The m6A Reader ECT2 Controls Trichome Morphology byAffecting mRNA Stability in Arabidopsis[OPEN]

Lian-Huan Wei,a Peizhe Song,a Ye Wang,a Zhike Lu,a Qian Tang,a Qiong Yu,a Yu Xiao,a Xiao Zhang,a

Hong-Chao Duan,a and Guifang Jiaa,b,1

a Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of BioorganicChemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University,Beijing 100871, ChinabNational Engineering Research Center of Pesticide (Tianjin), Nankai University, Tianjin 300071, China

ORCID ID: 0000-0002-4186-6922 (G.J.)

The epitranscriptomic mark N6-methyladenosine (m6A) can be written, read, and erased via the action of a complex network ofproteins. m6A binding proteins read m6A marks and transduce their downstream regulatory effects by altering RNA metabolicprocesses. The characterization of m6A readers is an essential prerequisite for understanding the roles of m6A in plants, but theidentities of m6A readers have been unclear. Here, we characterized the YTH-domain family protein ECT2 as an Arabidopsisthaliana m6A reader whose m6A binding function is required for normal trichome morphology. We developed the formaldehydecross-linking and immunoprecipitation method to identify ECT2-RNA interaction sites at the transcriptome-wide level. Thisanalysis demonstrated that ECT2 binding sites are strongly enriched in the 39 untranslated regions (39 UTRs) of target genes andled to the identification of a plant-specific m6A motif. Sequencing analysis suggested that ECT2 plays dual roles in regulating 39UTR processing in the nucleus and facilitating mRNA stability in the cytoplasm. Disruption of ECT2 accelerated the degradationof three ECT2 binding transcripts related to trichome morphogenesis, thereby affecting trichome branching. The results shedlight on the underlying mechanisms of the roles of m6A in RNA metabolism, as well as plant development and physiology.

INTRODUCTION

The epitranscriptomic mark N6-methyladenosine (m6A) is themost prevalent dynamic internal mRNA modification in eukar-yotes. In this process, analogous toDNAmethylation, m6A can bedynamically written, erased, and read; these events are nowknown to function in gene regulation (Bokar et al., 1997; Jia et al.,2011;Zhengetal., 2013;Wanget al., 2014, 2015;Xiaoet al., 2016).Mammals have at least two types of proteins that can write m6Amodifications: methyltransferases like 16 (METTL16) and multi-protein writer complexes from which several proteins have beencharacterized (e.g.,METTL3,METTL14,Wilms tumor1-associatingprotein [WTAP],KIAA1429,andRNAbindingmotifprotein15) (Bokaret al., 1997; Liu et al., 2014; Ping et al., 2014; Schwartz et al., 2014;Patil et al., 2016;Pendletonetal., 2017).While less isknownaboutm6

A methylation in plants, two major transcriptome-wide sequencingstudies in Arabidopsis thaliana (Can-0 and Hen-16) have demon-strated that m6A is a highly conserved and dynamic modificationwith functional roles inmRNAmetabolism inplants (Luoetal., 2014).Several m6A methyltransferase subunits have been characterizedin Arabidopsis (e.g.,N6-adenosine-methyltransferase MT-A70-like[MTA], the plant homolog of human METTL3; Methyltransferase

MT-A70 family protein, a homolog of human METTL14; FKBP12interacting protein 37 [FIP37], a homolog of human WTAP; VIRIL-IZER, a homolog of human KIAA1429; and the E3 ubiquitin ligaseHAKAI) (Zhong et al., 2008; Bodi et al., 2012; Shen et al., 2016;Ruzi�ckaetal.,2017).m6Amodification inplants isreversibleandcanbe erased by two Arabidopsis homologs of the human m6A de-methylase ALKBH5, ALKBH9B, and ALKBH10B (Duan et al., 2017;Martínez-Pérez et al., 2017). ALKBH9B has m6A demethylaseactivity and involved in defense against viral infection (Martínez-Pérez et al., 2017); ALKBH10B is an mRNA m6A demethylase af-fecting thefloral transitionandvegetativegrowth (Duanetal., 2017).The characterization of m6A readers is thus important for

deepening our understanding of how m6A functions in mRNAprocessing and thus functions in many biological processes.Fundamental studies have established that five human YTH(YT512-B Homology)-domain family proteins are m6A readersthat function in or affect pre-mRNA splicing, nuclear export,mRNA degradation, and mRNA translation efficiency (Wanget al., 2014, 2015; Xiao et al., 2016; Hsu et al., 2017; Roundtreeet al., 2017; Shi et al., 2017). Structural studies of the YTH do-mains from reader proteins in complexwithm6A-containingRNA(e.g., YTHDF1 and YTHDC1) have revealed that all YTH domainproteins employ a highly conserved aromatic cage to recognizem6A (Xu et al., 2014, 2015). In conjunctionwithm6A readers,m6Amodifications function in the regulation of stem cell fate, sexdetermination inDrosophilamelanogaster, thematernal-to-zygotictransition in zebrafish (Danio rerio), and femaleoocytematuration inmammals, amongother roles (Aguilo et al., 2015;Geula et al., 2015;Lence et al., 2016; Ivanova et al., 2017; Kan et al., 2017; Zhao et al.,

1 Address correspondence to guifangjia@pku.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Guifang Jia (guifangjia@pku.edu.cn).[OPEN] Articles can be viewed without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.17.00934

The Plant Cell, Vol. 30: 968–985, May 2018, www.plantcell.org ã 2018 ASPB.

2017). To date, although a family of 13 proteins with putative YTHdomains have been reported in Arabidopsis (Li et al., 2014), it re-mains unclear which plant proteins function as m6A readers. Toexplore and come to understand the roles of m6A in plants, thecharacterizationofplantm6A readers isessential andwill likelyshedlight on important regulatory mechanisms related to these ex-tremely basic processes of RNA metabolism, including whetherplantssharesimilarm6Areadingmechanismswithothereukaryotesor perhaps employ unique, plant-specific processes.

Here, we demonstrate that EVOLUTIONARILY CONSERVEDC-TERMINAL REGION2 (ECT2) is an m6A reader protein. We es-tablish that its m6A binding function is required for normal trichomemorphology. Subcellular localization analysis showed that ECT2 ispresent in both the nucleus and the cytoplasm. We developeda method, formaldehyde cross-linking and immunoprecipitation(FA-CLIP), which allowed us to identify the binding sites of ECT2.Analysis of the positions of these sites not only showed dramatic 39untranslated region (UTR) enrichment, but also led to the identifi-cation of them6A target motif, UGUA, which is consistently locatedinapositioncharacteristicofso-calledfarupstreampolyadenylationsignals for alternative polyadenylation, suggesting that ECT2mightregulate 39 UTR processing in the nucleus. We also show that thelack of ECT2 results in significantly reduced cellular populations ofECT2 target transcripts, indicating that, unlike the human m6Areader YTHDF2, the m6A reader ECT2 does not prepare m6A-modified transcripts for degradation in the cytoplasm, but it insteadmight facilitatemRNA stability in the cytoplasm.Disruption ofECT2decreased the expression levels of three ECT2 binding transcriptsrelatedtotrichomemorphogenesisthroughacceleratingtheirmRNAdegradation, thereby affecting trichomebranching. Collectively, our

work demonstrates that the m6A binding function of ECT2 controlstrichomemorphologyviaaffectingmRNAstability andsuggests thatECT2 functions in basic RNA metabolism, specifically in 39 UTRprocessing and mRNA stability.

