3A displayed only weak activity (Figure 7B). We suspected that like some other E3 ligases, MAC3A activity might depend on posttranslational modification (Wang et al., 2015). Thus, we treated MBP-MAC3A and MBP protein with total protein extracts from inflorescences of mac3a mac3b. The use of mac3a mac3b was to avoid contamination from endogenous MAC3A/3B, since MAC3A potentially interacts with MAC3B. The treatment greatly improved MAC3A activity (Figure 7B). Notably, alkaline phosphatase (calf intestinal phosphatase [CIP]) treatment of MAC3A after incubation with mac3a mac3b protein extracts completely eliminated MAC3A activity (Figure 7B). These results demonstrate that MAC3A is a bona fide ubiquitin E3 ligase and that its activity depends on protein phosphorylation.
The Ubiquitin Ligase Activity of MAC3A Is Required for miRNA and pri-miRNA Accumulation
Since MAC3A is a ubiquitin ligase, we next asked if its function in miRNA biogenesis requires this activity. Based on the fact that the U-box domain of Prp19-like family is conserved in eukaryotes (Ohi et al., 2003), we generated two mutant versions of MAC3A in U-box domain through site-directed mutagenesis. In one mutant, the conserved amino acids of tyrosine (Y) at position 23 and glutamic acid (E) at position 24 were replaced with glycine (G) and alanine (A) (MAC3AMut1), respectively, while in the other one, the conserved amino acids of histidine (H) at position 31 and aspartic acid (D) at position 34 were replaced with alanines (AA) (MAC3AMut2) (Figure 8A). These two mutations disrupted the ubiquitin ligase activity of MAC3A (Supplemental Figure 8A). To evaluate the effect of MAC3AMut1 and MAC3AMut2 on miRNA biogenesis, we generated stable transgenic lines in mac3a mac3b expressing MAC3AMut1 or MAC3AMut2 under the control of 35S promoter. The expression MAC3AMut1 or MAC3AMut2 did not rescue the developmental defects of mac3a mac3b (Supplemental Figure 8B; Figure 8B). Agreeing with this observation, the accumulation of both pri-miRNAs and miRNAs in mac3a mac3b was not recovered by MAC3AMut1 or MAC3AMut2 (Figures 8C and 8D). These results suggest that the ubiquitin ligase activity of MAC3A is required for miRNA biogenesis.
E3 Ubiquitin Ligase Activity Is Required for MAC3A Function in miRNA Biogenesis.
(A) The aligned sequences of conserved U-box domain in MAC3A orthologs. The two mutated sites are shown in blue and red boxes, respectively.
(B) Fourteen-day-old seedlings of Col, mac3a mac3b, and transgenic mac3a mac3b harboring pro35S:MAC3AMut1-GFP or pro35S:MAC3AMut2-GFP constructs. Two individual transgenic lines of each construct are shown. The transgenic line harboring pro35S:MAC3A-GFP is shown as control. Bar = 1 cm.
(C) and (D) The pri-miRNA (C) and miRNA (D) levels in Col, mac3a mac3b, 35S:MAC3AMut1-GFP transgenic plants, and pro35S:MAC3AMut2-GFP transgenic plants detected by RT-qPCR. pri-miRNA levels in mac3a mac3b and transgenic plants were normalized to those of UBQ5 and compared with Col (values were set as 1). miRNA levels in mac3a mac3b and transgenic plants were normalized to those of U6 RNA and compared with Col (value set as 1). Error bars indicate sd of three replicates (*P < 0.05 and **P < 0.01 by Student's t test, compared with Col-0 value).
MAC3A and MAC3B are conserved U-box type ubiquitin E3 ligases. In plants, MAC3A and MAC3B play important roles in plant immunity and development, and their counterparts in other organisms are required for splicing. In Arabidopsis, the MAC also associates with the spliceosome. However, only a few genes display moderated splicing in defects in mac3a mac3b (Monaghan et al., 2010; Xu et al., 2012). Consequently, how MAC3A and MAC3B regulate development and immunity remains elusive. In this study, we show that the accumulation of miRNA is reduced in mac3a mac3b. Furthermore, MAC3A and MAC3B associate with the DCL1 complex and pri-miRNAs. These results suggest that MAC3A and MAC3B are important players in miRNA biogenesis, in addition to their role in splicing. Impaired miRNA biogenesis may partially explain the pleiotropic developmental defects of mac3a mac3b, since miRNAs target many genes that are required for proper development.
There are at least three possible explanations for the decreased pri-miRNA levels in mac3a mac3b. First, mac3a mac3b may have reduced MIR transcription. The facts that mac3a mac3b does not show altered MIR promoter activity and that MAC3A does not co-IP with Pol II suggest that MAC3A and MAC3B may not affect MIR transcription. However, we cannot rule out the possibility that MAC3A and MAC3B influence MIR elongation or termination. Second, enhanced pri-miRNA processing in mac3a mac3b may also decrease pri-miRNA accumulation. However, reduced pri-miRNA processing is observed in mac3a mac3b, arguing against this possibility. Third, mac3a mac3b may have reduced stability of pri-miRNAs (Figure 9). We give this option more weight, given the observations that MAC3A associates with pri-miRNAs in vivo and interacts with PRL1, which protects pri-miRNAs from degradation. It is reasonable to speculate that MAC3A may stabilize pri-miRNAs through modulating the function of PRL1. Indeed, it has been observed that the interaction between PRP19 (a MAC3A ortholog) and the RNA binding protein CWC2 is required for the stabilization of small nuclear RNAs related to splicing in yeast (McGrail et al., 2009; Vander Kooi et al., 2010).
Proposed Model for MAC Function in miRNA Biogenesis.
MAC is required for pri-miRNA transcription, processing, and stability. Some MAC components such as CDC5 interact with the MIR promoter and Pol II to positively regulate MIR transcription. Following transcription, MAC binds pri-miRNAs to prevent their turnover and functions as a cofactor to promote pri-miRNA processing. The MAC may also facilitate the recruitment of the DCL1 complex to the processing sites. Because the MAC contains subunits with diversified functions, individual MAC components may contribute distinctly and synergistically to miRNA biogenesis. Lack of the MAC results in reduced pri-miRNA transcription, stability, and/or processing.