RESULTS

The Ubiquitously Expressed YTH-Domain Family ProteinECT2 Is Strongly Expressed in Rapidly Developing Tissuesand Has a Morphogenesis-Related Function

Exciting recent discoveries about how human YTH-domain familyproteins mediate m6A modifications and thereby influence mRNAprocessing and biological processes motivated us to explore thepotential regulatory roles of evolutionarily related proteins in plants(Wangetal.,2014,2015;Xiaoetal.,2016;Hsuetal.,2017;Roundtreeetal.,2017;Shietal.,2017).Thereare13YTH-domain familyproteinsin Arabidopsis (Li et al., 2014). qPCR analysis of RNA samples fromArabidopsis seedlings revealed that the mRNA abundance of ECT2was higher than that of the other YTH-domain family genes(Supplemental Figure 1).We also detectedECT2 transcripts inmostArabidopsis vegetative and reproductive organs that we examined,highlighting the apparently ubiquitous transcription of this gene inArabidopsis (Figure 1A). Histochemical assays based on a ECT2promoter-driven GUS construct in transgenic Arabidopsis plants(ECT2pro:GUS) indicated thatECT2expression is strongest in rapidlygrowing tissuessuchasapicalmeristems, lateral rootprimordia, roottips, trichomes, and pollen (Figure 1B to 1G), suggesting that ECT2functions in these actively developing tissues.

m6A Read by ECT2 Affects Trichome Morphology 969

Topursue this idea,weobtained two independent homozygousT-DNA insertion lines for the ECT2 gene, ect2-1 (SALK_002225)and ect2-2 (SAIL_11_D07) (Supplemental Figures 2A and 2B)(Alonso et al., 2003). The T-DNA insertion is located in intron 3 ofthe ECT2 locus in ect2-1 and in exon 6 in ect2-2. RT-PCR showedthat full-length ECT2 transcript was not expressed in ect2-1,whereas ect2-2 retained ;26% of wild-type levels of full-lengthECT2 transcript (Supplemental Figures 2C and 2H). Examinationof ect2-1 and ect2-2 plants showed an obvious trichome mor-phological phenotype. Cryo-scanning electronmicroscopy (cryo-

SEM) revealed that the trichomes of both ect2-1 and ect2-2plantsweremore extensively branched thanwild-type trichomes (Figure2A).Whereas83%ofwild-type trichomeshad threebranches, andnone of these plants had trichomes with more than four branches(8%had fourbranches),;50%ofect2 trichomes (50.6% inect2-1and 43.6% in ect2-2) had four branches, and 1 to 2% of ect2trichomes had five branches (Figure 2B). This phenotype, whichwas exhibited in two independent homozygous ect2 mutants,strongly suggests that ECT2 has a function related to trichomemorphogenesis.

Figure 1. ECT2 Is Ubiquitously Expressed, with Highest Expression Levels in Rapidly Developing Tissues.

(A) Relative gene expression was measured using qPCR with ACTIN2 as a reference gene, prior to normalization to ECT2 expression levels in 14-d-oldseedlings. Data are represented as means 6 SE, n = 2 biological replicates 3 3 technical replicates. Biological replicates are parallel measurements ofbiologically distinct samples, and technical replicates are repeated measurements of the same sample.(B) to (G) GUS staining analysis of ECT2pro:GUS transgenic Arabidopsis plants. Blue bar = 1 mm; purple bar = 500 mm; red bar = 100 mm.(B) A 12-d-old seedling and its meristem (shown on the right).(C) Stage VII lateral root of a 12-d-old seedling.(D) Main root of 12-d-old seedling.(E) Trichomes of a 12-d-old seedling.(F) Three-week-old rosette leaves.(G) A 5-week-old flower and its pollen (stamens with pollen shown on the right).

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ECT2 Is an m6A Reader Protein Whose m6A BindingFunction Is Required for Normal Trichome Morphology

ECT2 is a homolog of humanYTHDF1/YTHDF2/YTHDF3 proteins(eachcontaining aYTHdomain) (Supplemental Figure3).Weuseda combination of in vitro, bioinformatics, and in vivo methods toexamine thepotentialm6AbindingactivityofECT2and toevaluateany possible relationship between this activity and the observedmorphogenesis-related phenotypes. We expressed and purifiedrecombinant GST-tagged ECT2 protein from Escherichia coli(Supplemental Figure 4A) and used it to perform the in vitro RNAimmunoprecipitation LC-MS/MS assays that are typically used instudies of m6A readers (Wang et al., 2014). We isolated poly(A)-

tailed RNA from 14-d-old Arabidopsis seedlings and incubated itwith purified GST-ECT2 protein. We used LC-MS/MS to examinethem6A/A ratios of RNAmolecules that were immunoprecipitatedbyECT2 (separated basedonGSTaffinity beads), RNAmoleculesin the flow-through eluate from this separation, and RNA mole-cules from the input (i.e., unincubated) samples. The ECT2-boundRNAs were highly enriched for m6A modifications compared withRNAmolecules in theflow-throughand input samples, suggestingthat ECT2 binds to m6A sites (Figure 2C). To verify that ECT2recognizes m6A sites in planta, we generated transgenic Arabi-dopsis plants (ECT2pro:ECT2-Flag/ect2-1) expressing an ECT2-Flag fusion protein driven by the ECT2 promoter in the ect2-1

Figure 2. ECT2 Is an m6A Reader Protein Whose m6A Binding Function Is Required for Normal Trichome Morphology.

(A) Cryo-SEM analysis of trichome morphology in wild-type, ect2-1, ect2-2, ECT2pro:ECT2-Flag/ect2-1, and ECT2pro:ECT2m-Flag/ect2-1 plants. Tri-chomes are from the third and fourth leaves of 3-week-old Arabidopsis plants. Bar = 100 mm.(B) Statistical analysis of trichome branch number in wild-type, ect2-1, ect2-2, ECT2pro:ECT2-Flag/ect2-1, and ECT2pro:ECT2m-Flag/ect2-1 plants. Threehundred trichomes from the third and fourth leaves of 3-week-old Arabidopsis plants were analyzed.(C) In vitro RIP-LC-MS/MS assay showing that m6A modification is enriched in GST-ECT2-bound mRNA compared with the flow-through and inputsamples. GST-ECT2 was expressed in E. coli, andmRNAwas isolated from 14-d-old wild-type Arabidopsis seedlings. Data are presented asmeans6 SE,n = 3 biological replicates 3 2 technical replicates. ****P < 0.0001 by t test (two-sided).(D) In vivo FA-RIP-LC-MS/MS showing that m6A modification is enriched in ECT2-Flag-bound RNA compared with IgG-bound RNA. Fourteen-day-oldECT2pro:ECT2-Flag/ect2-1 seedlings were used for the experiment. Data are presented as means6 SE, n = 2 biological replicates3 3 technical replicates.**P < 0.01 by t test (two-sided).