MAC3A/MAC3B interacts with DCL1 and SE but appears to have weak or no association with HYL1. Interestingly, a lack of MAC3A and MAC3B impairs the localization of HYL1 at the D-body. How does this happen? One possibility is the decreased pri-miRNAs in mac3a mac3b may affect the formation of D-body. However, loss-of-function mutants mos2 and pinp1, in which the levels of pri-miRNAs are increased or unaltered, respectively, also display impaired HYL1 localization or D-body assembly, arguing against this possibility. In human, PRP19-mediated ubiquitination regulates the protein-protein interaction of the spliceosome, which is important for the spliceosome assembly (Das et al., 2017). In addition, PRP19 also promotes the recruitment of ATRIP (a kinase) to the DNA damage site through modifying DNA replication protein A (Maréchal et al., 2014). Thus, it is possible that MAC3A and MAC3B may influence the recruitment of HYL1 through modifying proteins involved in D-body assembly. Alternatively, they may cotranscriptionally facilitate the recruitment of the D-body to the processing site of pri-miRNAs (Figure 9). The association of MAC3A/3B with the DCL1 complex is consistent with these hypotheses.
pri-miRNA processing is also reduced in mac3a mac3b. This cannot be attributed to altered expression of genes involved in miRNA biogenesis, as the levels of these genes are either slightly increased or unaltered in mac3a mac3b. We have shown the CDC5 promotes DCL1 activity through its interaction with the regulatory domains of DCL1 (Zhang et al., 2013), while PRL1 functions an accessory factor to facilitate CDC5 function in modulating DCL1 activity (Zhang et al., 2014). By analogy, MAC3A and MAC3B may function as components of the MAC to directly or indirectly enhance the DCL1 activity (Figure 9). Alternatively, impaired HYL1 localization or D-body formation may affect the DCL1 activity.
In summary, we find that MAC3A and MAC3B, two core components of the MAC, act redundantly in miRNA biogenesis. They associate with the DCL1 complex, positively modulate pri-miRNA accumulation, facilitate HYL1 localization at the D-body, and enhance DCL1 activity. More importantly, we show that MAC3A is a phosphorylation-dependent ubiquitin ligase and that this ligase activity is required for miRNA biogenesis. This result indicates that certain signals may modulate MAC3A activity through phosphorylation and thereby regulate miRNA accumulation. The involvement of four MAC core components in miRNA biogenesis suggests that the MAC functions as a complex to promote miRNA biogenesis.
Besides core components, the MAC also contains at least 13 accessory components. The core and accessory components of the MAC are proteins with diversified functions, such as transcription factors, RNA binding proteins, ubiquitin ligase, helicases, chromatin protein, WD proteins, protein-protein interaction regulators, coiled-coil domain-containing proteins, and zinc-finger-domain-containing proteins. Moreover, the accessory components are dynamically associated with the core complex, and subcomplexes with different functions are often formed. Thus, it is likely that various MAC components act individually and coordinately in miRNA biogenesis through influencing pri-miRNA transcription, processing, and stability and/or likely have a role in the assembly of D-body (Figure 9), which resembles the diversified function of PRP19 in splicing. Consistent with this notion, CDC5 and PRL1 contribute differently to pri-miRNA accumulation but act as a complex to regulate DCL1 activity. It will be interesting to further determine the functional mechanism of these proteins as individual components and as a complex in miRNA biogenesis. The functions of the PRP19 complex from metazoans in splicing, transcription, chromatin stability, and lipid droplet biogenesis have been well documented (Chanarat and Straßer, 2013). However, its function in metazoan miRNA biogenesis is unknown. Given the fact that all four MAC components associate with SE, an ortholog of ARS2, which is a key component of miRNA biogenesis in metazoa, it will not be surprising if the PRP19 complex plays a role in metazoan miRNA biogenesis.
Plant Materials and Growth Conditions
SALK_089300 (mac3a) (Monaghan et al., 2009) and SALK_050811 (mac3b) were obtained from the ABRC. They are in the Columbia (Col) genetic background. Transgenic lines containing a single copy of proMIR167a:GUS or pro35S:HYL1-YFP were crossed to mac3a mac3b. In the F2 generation, wild-type plants or mac3a mac3b harboring proMIR167a:GUS or pro35S:HYL1-YFP were selected through PCR-based genotyping for mac3a, mac3b, GUS, or GFP. Approximately fifteen wild-type or mac3a mac3b plants were pooled for GUS transcript level analyses. All plants were grown at 22°C with 16-h-light (cool white fluorescent lamps, 25-W Sylvania 21942 FO25/741/ECO T8 linear tube) and 8-h-dark cycles.
Plasmid Construction
A DNA fragment containing 2066-bp promoter and 3841-bp coding region of MAC3A was PCR amplified using DNAs from Col as templates with the primers of proMAC3A-4F and MAC3Acds-1R. The resulting PCR product was cloned into pENTR/D-TOPO vector and subsequently cloned into pMDC163 binary vector to generate the proMAC3A:MAC3A-GUS plasmid. The MAC3A full-length cDNA was RT-PCR amplified with the primers of MAC3Acds-1F and MAC3Acds-1R, cloned into pENTR/D-TOPO vector, and subcloned into pEarleyGate203 or pMDC83 to generate the pro35S:MYC-MAC3A construct or the pro35S:MAC3A-GFP construct. The MAC3B full-length cDNA was amplified with the primers of MAC3Bcds-1F and MAC3Bcds-1R by RT-PCR and cloned into pENTR/D-TOPO vector and subcloned into pMDC83 to generate the pro35S:MAC3B-GFP construct. To construct cCFP-MAC3A or cCFP-MAC3B, MAC3A cDNA or MAC3B cDNA was PCR amplified using the primer pair MAC3A-3F/2R or MAC3B-3F/2R, respectively, and cloned into pSAT4-cCFP-C vector. Then, the pro35S:cCFP-MAC3A fragment or the pro35S:cCFP-MAC3B fragment was released by I-SceI restriction enzyme digestion and subcloned to pPZP-RCS2-ocs-bar-RI vector. The constructs cCFP-SE, nVenus-DCL1, nVenus-HYL1, nVenus-SE, and nVenus-AGO1 were described previously (Ren et al., 2012). To construct MBP-MAC3A, the MAC3A cDNA sequence was amplified with primer MAC3A-5F(Not1) and MAC3A-5R(Sal1) and subsequently inserted into the pMAL-C5X vector. Site-mutagenesis of MAC3A was performed according the protocol of QuikChange II site-directed mutagenesis kit (Agilent). The primers are list in Supplemental Table 1.
Plant Complementation
The proMAC3A:MAC3A-GUS, pro35S:MYC-MAC3A, pro35S:MAC3AMut1-GFP, pro35S:MAC3AMut2-GFP, and pro35S:MAC3B-GFP plasmids were transformed into mac3a mac3b using Agrobacterium tumefaciens-mediated floral dip method. The transgenic plants harboring proMAC3A:MAC3A-GUS, pro35S:MAC3AMut1-GFP, pro35S:MAC3AMut2-GFP, or pro35S:MAC3B-GFP were selected on MS medium containing hygromycin (30 μg/mL). pro35S:MYC-MAC3A transformants were selected by spraying seedlings with 120 mg/L BASTA solution.
E3 Ubiquitin Ligase Activity Assay
MBP-tagged fusion proteins were expressed in Escherichia coli strain BL21 (DE3) and purified with Amylose Resin (E8021S; NEB) by following the protocol provided by the manufacturer. The purified proteins were further desalted and concentrated using the Amicon Centrifugal Filter (Millipore). The concentration of purified protein was determined using protein assay agent (Bio-Rad).
The in vitro ubiquitination assay was performed as described (Zhou et al., 2017). Briefly, the components of 3 μg FLAG-ubiquitin, 40 ng E1 (GST-SlUBA1), 120 ng 6xHIS-AtUBC8 with 4 μg MBP, MBP-MAC3A, MBP-MAC3AMut1, or MBP-MAC3AMut2 proteins were added to a 30 μL reaction buffer (50 mM Tris-HCl, pH 7.5, 5 mM ATP, 5 mM MgCl2, 2 mM DTT, 3 mM creatine phosphate, and 5 µg/mL creatine phosphokinase). To detect the influence of protein modification on MAC3A activity, the recombinant proteins were treated as previously described with modifications (Wang et al., 2015). Briefly, 4 µg MBP, MBP-MAC3A, MBP-MAC3AMut1, or MBP-MAC3AMut2-bound amylose resin were incubated with the total protein extracts from mac3a mac3b for 1 h at room temperature followed by extensively washing for three times. Following treatment, half of protein-bound resin was treated with CIP (NEB) for 30 min, while the other half was incubated with reaction buffer without CIP. After washing, protein-bounded resins were used to perform ubiquitin assay as described above. The reaction was terminated by addition of SDS sample loading buffer with 100 mM DTT. FLAG-ubiquitin and MBP-MAC3A were then detected with a mouse monoclonal anti-FLAG M2-peroxidase-conjugated antibody (A8592; Sigma-Aldrich) and anti-MBP antibody (E8030; NEB), respectively.
Co-IP Assay
To test the interaction between MAC3A and RPB2, anti-RPB2 antibody was used to perform IP on the protein extracts from inflorescences of transgenic plants harboring pro35S:MAC3A-GFP (Ren et al., 2012). After IP, MAC3A-GFP and RPB2 were detected by immunoblot using an anti-GFP monoclonal antibody (B230720; Biolegend) and anti-RPB2 antibodies (ab10338; Abcam). To examine the co-IP of MAC3A with CDC5 and PRL1, MYC-MAC3A was coexpressed with YFP, CDC5-YFP, or PRL1-YFP in Nicotiana benthamiana as described (Ren et al., 2012). To examine the co-IP of MAC3A with DCL1 and SE, MAC3A-YFP was coexpressed with MYC-DCL1 or MYC-SE in N. benthamiana. IP was performed on protein extracts using anti-GFP or anti-MYC antibodies coupled to protein G agarose beads as described (Ren et al., 2012). After IP, proteins were detected with immunoblotting using monoclonal antibodies against YFP (B230720; Biolegend) or MYC (06-340; Millipore).
ChIP Assay
ChIP was performed using 14-d-old seedlings from Col-0 and mac3a mac3b as described (Kim et al., 2011). Three biological replicates were performed. Anti-RPB2 antibody (ab10338; Abcam) was used for immunoprecipitation. qPCR was performed using primers listed in Supplemental Table 1.
Dicer Activity Assay
In vitro MIR162b processing assay was performed as described (Qi et al., 2005; Ren et al., 2012). DNA templates used for in vitro transcription were generated through PCR with primers listed in Supplemental Table 1. In vitro transcription of MIR162b, N-UBQ5, and antisense N-UBQ5 were performed using T7 RNA polymerase in the presence of [α-32P]UTP, ATP, CTP, GTP, and unlabeled UTP. MIR162b was processed in reaction buffer (100 mM NaCl, 1 mM ATP, 0.2 mM GTP, 1.2 mM MgCl2, 25 mM creatine phosphate, 30 μg/mL creatine kinase, and 4 units of RNase inhibitor) containing 30 μg protein at 25°C. After the reaction was stopped at 50 or 100 min, RNAs were extracted and separated on a PAGE gel. ImageQuant was used to quantify the radioactive signals detected by a phosphor imager.
Morphological Analyses and GUS Histochemical Staining
Morphological and cellular analyses were performed according to the previously reported methods (Li et al., 2012). GUS staining was performed as described (Zhang et al., 2013). Briefly, tissues from plants of mac3a mac3b harboring proMAC3A:MAC3A-GUS or plants harboring proMIR167a:GUS were incubated with staining solution at 37°C for 5 h. 70% ethanol was used for tissue clearing before imaging.
BiFC Assay
BiFC assay was performed as described (Zhang et al., 2013). Paired cCFP and nVenus fusion proteins were coexpressed in N. benthamiana leaves. After 40 h expression, a confocal microscope (Fluoview 500 workstation; Olympus) was used to detect YFP and chlorophyll autofluorescence signals at 488 nm with a narrow barrier (505–525 nm; BA505-525; Olympus).
RNA Gel Blot and RT-qPCR Analyses
RNA gel blotting was performed as described (Ren et al., 2012). Approximately fifteen micrograms of total RNAs extracted from inflorescences was resolved on 16% PAGE gel and transferred to nylon membranes. 32P-labeled antisense DNA oligonucleotides were used to detect small RNAs. Radioactive signals were detected with a phosphor imager and quantified with ImageQuant. Inflorescences of plants grown on three different growth rooms at the same condition (22°C with 16-h-light and 8-h-dark cycles) were harvested as three replicates. The levels of pri-miRNAs, miRNA target transcripts, and GUS mRNA were determined using RT-qPCR. One microgram of total RNAs from inflorescences was used to generate cDNAs using the SuperScript III reverse transcriptase (Invitrogen) and an oligo dT18 primer. cDNAs were then used as templates for qPCR on an iCycler apparatus (Bio-Rad) with the SYBR green kit (Bio-Rad). The primers used for PCR are listed on Supplemental Table 1.