m6A Read by ECT2 Affects Trichome Morphology 971

mutant background and performed in vivo formaldehyde RNAimmunoprecipitation (FA-RIP)-LC-MS/MS assays. The proportionof RNA molecules with m6A modifications was dramatically higheramong the ECT2-Flag immunoprecipitation (IP) RNAs comparedwithcontrol IgG IPRNAs (Figure2D). To furtherconfirmthat them6Abinding function of ECT2 depends on the presence of the YTHdomain, we designeda putative binding function-abolished formofECT2 W521A/W534A (named ECT2m, which contains two muta-tions: Trp-521 to Ala and Trp-534 to Ala) based on sequencealignments between five human YTH-domain proteins (amongthese, YTHDF1/YTHDF2/YTHDC1, with m6A binding function li-gands that have been identified in crystal structures) and 13 Ara-bidopsis YTH-domain family proteins, which strongly suggestedthat three tryptophan residues in theYTHdomainofECT2 (Trp-464,Trp-521, and Trp-534) are likely directly involved in the binding ofECT2 to m6A sites (Supplemental Figure 3). We expressed andpurified recombinant GST-ECT2 and GST-ECT2m protein fromE. coli (Supplemental Figure 4A) and performed electrophoreticmobility shift assay (EMSA)with synthetic 42-mer RNAs containingeither m6A or A. The EMSA analysis showed that GST-ECT2m hada completely abolished m6A binding function compared with wild-type GST-ECT2 (Supplemental Figure 4B). Collectively, these re-sults indicate that ECT2 binds to RNA transcripts that harbor m6Asites, establishing that ECT2, like human YTH-domain familyproteins, is an m6A reader protein.

Having established that ECT2 is an m6A reader, we performedgenetic complementation to determine whether ECT2’s m6Abinding activity, per se, functions inmorphogenesis. Here, we used

two complementation lines in the ect2-1 mutant background. TheECT2pro:ECT2m-Flag/ect2-1 line expressed an m6A binding func-tion-abolished form of ECT2,W521A/W534A, whileECT2pro:ECT2-Flag/ect2-1 plants expressed a FLAG-tagged but otherwiseunalteredECT2protein (FLAG tag) (Supplemental Figure 2I). In bothlines, expression was driven by the native ECT2 promoter. Con-sistent with the conclusion that the m6A binding activity of ECT2 isrequired for normal morphogenesis, the ect2-1mutant phenotypewas restored by the expression of wild-type ECT2, but not by theexpression of the m6A binding function-abolished form of ECT2,W521A/W534A (Figures 2A and 2B). Thus, the m6A site bindingactivity of the m6A reader protein ECT2 is required for normal tri-chome morphogenesis in Arabidopsis.

ECT2 Is Present in Both the Nucleus and Cytoplasm

As thesubcellular localizationofm6A readers isknown to influencethe types of regulatorymechanisms they utilize (Wanget al., 2014,2015; Xiao et al., 2016; Roundtree et al., 2017), we characterizedthe subcellular localization of ECT2. Analysis of the ECT2 se-quence using the TargetP (Emanuelsson et al., 2007) identified noobvious localization tags. We generated transgenic Arabidopsisplants (ECT2pro:ECT2-eGFP/ect2-1) and analyzed cells in the roottipusingconfocalmicroscopy,findingthatECT2ispresent inboththenucleus and cytoplasm. Overlay of ECT2-eGFP and 49,6-diamidino-2-phenylindole-staining images indicated colocalization of the ECT2fusion signal with the nucleus, but there was also extensive eGFPsignal in the cytoplasm (Figure 3). We also used wild tobacco

Figure 3. ECT2 Is Expressed in Both the Nucleus and Cytoplasm.

Confocal microscopy showing the subcellular localization of ECT2 in ECT2pro:ECT2-eGFP/ect2-1 transgenic Arabidopsis root tips. ECT2 is found in both thenucleus and cytoplasm. Magnified images of cells in the white boxes in (A) are shown in (B), and further magnifications are shown in (C). Bars = 10 mm.

972 The Plant Cell

(Nicotiana benthamiana) for the transient expression of the ECT2-eGFP fusion protein and again found that ECT2 was present inboth the nucleus and cytoplasm (Supplemental Figure 5). Thesubcellular localization of ECT2 suggested that ECT2might playdual roles in regulating pre-mRNAprocessing in the nucleus andother aspects of mRNAmetabolism involvingmature transcriptsin the cytoplasm.

Development of the FA-CLIP Method Enables theIdentification of Transcriptome-Wide ECT2-RNA InteractionSites, Revealing an m6A Motif and 39 UTR Enrichment

UV-cross-linking and immunoprecipitation (UV-CLIP) and pho-toactivatable ribonucleoside-enhanced cross-linking and immu-noprecipitation (PAR-CLIP) methods have been widely used tomap protein-RNA interaction sites in human cells (Ule et al., 2003;Hafner et al., 2010). Unlike the UV cross-linking used in UV-CLIP,PAR-CLIP involves the incorporation of 4-thiouridine into RNA forcross-linking. However, these methods have been ineffective inplants due to technical issues relating to cross-linking effi-ciency (Meyer et al., 2017). Formaldehyde cross-linking has beenwidely used to examine protein-DNA interactions in chromatinimmunoprecipitation, protein-RNA interactions in plant FA-RIP,protein-protein interaction, and chromatin structure (Terzi andSimpson, 2009; Hoffman et al., 2015). Traditional FA-RIP used inplants can only identify the binding of transcripts to proteins andnot protein-RNA interaction sites (Terzi and Simpson, 2009; Xinget al., 2015; Meyer et al., 2017). To enable the identification of thebinding sites on transcripts targeted by ECT2, we developeda convenient FA-CLIPmethod that combines FA-RIP and CLIP. Aconceptual diagramof the FA-CLIPmethod is presented in Figure4A. The key innovation of FA-CLIP is the use of formaldehyde tocross-link proteins and RNA; this facilitates the chopping up oftranscripts into protein binding regions and enables us to inducemutationsusing reverse transcription. Inorder tominimize thebiasof formaldehyde cross-linking and to ensure the accuracy of ouridentified binding sites, our method uses IP enrichment andmutation to identify binding sites; by contrast, UV-CLIP and PAR-CLIP identify binding sites based only onmutation.We performedFA-CLIP to identify ECT2 binding sites (i.e., ECT2 binding peaks,termed FA-CLIP peaks) on transcripts using 14-d-old ECT2pro:ECT2-Flag/ect2-1 and wild-type seedlings (termed FA-CLIP-ECT2 and Mock, respectively). Total RNA with rRNA depletionfrom ECT2pro:ECT2-Flag/ect2-1 seedlings was used for RNA-seq(termed “Input”). The enrichment peaks were filtered using thecriteria of enrichment fold (FA-CLIP-ECT2 [or Mock]/Input)$ 2 andP value < 0.01. IP enrichment-based ECT2 binding peaks (termedFA-CLIP enrichment peak) were obtained by subtracting the en-richment peaks in Mock (Mock versus Input) from the enrichmentpeaks in FA-CLIP-ECT2 (FA-CLIP-ECT2 versus Input). Mutationpeaks were identified using PARAlyzer (Corcoran et al., 2011). Themutation-based ECT2 binding peaks (termed FA-CLIP mutationpeaks) were identified as the mutation peaks in the FA-CLIP-ECT2results while excluding the mutation peaks in Mock. ECT2 bindingpeaks (termed FA-CLIP peaks) were defined as overlapping peaksof FA-CLIP mutation peaks and FA-CLIP enrichment peaks.

We conducted two biological replications (repeat experiments)of this FA-CLIP analysis and retained the FA-CLIPpeaks common

to both replications as high-confidence ECT2 binding peaks forsubsequentanalysis.Comparedwith themutationpeaks (6562and7111) in duplicate Mock samples, the mutation-based ECT2bindingpeaks (termed “FA-CLIPmutation peak”) contained37,614and 37,694 peaks in two biological replications (SupplementalFigures 6A and 6B). By subtracting the enrichment peaks in Mock(20,794) from the enrichment peaks in FA-CLIP-ECT2 (34,679),20,040peakswere identifiedas IPenrichment-basedECT2bindingpeaks (termed FA-CLIP enrichment peak) (Supplemental Figure6C). Overlapping FA-CLIP mutation peaks with FA-CLIP enrich-ment peaks, the first replication identified ;8700 FA-CLIP peaksand the second identified;10,400;;6000 peakswere common toboth. These ;6000 high-confidence ECT2 binding sites corre-spondedto3680uniquetranscripts/genes (Figure4B;SupplementalData Set 1). Almost all of the transcripts targeted by ECT2 weremRNAmolecules, but;0.7%were noncoding RNA or other RNAs(Figure 4C). Moreover, when we looked at the overlap of theFA-CLIP-identified ECT2 targeted sites with them6A sites identifiedfromaseparatem6A-seqanalysisofpoly(A)-tailedRNAfromwild-typeCol-0 seedlings (Duan et al., 2017), we found that 49.9% (3016 out of6047) of ECT2-targeted peaks were modified with m6A (Figure 4D;Supplemental Data Set 2). Analysis of the distance betweenm6A andECT2 binding sites revealed that m6A frequently overlaps with thebinding site of ECT2 (Supplemental Figure 6D). These results confirmthat ECT2 recognizes m6A on RNA transcripts in plants.We then looked for other trends among the ECT2 targeted

transcripts and found that the majority (90%) of ECT2-targetedsites occur within 39 UTR of RNA transcripts (Figure 4E). We alsosystematically examined the distribution of the ECT2binding sitesalong transcripts, revealing strong 39 UTR enrichment of ECT2target sites (Figure 4F). Clustering all ECT2 binding peaks usingHOMER (Hypergeometric Optimization of Motif Enrichment) didnot identify any previously reported signature m6A motifs in ourdata set (e.g., GGACU, and so on), but we did identify a stronglyconservedmotif among theECT2bindingpeaks:URUAY (R=G>A,Y=U>A, where the majority [over 90%] is UGUAY) (Figure 4G).Importantly, this motif was also clearly identified in an analysis ofthe ECT2 binding peaks common to both the m6A-seq andFA-CLIP data sets (Supplemental Figure 7). Collectively, weidentified 3680 unique transcripts/genes containing over 6000ECT2binding sites,whichweremainly locatedwithin 39UTRs andwere clustered into the distinct binding motif, URUAY (R=G>A,Y=U>A, where the majority [over 90%] is UGUAY).

The URUAY Motif Is a Plant-Specific m6A Motif

OurFA-CLIP results showed that theECT2m6A reader recognizesa highly conserved motif, URUAY (R=G>A, Y=U>A, where themajority [over 90%] is UGUAY), that is distinct from the RRACHm6Amotif thatwascharacterizedfromothereukaryotes(Dominissiniet al., 2012). To demonstrate that this URUAY motif is a genuinem6Asite (that is, itcanbemethylatedbyanendogenousm6Awriter),weperformedinvitromethylationassaysusingcellextracts (nuclearfraction proteins) prepared from Arabidopsis seedlings. First, werandomly chose two m6A peaks that contained the UGUA motiffromourm6Asequencingdata touseas templates tobuild twoRNAoligos: using splint ligation (Moore and Query, 2000), we intro-duced [g-32P] into theUGUAmotifs, ensuringuniformdistributionof

m6A Read by ECT2 Affects Trichome Morphology 973

Figure 4. Transcriptome-Wide ECT2-RNA Interaction Sites Reveal a Unique Binding Motif and 39 UTR Enrichment.

(A) Schematic diagram of the FA-CLIP method. FA-CLIP procedure: A cell extract from formaldehyde-cross-linked 14-d-old ECT2pro:ECT2-Flag/ect2-1seedlings was subjected to immunoprecipitation using anti-Flag M2 magnetic beads. Two on-bead digestions using RNase T1 and Protease K wereperformed sequentially: ECT2 protein-bound transcripts were digested into protein binding regions (;30 nucleotides) using RNase T1, and ECT2 protein

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[g-32P]-adenosine in these two RNA oligos (oligo 1 and oligo 2,see figure legends for sequences). Subsequently, we incubatedthese uniformly adenosine-labeled oligo substrates either with orwithout nuclear protein extracts for in vitro methylation reactions.Finally, we monitored the m6A modification status using nucleaseP1 treatment and thin-layer chromatography (TLC) (Figure 5A).

These in vitro methylation assays with plant nuclear proteinsclearly showed that an endogenous plant m6A writer(s) couldmethylate the UGUA sites of the two transcripts we had selectedrandomly fromamong theECT2binding sites common to both them6A-seq and FA-CLIP data sets. To explore the in vitro methyl-ation activity in greater detail, we built three additional RNAmoleculescontaininguniformly [g-32P]-adenosine-labeledmotifs:(1) UGUA, (2) GGACU, and (3) the randomly generated sequenceCUAUG. Recall that the GGACU motif is the known m6A con-sensus sequence in humans and other eukaryotic organisms.Assays with these three RNA substrates revealed that the en-dogenous Arabidopsis m6A writers present in nuclear proteinextracts have more efficient methylation activity for the UGUAsequence than for GGACU or the random sequence CUAUG(Figures 5B and 5C). Collectively, these results demonstrate thattheUGUAsequence is a uniquem6Amotif that canbemodifiedbyendogenous Arabidopsis m6A writer(s).

Given that human YTH-domain family proteins can read m6Amodified RRACH sites (especially the GGAC sequence) (Wanget al., 2014, 2015; Xiao et al., 2016), we next compared ECT2’sbinding affinity toward the UGUA and GGACU motifs. We usedEMSA assays with synthetic 42-mer RNAs to analyze the bindingaffinitiesof theECT2m6Areaderproteinwith theURUAYmotif, thepreviously characterized GGACU m6A motif, and the randomlygenerated sequence CUAUG. A total of six 42-mer RNAs weresynthesized: all shared the same sequence, except for a central5-nucleotide region comprising UGUXA, GGXCU, or the randomCUXUG,whereX represents anadenosinewith orwithout them6Amodification.Whenwe exposed these 42-mer RNAs to a range ofconcentrations of recombinant GST-ECT2 protein, we found thatECT2 only bound to the 42-mer RNAs harboring the m6A modi-fication. ECT2wasable to recognizebothmethylatedUGUAAandmethylated GGACUwith similar binding affinities; ECT2’s bindingaffinity for them6A-modified randomsequencewas;3-fold lower

than that for the two m6A target motifs (Figure 5D). Human YTH-domain proteins read the m6A-modified GGAC sequence; wefound that the Arabidopsis YTH-domain family protein ECT2can bind to the m6A-modified UGUA motif. ECT2’s bindingaffinity for m6A-modified UGUA is similar to human YTH-do-main family proteins’ binding affinity for m6A-modified GGACU(Wang et al., 2014), suggesting that ECT2 may function throughspecific recognition of m6A-methylated UGUA in plant cells. Col-lectively, our results demonstrate that ECT2 reads the plant-specificm6A motif, URUAY, which is present in the 39 UTRs of mRNAmolecules in Arabidopsis.