RIP Analyses
RIP was performed according to Wierzbicki et al. (2008) and Ren et al. (2012). Approximately two grams of seedlings of transgenic plants harboring the pro35S:MYC-MAC3A transgene was used to examine the association of MAC3A with pri-miRNAs in vivo. After cross-linking with 1% formaldehyde for 10 min, glycine was added to quench the reaction for 10 min. Nuclei were extracted and lysed in the buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1% SDS) by sonication five times. After debris was removed by centrifugation at 16,000g for 10 min, equal amounts of proteins from various samples were diluted with RIP dilution buffer and incubated with anti-GFP antibodies conjugated to protein-G agarose beads. The immunoprecipitates were then eluted with elution buffer (100 mM NaHCO3 and 1% SDS) at 65°C. Following reverse cross-linking with proteinase K (Invitrogen) and 200 mM NaCl at 65°C, RNAs were extracted and used as templates for RT-PCR analyses. All the primers are listed in Supplemental Table 1.
In Vitro RNA Pull-Down Assay
In vitro RNA pull-down assay was performed as described (Ren et al., 2012). The amylose resin beads containing MBP or MBP-MAC3A were incubated with [32P]-labeled probes at 4°C for 1 h. After the beads were washed four times, RNAs were extracted and resolved on PAGE gels. Radioactive signals were detected with a phosphor Imager and quantified by ImageQuant.
Inflorescences of Col, mac3a mac3b, and cdc5-1 grown on two separate growth rooms at the same condition (22°C with 16-h-light and 8-h-dark cycles) were harvested as two biological replicates and used for RNA extraction and small RNA library preparation following standard protocol. The data set was deposited into the National Center for Biotechnology Information Gene Expression Omnibus (Col accession nos. GSM2829820 and GSM2829821; mac3a mac3b accession nos. GSM2829822 and GSM2829823; Col accession nos. GSM2805383 and GSM2805384; cdc5-1 accession nos. GSM2805385 and GSM2805386). The sequencing data (Col access nos. GSM2257315, GSM2257316, and GSM2257317; dcl1 accession nos. GSM2257321, GSM2257322, and GSM2257323) generated by Wu et al. (2016) were used to analyze the effect of DCL1 on miRNA accumulation. After sequencing, miRNA analysis was performed after removing reads aligned to t/r/sn/snoRNA according to Ren et al. (2012). Normalization was done using the total numbers of perfectly aligned reads (Nobuta et al., 2010). The mean values of miRNA abundance from biological replicates were compared by using EdgeR with trimmed mean of M values (TMM) normalization method (Robinson et al., 2010). Downregulated miRNAs with confidence (P < 0.1; folder < 0.7) were used to identify the overlapping effect of mac3a mac3b, cdc5-1, and dcl1-9. The Venn diagram was plotted with the VennDiagram from the R package (Chen and Boutros, 2011).
Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: MAC3A (AT1G04510), MAC3B (AT2G33340), CDC5 (AT1G09770), PRL1 (AT4G15900), DCL1 (AT1G01040), SE (AT2G27100), HYL1 (AT1G09700), DDL (AT3G20550), CBP20 (AT5G44200), CBP80 (AT2G13540), HEN1 (AT4G20910), AGO1 (AT1G48410), ARF4 (AT5G60450), ARF8 (AT5G37020), CKB3 (AT3G60250), CUC1 (AT3G15170), MYB33 (AT5G06100), PHV (AT1G30490), PHO2 (AT2G33770), PPR (AT1G62670), SPL9 (AT2G42200), SPL10 (AT1G27370), SPL13 (AT5G50570), and UBIQUITIN5 (AT3G62250). Protein sequences of MAC3 homologs in other species can be obtained in National Center for Biotechnology Information under the following accession numbers: AAN13133 (MAC3A, AT1G04510, Arabidopsis thaliana), FJ820118 (MAC3B, AT2G33340, Arabidopsis), XP_009143870 (Brassica rapa), XP_009141306 (B. rapa), XP_004247768 (Solanum lycopersicum), XP_003555746 (Glycine max), XP_003535988 (G. max), XP_015614850 (Os10g32880, Oryza sativa), KXG38386 (SORBI_3001G226000, Sorghum bicolor), ONM06005 (ZEAMMB73_Zm00001d032763, Zea mays), AQK65171 (ZEAMMB73_Zm00001d014078, Z. mays), XP_001701820 (Chlamydomonas reinhardtii), NP_055317 (HsPRP19, Homo sapiens), NP_598890 (MmPRP19, Mus musculus), CAB10135 (SpPRP19, Shizosaccharomyces pombe), and CAA97487 (ScPRP19, Saccharomyces cerevisiae). Small RNA deep sequencing data sets are available from the National Center for Biotechnology Information Gene Expression Omnibus under the following reference numbers: Col accession numbers GSM2829820 and GSM2829821; mac3a mac3b accession numbers GSM2829822 and GSM2829823; Col accession numbers GSM2805383 and GSM2805384, cdc5-1 accession numbers GSM2805385 and GSM2805386; Col accession numbers GSM2257315, GSM2257316, and GSM2257317; dcl1 accession numbers GSM2257321, GSM2257322, and GSM2257323.
Supplemental Figure 1. Small RNA sequencing analyses of mac3a mac3b, cdc5 and dcl1-9.
Supplemental Figure 2. Expression of MAC3A and MAC3B complements the defects of mac3a mac3b.
Supplemental Figure 3. Effect of MAC3A and MAC3B on the expression levels and splicing of genes involved in miRNA biogenesis.
Supplemental Figure 4. Interaction of MAC3B with CDC5, PRL1, and the DCL1 complex detected by BiFC analysis.
Supplemental Figure 5. RNA binding activity of MAC3A.
Supplemental Figure 6. HYL1-YFP localization in root tips in Col and mac3a mac3b mutant.
Supplemental Figure 7. Phylogenetic analysis of MAC3A orthologs.
Supplemental Figure 8. Ubiquitin ligase activity of MAC3A is required for miRNA biogenesis.
Supplemental Table 1. The sequences of oligonucleotides.
Supplemental Data Set 1. miRNA profile change in mac3a mac3b, cdc5-1, and dcl1-9 relative to wild-type plants as determined by small RNA sequencing.
Supplemental Data Set 2. Text file of the alignment used for the phylogenetic analysis shown in Supplemental Figure 7.
Supplemental Data Set 3. Results of statistical analyses.
This work was supported by the Nebraska Soybean Board (Award 1727 to B.Y.), by the National Science Foundation (Awards OIA-1557417 to B.Y. and IOS-1645659 to L.Z.), and by the Pioneer Hundred Talents Program of Chinese Academy of Sciences (to S.L.).
S.L. and B.Y. designed the experiments and prepared the manuscript. S.L., B.Y., K.L., B.Z., M.L, S.Z., L.Z., and C.Z. performed the experiments. S.L., C.Z., and B.Y. analyzed the data.
www.plantcell.org/cgi/doi/10.1105/tpc.17.00953
The author responsible for distribution of materials integral to the findings presented in this manuscript in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Bin Yu (byu3{at}unl.edu).