ECT2 Is Involved in 39 UTR Processing and mRNA Stability

For context, polyadenylation—the additionof apoly(A) tail to amRNAmolecule—is initiated by so-called poly(A) signals, which are dividedinto four different groups: cleavage elements at the poly(A) site, nearupstream elements located at positions ;20 to 30 nucleotides up-stream of the poly(A) site, far upstream elements (FUEs) that occur ina window from;40 to 150 nucleotides upstream of the poly(A) site,and downstream elements located 20 to 40 nucleotides beyond thepoly(A) site (Shen et al., 2008). Having established that ECT2binds totheURUAYm6Amotif (R=G>A,Y=U>A,wherethemajority [over90%]is UGUAY), we noted that thisUGUA sequence has been reported tobe a poly(A) signal for polyadenylation (Shen et al., 2008; Yang et al.,2011; Elkon et al., 2013; Masamha et al., 2014). In Arabidopsis, thissequencetypicallyoccurs inFUEs [;40–150nucleotidesupstreamofthe poly(A) site] (Loke et al., 2005; Shen et al., 2008).To investigate whether the UGUA sites that are bound by the

ECT2 reader are somehow related to poly(A) signal(s), we cal-culated the distance between poly(A) site and either the ECT2binding sites (FA-CLIP peaks) or mutation peaks inMock. Unlikein the Mock results, the positions of the ECT2 binding sites areconsistently located ;30 to 150 nucleotides upstream of thepoly(A) sites in a region characteristic of FUE polyadenylationsignals (Supplemental Figure 8A). Subcellular localization as-says showed that ECT2 occurs in the nucleus and cytoplasm(Figure 3).We thus propose that ECT2 binds to them6A-modifedpoly(A) signal sequence UGUA in the FUEs of its target tran-scripts and thereby promotes their polyadenylation, suggesting

Figure 4. (continued).

was digested by ProteaseK to leave an amino acid tag (that is, cross-linked residue) on ECT2-boundRNA fragments, whichwill inducemutation by reversetranscription. ECT2-bound RNA fragments were isolated, converted to cDNA (harboring mutations), and sequenced (termed FA-CLIP-ECT2). FA-CLIP ofwild-type Col-0 (termed Mock) was also performed as described for FA-CLIP-ECT2. Total RNA with rRNA depletion from ECT2pro: ECT2-Flag/ect2-1seedlings was used for RNA-seq (termed Input). IP enrichment-based ECT2 binding peaks (termed FA-CLIP enrichment peaks) were obtained by sub-tracting theenrichment peaks inMock (Mock versus Input) from the enrichment peaks in FA-CLIP-ECT2 (FA-CLIP-ECT2 versus Input). Themutation-basedECT2 binding peaks (termed FA-CLIP mutation peaks) are determined by subtracting the mutation peaks in Mock from the mutation peaks in FA-CLIP-ECT2. ECT2 binding peaks (termed “FA-CLIP peaks”) are defined as overlapping FA-CLIP mutation peaks and FA-CLIP enrichment peaks.(B) Overlap of two biological replicates of the number of FA-CLIP peaks identifying ;6000 high-confidence ECT2 binding sites (i.e., ECT2 targets)corresponding to 3680 unique transcripts/genes. Biological replicates are parallel measurements of biologically distinct samples.(C) Pie chart presenting RNA types (that is, transcript species) of ECT2-bound transcripts.(D) Overlap of the identified ECT2 binding peaks (FA-CLIP peaks) and m6A peaks.(E)Piechart presenting the fractionofECT2bindingpeaks ineachof the threenon-overlapping transcript segments (59UTR, codingsequence [CDS], and39UTR).(F)Metageneprofilesof thedistributionofECT2bindingpeaksalonganormalized transcriptcomposedof three rescalednonoverlappingsegments listedonthe x axis.(G) Binding motif identified by HOMER based on all identified ECT2 binding peaks (6047 peaks; P = 1 3 102215).

m6A Read by ECT2 Affects Trichome Morphology 975

that ECT2might regulate alternative polyadenylation and 39UTRprocessing in the nucleus. ECT2 may selectively bind m6A-containing poly(A) signal FUEs, thereby recruiting the poly-adenylation machinery to undertake alternative polyadenylationand further 39 UTR processing (Supplemental Figure 8C).

Given that ECT2 is localized to both the nucleus and cytoplasmand that the cytoplasmic m6A reader YTHDF2 degrades m6A-modified transcripts in human, we thought it might be informativeto examinewhether ECT2 influencesmRNAstability; we thereforeperformedmRNA sequencing (mRNA-seq) in wild-type Col-0 and

Figure 5. ECT2 Recognizes URUAY, a Plant-Specific 39 UTR m6A Motif That Can Be Methylated by Arabidopsis Endogenous m6A Writer Proteins.

(A)TLCresults froman invitromethylationassayusingnuclearextractswithsitespecific labeledsubstrates. pA indicates [g-32P]-labeledadenosine.Oligo1,59-CUCGAUCCUUUUUGUpAGUUUCCGAC-39; Oligo 2, 59-UAUGCGUCUACUGUpACGGUUGAAUUU-39.(B) TLC results from an in vitro methylation assay using nuclear extracts with site-specific labeled oligo RNA substrates. The RNA probes were as follows,and pA indicates [g-32P]-labeled adenosine. UGUpA, 59-CUCGAUCCUUUUUGUpAGUUUCCGAC-39; GGpACU, 59-CUCGAUCCUUUUGGpACU-GUUUCCGAC-39; CUpAUG, 59-CUCGAUCCUUUUCUpAUGGUUUCCGAC-39.(C) Quantification of m6A/A ratios in (B), as calculated by densitometry using Image J.(D) EMSAmeasuring the dissociation constant (Kd, nM) of GST-ECT2 with methylated and unmethylated RNA probes. The 4 nmol RNA probe was labeledwith [g-32P], and GST-ECT2 concentration ranged from 10 to 2000 nM. Oligo RNA, 59-AUGGGCCGUUCAUCUGCUAAAA(GGXCU/UGUXA/CUXUG)GCUUUUGGGGCUU*G*U-39, X = A/m6A. The asterisk indicates that thiol-protected bases were used for the experiment.

976 The Plant Cell

Figure 6. ECT2’s m6A Binding Function Increases mRNA Stability of Three Trichome Morphogenesis-Related Genes.