Revised January 16, 2018.
Accepted January 31, 2018.
Published February 5, 2018.
Allen, E.,
Xie, Z.,
Gustafson, A.M.,
Carrington, J.C.
(2005). MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121: 207–221.
Axtell, M.J.
(2013). Classification and comparison of small RNAs from plants. Annu. Rev. Plant Biol. 64: 137–159.
Axtell, M.J.,
Jan, C.,
Rajagopalan, R.,
Bartel, D.P.
(2006). A two-hit trigger for siRNA biogenesis in plants. Cell 127: 565–577.
Baulcombe, D.
(2004). RNA silencing in plants. Nature 431: 356–363.
Baumberger, N.,
Baulcombe, D.C.
(2005). Arabidopsis ARGONAUTE1 is an RNA Slicer that selectively recruits microRNAs and short interfering RNAs. Proc. Natl. Acad. Sci. USA 102: 11928–11933.
Ben Chaabane, S.,
Liu, R.,
Chinnusamy, V.,
Kwon, Y.,
Park, J.H.,
Kim, S.Y.,
Zhu, J.K.,
Yang, S.W.,
Lee, B.H.
(2013). STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis. Nucleic Acids Res. 41: 1984–1997.
Bologna, N.G.,
Schapire, A.L.,
Zhai, J.,
Chorostecki, U.,
Boisbouvier, J.,
Meyers, B.C.,
Palatnik, J.F.
(2013). Multiple RNA recognition patterns during microRNA biogenesis in plants. Genome Res. 23: 1675–1689.
Chanarat, S.,
Sträßer, K.
(2013). Splicing and beyond: the many faces of the Prp19 complex. Biochim. Biophys. Acta 1833: 2126–2134.
Chen, H.,
Boutros, P.C.
(2011). VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinformatics 12: 35.
Chen, T.,
Cui, P.,
Xiong, L.
(2015). The RNA-binding protein HOS5 and serine/arginine-rich proteins RS40 and RS41 participate in miRNA biogenesis in Arabidopsis. Nucleic Acids Res. 43: 8283–8298.
Cho, S.K.,
Shah, P.,
Poulsen, C.P.,
Yang, S.W.
(2014). COP1 E3 ligase protects HYL1 to retain microRNA biogenesis. Nat. Commun. 5: 5867.
Das, T.,
Park, J.K.,
Park, J.,
Kim, E.,
Rape, M.,
Kim, E.E.,
Song, E.J.
(2017). USP15 regulates dynamic protein-protein interactions of the spliceosome through deubiquitination of PRP31. Nucleic Acids Res. 45: 4866–4880.
Deng, X.,
Lu, T.,
Wang, L.,
Gu, L.,
Sun, J.,
Kong, X.,
Liu, C.,
Cao, X.
(2016). Recruitment of the NineTeen Complex to the activated spliceosome requires AtPRMT5. Proc. Natl. Acad. Sci. USA 113: 5447–5452.
Dong, Z.,
Han, M.H.,
Fedoroff, N.
(2008). The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1. Proc. Natl. Acad. Sci. USA 105: 9970–9975.
Earley, K.W.,
Poethig, R.S.
(2011). Binding of the cyclophilin 40 ortholog SQUINT to Hsp90 protein is required for SQUINT function in Arabidopsis. J. Biol. Chem. 286: 38184–38189.
Fang, X.,
Cui, Y.,
Li, Y.,
Qi, Y.
(2015a). Transcription and processing of primary microRNAs are coupled by Elongator complex in Arabidopsis. Nat. Plants 1: 15075.
Shi, Y.,
Liu, X.,
Chen, Z.,
(2015b). CMA33/XCT regulates small RNA production through modulating the transcription of Dicer-like genes in Arabidopsis. Mol. Plant 8: 1227–1236.
Fang, Y.,
Spector, D.L.
(2007). Identification of nuclear dicing bodies containing proteins for microRNA biogenesis in living Arabidopsis plants. Curr. Biol. 17: 818–823.
Francisco-Mangilet, A.G.,
Karlsson, P.,
Kim, M.H.,
Eo, H.J.,
Oh, S.A.,
Kim, J.H.,
Kulcheski, F.R.,
Park, S.K.,
Manavella, P.A.
(2015). THO2, a core member of the THO/TREX complex, is required for microRNA production in Arabidopsis. Plant J. 82: 1018–1029.
Fujioka, Y.,
Utsumi, M.,
Ohba, Y.,
Watanabe, Y.
(2007). Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in Arabidopsis. Plant Cell Physiol. 48: 1243–1253.
Gregory, B.D.,
O'Malley, R.C.,
Lister, R.,
Urich, M.A.,
Tonti-Filippini, J.,
Millar, A.H.,
Ecker, J.R.
(2008). A link between RNA metabolism and silencing affecting Arabidopsis development. Dev. Cell 14: 854–866.
Hajheidari, M.,
Farrona, S.,
Huettel, B.,
Koncz, Z.,
Koncz, C.
(2012). CDKF;1 and CDKD protein kinases regulate phosphorylation of serine residues in the C-terminal domain of Arabidopsis RNA polymerase II. Plant Cell 24: 1626–1642.
Christie, M.D.,
Seymour, D.K.,
Wang, H.,
Wang, X.,
Hagmann, J.,
Kulcheski, F.,
(2015). KH domain protein RCF3 is a tissue-biased regulator of the plant miRNA biogenesis cofactor HYL1. Proc. Natl. Acad. Sci. USA 112: 14096–14101.
Kim, W.,
Benhamed, M.,
Servet, C.,
Latrasse, D.,
Zhang, W.,
Delarue, M.,
Zhou, D.X.
(2009). Histone acetyltransferase GCN5 interferes with the miRNA pathway in Arabidopsis. Cell Res. 19: 899–909.
Kim, Y.J.,
Zheng, B.,
Yu, Y.,
Won, S.Y.,
Mo, B.,
Chen, X.
(2011). The role of Mediator in small and long noncoding RNA production in Arabidopsis thaliana. EMBO J. 30: 814–822.
Köster, T.,
Meyer, K.,
Weinholdt, C.,
Smith, L.M.,
Lummer, M.,
Speth, C.,
Grosse, I.,
Weigel, D.,
Staiger, D.
(2014). Regulation of pri-miRNA processing by the hnRNP-like protein AtGRP7 in Arabidopsis. Nucleic Acids Res. 42: 9925–9936.
Lau, C.K.,
Bachorik, J.L.,
Dreyfuss, G.