(A) Three trichome morphogenesis-related genes are m6A methylated and are ECT2 targets.(B) FA-RIP-qRT-PCR validation of the binding affinity of ECT2 to TTG1, ITB1, and DIS2. AT2G07689 is the internal control gene. Data are represented asmeans 6 SE, n = 2 biological replicates 3 3 technical replicates.

m6A Read by ECT2 Affects Trichome Morphology 977

ect2-1 seedlings (Supplemental Data Set 3). The transcripts in thedata set were analyzed as reads per kilobase per million reads(RPKM), and any transcript with an RPKM value <1 was ex-cluded. The filtered transcripts were classified into separategroups defined based on our previous experimental results fromthis study: the ECT2 FA-CLIP targets (i.e., ECT2 binding tran-scripts, 3554genes), the FA-CLIPpeaks that overlappedwithm6

A peaks from our m6A methylome analysis (FA-CLIP+m6A tar-gets, 2007genes), and the 1126 remaining genes that hadRPKMvalues >1 but were not among the ECT2 binding transcripts fromthe FA-CLIP analysis (termed “non-targets”). Analysis of tran-script accumulation in ect2-1 and wild-type plants revealed thatthe mutant plants had significantly reduced accumulation of themajority of ECT2 binding transcripts relative to non-targets. Asimilar decrease was observed among the majority of ECT2binding transcripts containingm6Amodifications (SupplementalFigure 8B). These exciting results directly suggest that ECT2does not function as the same type of reader as human YTHDF2,which has been shown to promotemRNAdegradation. Rather, itindicates that ECT2 might function in facilitating m6A-mediatedmRNAstability in thecytoplasm (Supplemental Figure8C).Giventhat the length of the 39UTR of a transcript affects the binding ofmicroRNA and RNA binding proteins for mRNA degradation(Tian and Manley, 2017), we speculate that this observed in-creased ECT2 target mRNA abundance (or stability) could alsobe a consequence of ECT2’s role in promoting m6A-mediatedalternative polyadenylation of 39 UTRs (Supplemental Figure8C). The detailed mechanisms remain to be investigated in thefuture.

ECT2 Functions in Important Biological Pathways, IncludingTrichome Morphogenesis

OurmRNA-seqdataset (SupplementalDataSet1) shows that therewere 105 significantly upregulated genes and 92 significantlydownregulated genes in the ect2-1mutant (cutoff criteria of FPKMfold change$ 2 andP value< 0.05) (Supplemental Data Sets 4 and5). To explore which cellular processes and signaling pathwaysECT2 may be involved in, we used DAVID to performed GeneOntology (GO) analysis of the ECT2-targeted genes identified byFA-CLIP analysis and the 197 differentially expressed genesidentified by mRNA-seq analysis (Supplemental Figure 9). GOanalysis revealed that the ECT2-targeted genes were positivelyenriched in several pathways, including mRNA processing, phos-phorylation, responses to temperature stimulus, and trichomemorphogenesis. GO analysis of differentially expressed genes in

ect2-1 based on mRNA-seq revealed strong enrichment for termsincluding, among others, response to temperature stimulus andresponse to endogenous stimulus. These predictions about genefunction were informative when viewed in the context of the ect2mutant phenotypes.

ECT2’s m6A Binding Function Stabilizes TrichomeMorphogenesis-Related Genes

Next,we further explored themechanismunderlying theabnormaltrichome branching phenotype of ect2 plants. In the GO termanalysis of ECT2 FA-CLIP binding transcripts, we found that12 ECT2-targeted genes were related to trichomemorphogenesis.We selected three such genes (TRANSPARENT TESTAGLABRA1[TTG1], IRREGULARTRICHOMEBRANCH1 [ITB1],andDISTORTEDTRICHOME2 [DIS2]), each of which contained overlapping ECT2binding sites and m6A sites in their 39 UTRs, as characterized in ourFA-CLIP andm6A-seq data sets (Figure 6A). We performed FA-RIP-qPCR and m6A-IP-qPCR assays using 14-d-old seedlings to verifythat thesethreegenesdid indeed(1)containm6Asites in their39UTRsand (2) were bound by ECT2 (Figures 6B and 6C), supporting thenotion that our FA-CLIP and m6A-seq data were both accurate androbust. We then measured the expression levels of these threetranscripts in 14-d-old seedlings. The expression levels of TTG1,ITB1, and DIS2 were significantly reduced in ect2-1 compared withthe wild type (Figure 6D), which is consistent with the abnormal tri-chomemorphology phenotype observed in themutants. Taking intoaccount the proposed functions of ECT2 (Supplemental Figure 8C),we reasonedthatECT2’sm6Abinding functionstabilizesTTG1, ITB1,andDIS2 transcripts. To investigate this possibility,wemeasured thelifetimes of these three transcripts by blocking transcription withactinomycin D. Transcription inhibition assays showed that TTG1,ITB1, and DIS2 transcripts were degraded more rapidly in ect2-1plants compared with wild-type plants, whereas the mRNA lifetimesof negativecontrol geneAT2G07689 inect2-1and thewild typeweresimilar (Figure 6E). Collectively, we demonstrated that ECT2’s m6Abinding function stabilizes TTG1, ITB1, andDIS2 transcripts, therebyaffecting trichome morphogenesis (Figure 6F). The subcellular lo-calization of ECT2 and high-throughput sequencing data analysissuggestedthatECT2mightplay roles in regulating39UTRprocessingin thenucleusanddirectly facilitatingmRNAstability in thecytoplasm(Supplemental Figure 8). The stabilization of TTG1, ITB1, and DIS2transcriptsmaybeaconsequenceof oneof the aforementioneddualfunctionsofECT2.TheexactpathwayunderlyinghowmRNAstabilityis affected by ECT2’s m6A binding function remains to be furtherinvestigated.

Figure 6. (continued).

(C) m6A-IP-qRT-PCR validation of the m6A peaks in TTG1, ITB1, and DIS2. AT2G07689 was used as the internal control gene. Data are represented asmeans 6 SE, n = 2 biological replicates 3 3 technical replicates.(D) Relative mRNA levels of TTG1, ITB1, and DIS2 in 14-d-old wild-type and ect2-1 Arabidopsis seedlings, with ACTIN2 as a reference gene. Data arerepresented as means 6 SE, n = 3 biological replicates 3 2 technical replicates. **P < 0.01 and ***P < 0.001 by t test (two-sided).(E) ThemRNA lifetime of TTG1, ITB1, andDIS2 in the wild type and ect2-1. ECT2 non-target AT2G07689 was used as the negative control. Seven-day-oldseedlings treated with actinomycin D were used for the transcription inhibition assays with 18S as the internal control gene. Data are represented asmeans 6 SE, n = 2 biological replicates 3 3 technical replicates. TI, transcription inhibition.(F) Proposed model describing how the m6A binding protein ECT2 regulates Arabidopsis trichome morphology.