(2009). Gemin5-snRNA interaction reveals an RNA binding function for WD repeat domains. Nat. Struct. Mol. Biol. 16: 486–491.
Laubinger, S.,
Sachsenberg, T.,
Zeller, G.,
Busch, W.,
Lohmann, J.U.,
Rätsch, G.,
Weigel, D.
(2008). Dual roles of the nuclear cap-binding complex and SERRATE in pre-mRNA splicing and microRNA processing in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 105: 8795–8800.
Li, S.,
Liu, K.,
Zhang, S.,
Rogers, K.,
Ren, G.,
Zhang, C.,
Yu, B.
(2017). STV1, a ribosomal protein, binds primary microRNA transcripts to promote their interaction with the processing complex in Arabidopsis. Proc. Natl. Acad. Sci. USA 114: 1424–1429.
Liu, Y.,
Zheng, L.,
Chen, L.,
Li, N.,
Corke, F.,
Lu, Y.,
Fu, X.,
Zhu, Z.,
Bevan, M.W.,
Li, Y.
(2012). The plant-specific G protein γ subunit AGG3 influences organ size and shape in Arabidopsis thaliana. New Phytol. 194: 690–703.
Manavella, P.A.,
Ott, F.,
Franz, M.,
Macek, B.,
(2012). Fast-forward genetics identifies plant CPL phosphatases as regulators of miRNA processing factor HYL1. Cell 151: 859–870.
Maréchal, A.,
Li, J.M.,
Ji, X.Y.,
Wu, C.S.,
Yazinski, S.A.,
Nguyen, H.D.,
Liu, S.,
Jiménez, A.E.,
Jin, J.,
Zou, L.
(2014). PRP19 transforms into a sensor of RPA-ssDNA after DNA damage and drives ATR activation via a ubiquitin-mediated circuitry. Mol. Cell 53: 235–246.
Mateos, J.L.,
(2010). Identification of microRNA processing determinants by random mutagenesis of Arabidopsis MIR172a precursor. Curr. Biol. 20: 49–54.
McGrail, J.C.,
Krause, A.,
O'Keefe, R.T.
(2009). The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing. Nucleic Acids Res. 37: 4205–4217.
Monaghan, J.,
Xu, F.,
Xu, S.,
Zhang, Y.,
Li, X.
(2010). Two putative RNA-binding proteins function with unequal genetic redundancy in the MOS4-associated complex. Plant Physiol. 154: 1783–1793.
Gao, M.,
Zhao, Q.,
Palma, K.,
Long, C.,
Chen, S.,
(2009). Two Prp19-like U-box proteins in the MOS4-associated complex play redundant roles in plant innate immunity. PLoS Pathog. 5: e1000526.
Nobuta, K.,
McCormick, K.,
Nakano, M.,
Meyers, B.C.
(2010). Bioinformatics analysis of small RNAs in plants using next generation sequencing technologies. Methods Mol. Biol. 592: 89–106.
Ohi, M.D.,
Vander Kooi, C.W.,
Rosenberg, J.A.,
Chazin, W.J.,
Gould, K.L.
(2003). Structural insights into the U-box, a domain associated with multi-ubiquitination. Nat. Struct. Biol. 10: 250–255.
Cheng, Y.T.,
Bi, D.,
Cheng, W.,
(2007). Regulation of plant innate immunity by three proteins in a complex conserved across the plant and animal kingdoms. Genes Dev. 21: 1484–1493.
Peragine, A.,
Yoshikawa, M.,
Wu, G.,
Albrecht, H.L.,
(2004). SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev. 18: 2368–2379.
Qi, Y.,
Denli, A.M.,
Hannon, G.J.
(2005). Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell 19: 421–428.
Qiao, Y.,
Shi, J.,
Zhai, Y.,
Hou, Y.,
Ma, W.
(2015). Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection. Proc. Natl. Acad. Sci. USA 112: 5850–5855.
Chen, X.,
(2014). Small RNAs meet their targets: when methylation defends miRNAs from uridylation. RNA Biol. 11: 1099–1104.
Xie, M.,
Dou, Y.,
(2012). Regulation of miRNA abundance by RNA binding protein TOUGH in Arabidopsis. Proc. Natl. Acad. Sci. USA 109: 12817–12821.
Robinson, M.D.,
McCarthy, D.J.,
Smyth, G.K.
(2010). edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26: 139–140.
Smith, M.R.,
Willmann, M.R.,
Berardini, T.Z.,
Möller, B.,
Weijers, D.,
(2009). Cyclophilin 40 is required for microRNA activity in Arabidopsis. Proc. Natl. Acad. Sci. USA 106: 5424–5429.
Song, L.,
Fedoroff, N.V.
(2010). RNA secondary structural determinants of miRNA precursor processing in Arabidopsis. Curr. Biol. 20: 37–41.
Lesicka, J.,
(2007). Arabidopsis primary microRNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body. Proc. Natl. Acad. Sci. USA 104: 5437–5442.
Willing, E.M.,
Rausch, S.,
Schneeberger, K.,
Laubinger, S.
(2013). RACK1 scaffold proteins influence miRNA abundance in Arabidopsis. Plant J. 76: 433–445.
Ren, L.,
Xu, P.,
Gould, K.L.,
Chazin, W.J.
(2010). The Prp19 WD40 domain contains a conserved protein interaction region essential for its function. Structure 18: 584–593.
Vaucheret, H.
(2008). Plant ARGONAUTES. Trends Plant Sci. 13: 350–358.
Voinnet, O.
(2009). Origin, biogenesis, and activity of plant microRNAs. Cell 136: 669–687.
Wang, J., et al
. (2015). The E3 ligase OsPUB15 interacts with the receptor-like kinase PID2 and regulates plant cell death and innate immunity. BMC Plant Biol. 15: 49.
Song, X.,
Li, X.,
Cao, S.,
Chu, C.,
Cui, X.,
(2013). NOT2 proteins promote polymerase II-dependent transcription and interact with multiple microRNA biogenesis factors in Arabidopsis. Plant Cell 25: 715–727.
Werner, S.,
Wollmann, H.,
(2010). Structure determinants for accurate processing of miR172a in Arabidopsis thaliana. Curr. Biol. 20: 42–48.
Wiborg, J.,
O'Shea, C.,
Skriver, K.
(2008). Biochemical function of typical and variant Arabidopsis thaliana U-box E3 ubiquitin-protein ligases. Biochem. J. 413: 447–457.
Wierzbicki, A.T.,
Haag, J.R.,
Pikaard, C.S.
(2008). Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell 135: 635–648.
Wu, C.,
Guo, S.,
Wong, S.M.