978 The Plant Cell

DISCUSSION

Previous studies ofm6Awriters and erasers in plants revealed thatthe m6A modification plays fundamental roles in plant de-velopment (Zhong et al., 2008; Shen et al., 2016; Duan et al., 2017;Martínez-Pérez et al., 2017; Ruzi�cka et al., 2017). The charac-terization of m6A readers is an essential prerequisite for exploringand coming to understand the roles ofm6A in plants; however, theidentities of plant m6A readers have been unclear. Here, weshowed that ECT2 is an m6A reader in Arabidopsis whose m6Abinding function is required for normal trichome morphology(Figure 2). Previous studies have reported increased trichomebranching in conditional knockout MTA plants and in plantsoverexpressing FIP37, suggesting that m6A is involved in regu-lating trichomemorphology (Vespa et al., 2004; Bodi et al., 2012).This study deepens our understanding of how m6A affects tri-chome cell development by demonstrating the functional impactof the m6A reader activity of ECT2 on trichome branching.

We developed the convenient FA-CLIP method, which allowedus to identify ECT2-RNA interaction sites at the transcriptome-wide level (Figure 4A). Using these methods, we characterizedtranscriptome-wide ECT2 binding sites; our findings support thenotion that ECT2 recognizes m6A-modified mRNA in plant cells(Figure 4D). Transcriptome-widecharacterizationof ECT2bindingsites revealed that ECT2 binding sites are dramatically enrichedwithin the 39 UTRs of mRNA transcripts and feature a highly con-served sequence motif: URUAY (R=G>A, Y=U>A, where the ma-jority [over90%] isUGUAY) (Figures4Eto4G),which isdistinct fromthe target motif of human YTH-domain family proteins (YTHDF1/YTHDF2/YTHDC1) (Wang et al., 2014, 2015; Xiao et al., 2016). Ourresults demonstrate that this UGUA motif can be methylated byendogenous Arabidopsis m6A writers and can be recognized byECT2. ECT2’s binding affinity for m6A-modified UGUA is similar tothe binding affinity of human YTH-domain family proteins for m6A-modified GGACU, suggesting that ECT2may function through thespecific recognition of m6A-methylated UGUA in plant cells. Ourexperimental evidence supports the notion that the m6A readerECT2bindingmotif UGUA sequencewe identified inArabidopsis isa plant-specific m6A consensus motif (Figure 5).

The subcellular localization of ECT2 in the nucleus and cyto-plasm indicates that ECT2 has at least two functions. The UGUAsequence has been confirmed as a poly(A) signal for poly-adenylation in many organisms (Graber et al., 1999). In Arabi-dopsis, theUGUAsignal is located in FUEs that occur in awindowfrom ;40 to 150 nucleotides upstream of the poly(A) site (Lokeet al., 2005). Our study identified the ECT2 binding motif UGUAand found the ECT2 binding sites are located ;30 to 150 nu-cleotides upstreamof poly(A) sites, strongly suggesting that ECT2binds to poly(A) signals in FUEs and functions in polyadenylation(Supplemental Figure 8A). Analysis of the humanm6Amethylomeand m6A writer subunit suggested that m6A functions in poly-adenylation (Ke et al., 2015; Molinie et al., 2016; Yue et al., 2018),but theunderlyingmechanismand the alternativepolyadenylationfunction-related m6A reader protein have not been clarified. Ourexperimental data suggest that the Arabidopsis m6A reader ECT2functions in polyadenylation and 39UTRprocessing in the nucleus.The molecular mechanism of m6A-meditaed polyadenylation inplants may be unique and distinct from that in human or other

eukaryotes. We proposed amodel for a mechanismwherein ECT2selectively binds to m6A-containing poly(A) signal FUEs, therebyrecruiting the polyadenylation machinery to undertake alternativepolyadenylation and further 39 UTR processing (SupplementalFigure 8C). Furthermore, the finding that plants with a functioningECT2 reader exhibit increased accumulation of ECT2-targetedtranscripts suggests that ECT2 in the cytoplasm might promotemRNA stability (Supplemental Figure 8B). On the other hand,considering that alternative polyadenylation of 39UTRs is known toaffectmRNAstabilitybyaffecting thebindingofmiRNAsand39UTRbinding proteins (Kedde et al., 2007; Chen, 2009; Tian andManley,2017), ECT2 in the nucleus might also influence mRNA stabilitythroughitspossible roleasamediatorofalternativepolyadenylationand 39 UTR processing (Supplemental Figure 8C).Ourmechanistic studies confirmed thatmRNAstability affected

by the m6A binding function of ECT2 controls trichome mor-phology. ECT2 bound to three trichome morphogenesis-relatedtranscripts, TTG1, ITB1, andDIS2, containing m6Amodifications.Disruption of ECT2 accelerated the mRNA degradation of TTG1,ITB1, and DIS2, thereby affecting trichome branching (Figure 6).In some ways similar to the demonstrated role of histone

methylation in the epigenetic regulation of the floral transition inArabidopsis (Henderson and Dean, 2004), the epitranscriptomicmodificationm6Ahasbeen found tocontrol embryodevelopment,flowering time,and trichomemorphology, amongotherprocesses(Zhong et al., 2008; Bodi et al., 2012; Duan et al., 2017). Ourcharacterization of the function of the Arabidopsis m6A readerECT2 in mRNA processing sheds light on the underlying mech-anisms through which m6A functions in RNA metabolism spe-cifically and more broadly in plant development and physiology.

METHODS

Plant Materials and Growth Conditions

The Arabidopsis thaliana genotypes used in this study include wild-type(Col-0) and two T-DNA insertion mutant lines, ect2-1 (SALK_002225) andect2-2 (SAIL_11_D07), which were obtained from the Arabidopsis Bi-ological Resource Center and confirmed as homozygotes (SupplementalFigures 2A and 2B). Transgenic plants (ECT2pro:ECT2-Flag/ect2-1,ECT2pro:ECT2m-Flag/ect2-1, ECT2pro:ECT2-eGFP/ect2-1, and ECT2pro:GUS) were obtained by transforming plasmids into either ect2-1 or wild-type (Col-0) plants. All Arabidopsis plants were grown on 0.53Murashigeand Skoog (1/2 MS) nutrient agar plates (PhytoTechnology Laboratories)for 14 d and then the seedlingswere transfer to soil. The plantswere grownunder the following conditions: 16 h light/8 h dark at 22°C, and light in-tensities of 90 to 120 mE m22 s21 (provided by fluorescent tubes witha white 4100K spectrum purchased from Bainuo).

Plasmid Construction

Total RNA was extracted using TRIzol reagent (Thermo Scientific) andreverse transcribed into cDNA using SuperScript III reverse transcriptase(ThermoScientific). The full-length ECT2 cDNAwas amplified via PCR andcloned into pGEX-6p-1 between the BamHI and XhoI sites for proteinexpression and purification. Two residue sites weremutated (SupplementalFigures 3 and 4) in the YTH domain of ECT2 to generate a putative bindingfunction-abolishedformofECT2,ECT2m(ECT2W521A/W534A).Full-lengthECT2 and ECT2m were cloned into a modified pCAMBIA1305 vector be-tween the SalI and PstI sites. The pCAMBIA1305 vector was modified by

m6A Read by ECT2 Affects Trichome Morphology 979

inserting the ECT2 native promoter (2 kb upstream of ECT2) between theEcoRI and XbaI sites and by adding a 33FLAG or eGFP tag sequencebetweentheHindIIIandBstEII sites.Thus, thePECT2-ECT2-Flag,PECT2-ECT2m-Flag, andPECT2-ECT2-eGFPconstructswereobtained, andanECT2promoterGUSconstruct (PECT2-GUS)wasgenerated toanalyzeECT2expression.ECT2wascloned intopCAMBIA1300 (C-terminaleGFPbetweentheHindIII andKpnIsites) between the SacI and HindIII sites to generate P35S-ECT2-eGFP.Schematic representations of the constructs are shown in SupplementalFigures2Dto2G.AllconstructswereconfirmedbySangersequencing,andtheprimers used in their generation are shown in Supplemental Table 1.