(2016). Analyses of RNA-Seq and sRNA-Seq data reveal a complex network of anti-viral defense in TCV-infected Arabidopsis thaliana. Sci. Rep. 6: 36007.
Wu, X.,
Li, J.,
Xu, L.,
(2013). A role for the RNA-binding protein MOS2 in microRNA maturation in Arabidopsis. Cell Res. 23: 645–657.
Fahlgren, N.,
Calamar, A.,
Givan, S.A.,
(2005). Expression of Arabidopsis MIRNA genes. Plant Physiol. 138: 2145–2154.
Wiermer, M.,
(2012). The cyclin L homolog MOS12 and the MOS4-associated complex are required for the proper splicing of plant resistance genes. Plant J. 70: 916–928.
Yan, J., et al
. (2017). The SnRK2 kinases modulate miRNA accumulation in Arabidopsis. PLoS Genet. 13: e1006753.
Park, M.Y.,
(2005). A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19: 2164–2175.
Yu, B.,
Bi, L.,
Ji, L.,
Chevalier, D.,
Agarwal, M.,
Ramachandran, V.,
Li, W.,
Lagrange, T.,
Walker, J.C.,
(2008). The FHA domain proteins DAWDLE in Arabidopsis and SNIP1 in humans act in small RNA biogenesis. Proc. Natl. Acad. Sci. USA 105: 10073–10078.
Zhai, J., et al
. (2013). Plant microRNAs display differential 3′ truncation and tailing modifications that are ARGONAUTE1 dependent and conserved across species. Plant Cell 25: 2417–2428.
Zhan, X.,
Wang, B.,
Li, H.,
Kalia, R.K.,
Chinnusamy, V.
(2012). Arabidopsis proline-rich protein important for development and abiotic stress tolerance is involved in microRNA biogenesis. Proc. Natl. Acad. Sci. USA 109: 18198–18203.
(2014). PRL1, an RNA-binding protein, positively regulates the accumulation of miRNAs and siRNAs in Arabidopsis. PLoS Genet. 10: e1004841.
(2015). New insights into pri-miRNA processing and accumulation in plants. Wiley Interdiscip. Rev. RNA 6: 533–545.
(2013). CDC5, a DNA binding protein, positively regulates posttranscriptional processing and/or transcription of primary microRNA transcripts. Proc. Natl. Acad. Sci. USA 110: 17588–17593.
Zhang, Z.,
Guo, X.,
Ge, C.,
Ma, Z.,
Jiang, M.,
Li, T.,
Koiwa, H.,
Zhang, X.
(2017). KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis. Proc. Natl. Acad. Sci. USA 114: 4011–4016.
Zhou, B.,
Mural, R.V.,
Oates, M.E.,
Connor, R.A.,
Martin, G.B.,
Gough, J.,
Zeng, L.
(2017). A subset of ubiquitin-conjugating enzymes is essential for plant immunity. Plant Physiol. 173: 1371–1390.
Zhu, H.,
Zhou, Y.,
Castillo-González, C.,
Lu, A.,
Zhao, Y.T.,
Duan, L.,
Li, Z.,
Wang, X.J.,
(2013). Bidirectional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1. Nat. Struct. Mol. Biol. 20: 1106–1115.
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text:
How Does What's In Print Affect Comics Studies?
Originally posted at The Hooded Utilitarian.
I have been thinking about access in comics studies lately, about the way the availability of resources can be used to steer critical interests in the field. Publishing houses and archivists, collectors and copyright holders, even cultural guardians – there are a lot of people and institutions involved in making decisions about which titles to seek out, preserve, and keep in print. Of course, anyone who works among the ephemera of popular culture faces similar challenges, but my question focuses on the implications for comics scholarship: How does access to materials shape the choices you make about the comics you study, teach, or review?
Last week, I visited the comic book archives at the Library of Congress in DC and at Virginia Commonwealth University's James Branch Cabell Library in Richmond. Although I arrived with a list of rare comics and fanzines that I wanted to read, the best part of spending time in archives was getting the chance to talk with the reference librarians who maintain the collections and know where to look for hidden gems. Thanks to Cindy Jackson at VCU, I got to turn the pages of The Adventures of Black Eldridge: The Panther, a newly-acquired underground comic produced by Ovid P. Adams in 1970. Megan Halsband at the Library of Congress introduced me to Joel Christian Gill's recent series of Strange Fruit Comics that uses satire and comics culture to dramatize obscure black historical figures (along with great bonus features like "Lil' Nino Brown in Slumland").
You may have to travel to Richmond to see Black Eldridge wield his machete against the villains of white supremacy, but Gill blogs about his work online and his Strange Fruit Comics are being collected in a new edition out this June. And it's a good thing too, because quite a few of the titles I regularly teach in my class on race and comics are now sadly out of print and I don't know how long I can keep asking students to hunt down used copies.
Questions of access must also take into account the way comics as a form continues to be disparaged ideologically by those in positions of power. Complaints that the College of Charleston forced last year's incoming students to endure lesbian "pornography" in Alison Bechdel's graphic novel, Fun Home, also prompted South Carolina State Representative Tommy Stringer to tweet: "Is the instructional ability of CofC teachers so low that they have to use comic books to teach freshman?" The material consequences of this kind of thinking are extremely serious; the state ultimately voted to slash the first-year reading program's dollars from the college's budget.
One wonders if Rep. Stringer would level the same accusations of teacher incompetency if the College of Charleston had selected March: Book One for their freshmen reading selection, as Michigan State and Marquette University are doing this fall. U.S. Congressman John Lewis has been signing copies of his graphic novel memoir to standing-room only crowds across the country. In interviews, Lewis along with his co-writer Andrew Aydin and artist Nate Powell have often praised the ability of comics to inspire social change and reviews celebrate the novel's success as a teaching tool. Furthermore, the 1958 comic book, Martin Luther King and The Montgomery Story, is available for purchase again due, in part, to a reference in Lewis's graphic novel and the publicity surrounding distribution of the comic book's translated Arabic edition during the Arab Spring in 2011. I suspect that high schools and colleges will keep these comics in print for a long time.
The research that I'm doing now on EC Comics greatly benefits from the initiatives of fans that have worked hard to keep reprints in circulation. The same can't be said, though, for comics like Fawcett's Jackie Robinson series, also from the 1950s. Last week, I had the chance to read three issues (three!) from the series, along with one about Joe Louis and Willie Mays, all incredibly fragile and rare. I'm eager to write about these comics, to try to make some kind of meaningful connections with other titles produced during this period. (Who knew that Robinson apprehended cat burglars in his spare time?) But now I'm realizing how important it is that we also work with publishers to make these comics more readily available. If readers can't get better access to titles like Jackie Robinson or The Adventures of Black Eldridge, how can they become a part of larger critical conversations in comics studies?

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text:
How usual is it to work from home?
How usual is it to work from home? - Products Eurostat News
Back How usual is it to work from home?
Print How usual is it to work from home?
In 2018, 5.2% of employed persons aged 15 to 64 in the European Union (EU) usually worked from home. This share has remained constant at around 5% throughout the last decade. However, over the same period, the share of those who sometimes work from home increased from 5.8% in 2008 to 8.3% in 2018.
With 14.0% of employed people usually working from home in 2018, the Netherlands topped the list of EU Member States, closely followed by Finland (13.3%), Luxembourg (11.0%) and Austria (10.0%). In contrast, very few people usually worked from home in Bulgaria (0.3%) and Romania (0.4%).
In the EU, the self-employed usually worked from home (18.5%) more often than employees (3.0%). This pattern was repeated in each Member State. The highest rates recorded were in Finland where more than 40% of self-employed persons usually worked from home (46.4%), the Netherlands (44.5%) and Austria (43.6%).
More women than men work from home
In 2018, a slightly higher share of women usually worked from home (5.5%) than men (5.0%). This was the case in most EU Member States, with the largest differences observed in France (8.1% of women, compared to 5.2% of men), Luxembourg (12.5% of women, 9.8% of men) and Malta (7.4% of women, 4.7% of men).
In contrast, in eight EU Member States, the situation was the reverse, with more men usually working from home than women. In the Netherlands (15.5% of men, compared to 12.3% of women), Denmark (8.5% of men, 7.0% of women) and Ireland (7.2% of men, 5.7% of women) this difference was especially large.
Data source: lfsa_ehomp
Older persons work from home more often than younger ones
The share of those working from home increases with age. In the EU, just 1.8% of 15-24 year-olds usually worked from home in 2018, compared to 5.0% among 25-49 year-olds and 6.4% among 50-64 year-olds. The highest share of 15-24 year-olds who usually worked from home was recorded in Luxembourg (8.7%). The next highest Member State was Estonia (5.2%).
For the other age categories, the Netherlands recorded the highest shares of those usually working from home (14.9% among 25-49 year-olds and 17.3% among 50-64 year-olds). They were followed by Finland (14.0% among 25-49 year-olds and 15.6% among 50-64 year-olds).
For more information on the statistics available in this area, take a look at the overview of labour market statistics (including the labour force survey (LFS)).


Melinda Looi has been featured in an article about "Living Moderately" in Malaysian Digest. The objective of the article was to highlight how living moderately isn't only the key to live a more fulfilling life, but can also help certain issues such as extremism. More often than not, most of us find ourselves overindulging, caving into our impulsiveness, or doing things excessively until it is on the brink of obsession. Many thanks to Malaysian Digest for this article feature!


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redpajama_set_name: RedPajamaGithub


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Burgoyne, A.D. and Drummond, P.D. (2008) Knowledge of HIV and AIDS in women in sub-Saharan Africa. African Journal of Reproductive Health, 12 (2). pp. 14-31.
Although most African people have heard of HIV and AIDS, there is still widespread misunderstanding about how HIV is spread, the consequences of infection, and how to protect against infection. The most vulnerable groups are poorly educated women, those from rural backgrounds, and women who are economically dependent on men. Lower levels of education, taboos associated with the discussion of sexuality and sexual health, the submissive role of women in a relationship, and male control of decision-making regarding sexual relations might explain why African women are less knowledgeable about HIV/AIDS than men. Although most African men and women are aware of the protective benefits of condoms, negative attitudes towards the acceptability and safety of condom use are widespread. More sexual health campaigns tailored to women, especially those with low education levels and those from rural areas, are needed to reduce the spread of HIV in Africa.


The Indian rupee continued to extend its winning streak and hit fresh 5-1/2 month high by breaking below 52 to a dollar. The rupee gained in the past one month on the back of government reforms and also due to improvement in the current account deficit, which has narrowed down from record highs. The rupee has also strengthened from dollar inflows in other assets like equities. Also, dollar sales are now coming in from exporters and banks on expectations of further appreciation in the unit. The sharp decline in the crude price is also adding to the rupee strength.
The World Bank signed a $100 million loan agreement with the Government of India and the Government of Himachal Pradesh (GoHP) to support the mountain State in its move towards an environmentally sustainable model of economic growth. The focus of this operation in Himachal Pradesh (HP) will be towards climate-smart development across the key engines of economic growth in the state – energy, watershed management, industry and tourism. Richly endowed with natural resources, HP provides economically valuable environmental services for much of the country. The state is home to three major river basins, and it serves as a watershed that is critical to the livelihood of more than 200 million people in Haryana, Punjab, Uttar Pradesh and Rajasthan. It is also one of the country's main sources of clean energy, generating hydropower that can help alleviate electricity shortages in much of North India. The forests of HP also act as an important carbon sink and are known to be global biodiversity hotspots.
The Union Finance Minister Shri P. Chidambaram approved a new tax saving scheme called "Rajiv Gandhi Equity Saving Scheme"(RGESS),exclusively for the first time retail investors in Securities Market. This Scheme would give tax benefits to new investors who invest up to Rs. 50,000 and whose annual income is below Rs. 10 lakh. The Scheme not only encourages the flow of savings and improves the depth of domestic capital markets, but also aims to promote an 'equity culture' in India. This is also expected to widen the retail investor base in the Indian securities markets.
a.Scheme is open to new retail investors, identified on the basis of their PAN numbers. This includes those who have opened the Demat Account but have not made any transaction in equity and /or in derivatives till the date of notification of this Scheme and all those account holders other than the first account holder who wish to open a fresh account.
b.Those investors whose annual taxable income is ≤ Rs. 10 lakhs are eligible under the Scheme.
c.The maximum Investment permissible under the Scheme is Rs. 50,000 and the investor would get a 50% deduction of the amount invested from the taxable income for that year.
d.To benefit the small investors, the investments are allowed to be made in instalments in the year in which tax claims are made.
e.The total lock-in period for investments under the Scheme would be three years including an initial blanket lock-in period of one year, commencing from the date of last purchase of securities under RGESS.


St. Nectarios is a wonderworking saint, born of a poor family. He worked as a teacher and writer. As a monk and bishop, he was know for his humility and prayer, and perseverance in difficult circumstances. This thirty-two page book includes eleven full page colour illustrations. It is suitable to be read to children of five years old and upwards.
Click the button below to add the Saint Nectarios of Aegina to your wish list.