Protein Expression and Purification

GST-ECT2 and GST-ECT2m proteins were purified using the samemethod.Plasmidswere transformed intoEscherichiacolistrainBL-21Goldcompetent cells. The E. coli cells were grown at 37°C to an OD600 of 0.6 to0.8, and recombinant protein expression was then induced with 0.3 mMIPTG. After 20 h of incubation at 16°C, the pellet from each 2-liter culturewas collected, resuspended in 30 mL of lysis buffer (10 mM Tris-HCl, pH8.0, 500 mM NaCl, 1 mM PMSF, 3 mM DTT, and 5% glycerol), and son-icated for10min.Thesamplewascentrifugedat13,000 rpmfor30min, andthe supernatant was filtered through a 0.45-mm filter membrane. The fil-tered supernatant was loaded onto a GST affinity column (GE Healthcare)that had been balanced with equilibrium buffer (10 mM Tris-HCl, pH 8.0,500 mM NaCl, and 3 mM DTT). After washing the column with 30 mL ofequilibrium buffer, the sample was eluted using 15 mL of elution buffer(10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM reduced glutathione, and3 mM DTT). The crude samples were further purified using a Superdex75 size exclusion column (GE Healthcare) with storage buffer (10 mM Tris-HCl, pH 7.5, 200 mM NaCl, 3 mM DTT, and 5% glycerol). The purifiedprotein was stored at 280°C.

Plant Transformation

Transformation of Arabidopsis was performed using the floral dip method(Clough and Bent, 1998) with Agrobacterium tumefaciens strain GV3101.The transgenic plants were screened on 1/2 MS medium containing hy-gromycin B and further identified by qPCR and protein immunoblotting.ECT2pro:GUS transgenic plantswere obtained by transforming thePECT2-GUSconstruct into 6-week-old wild-type Col-0 plants. The complementationlines ECT2pro:ECT2-Flag/ect2-1, ECT2pro:ECT2m-Flag/ect2-1, and ECT2pro:ECT2-eGFP/ect2-1 were generated by transforming the PECT2-ECT2-Flag,PECT2-ECT2m-Flag, andPECT2-ECT2-eGFPconstructs, respectively, into6-week-old ect2-1 plants.

GUS Staining Assay

TheGUS assaywas performed using a staining kit (Coolaber).ECT2pro:GUStransgenic plant tissues were incubated overnight at 37°C in staining buffer.After terminating the reaction, the tissueswereclearedwith70%ethanol andimaged.

Gene Expression Analysis by RT-qPCR

TotalRNAwasextracted from14-d-oldArabidopsis seedlingsusingTRIzolreagent, and the total RNA was treated with DNaseI (NEB). First-strandcDNA was synthesized using SuperScriptIII (Thermo Scientific) and ran-dom primers. qPCR was performed using Ultra SYBR Mixture with ROX(CWBIO) on a ViiA 7 Dx instrument (Applied Biosystems). All samples wereanalyzed in duplicate, and the relative expression levels were determinedbased on ACTIN2 as the internal control. The 22DDCT method was used tocalculate thegene expression levels. TheqPCRprimers used forECT2 andACTIN2 are listed in Supplemental Table 1; the qPCR primers used for the

12otherYTH-domain family geneswerepreviously reported (Li et al., 2014)and are also listed in Supplemental Table 1.

Subcellular Localization

Theroottipsof14-d-oldtransgenicArabidopsisECT2pro:ECT2-eGFP/ect2-1plantsandthe leavesof3-week-oldNicotianabenthamianaplants transientlyexpressingP35S-ECT2-eGFP (72 hafter infiltration)wereused toexamine thesubcellular localization of ECT2. The P35S-ECT2-eGFP plasmid was trans-formed into Agrobacterium GV1301 cells. Cultures were grown at 28°C toOD600 of;1.0. The cellswere resuspended in infiltration buffer (10mMMES,pH5.6,10mMMgCl2, and100mMacetosyringone) toadjust theOD600 to0.6and infiltrated into3-week-oldN.benthamiana leaves.Confocal imagesweretaken on an LSM 700 confocal laser scanning microscope (Zeiss).

Cryo-SEM Analysis of Arabidopsis Trichomes

Cryo-SEM was used to study the trichome branches of 3-week-old wild-type, ect2-1, ect2-2 ECT2pro:ECT2/ect2-1, and ECT2pro:ECT2m/ect2-1Arabidopsis leaves (the third and the fourth leaves). The analyses wereperformed as previously described (Esch et al., 2004) with minor mod-ifications. The equipment included the FEI Helios NanoLab G3 UCscanning electron microscope (Thermo Scientific) and the QuorumPP3010T workstation (Quorum Technologies), which had a cryo prepa-ration chamber connected directly to the microscope. The Arabidopsisleaveswere frozen in subcooled liquid nitrogen (2210°C) and transferred invacuo to the cold stage of the chamber, where sublimation (290°C, 5 min)andsputter coating (10mA,30s)withplatinumwereconducted.Finally, thesamples were transferred to another cold stage in the scanning electronmicroscope and imaged.

FA-CLIP

Fourteen-day-old wild-type and ECT2pro:ECT2-Flag/ect2-1 seedlingsgrownon1/2MSplateswereharvestedandsubjected toFA-CLIP toobtainthe Mock and FA-CLIP-ECT2 sequencing data sets. Total RNA isolatedfrom ECT2pro:ECT2-Flag/ect2-1 seedlings using TRIzol reagent wassubjected to rRNA removal with a RiboMinus Plant Kit for RNA-seq(ThermoScientific). The total RNAwith rRNA depletion (50 ng) was used togeneratea libraryusingaNEBNextUltraRNALibraryPrepKit for Illuminakit(NEB) to produce the Input.

The FA-CLIP procedure was performed as follows: (1) Formaldehydefixation and cross-linking. Col-0 (Mock) and ECT2pro:ECT2-Flag/ect2-1seedlings (FA-CLIP-ECT2)were fixedon ice for 30min in 1%formaldehydesupplemented with PMSF under a vacuum. After removing the 1%formaldehyde solution, 125 mM glycine solution was used to quench thereaction for 5 min under a vacuum. The tissues were washed three timeswith precooledwater, and all of thewaterwas then removed by blotting thetissue with a paper towel. The tissues were frozen in liquid nitrogen andstored at 280°C or used directly for the next step in the protocol.

(2) Immunoprecipitation. Three grams of seedling material was groundinto a fine powder in liquid N2. Immediately after the samples turned darkgreen, they were combined with 3 mL of lysis buffer (150 mM KCl, 50 mMHEPES, pH 7.5, 2 mM EDTA, 0.5% Nonidet P-40 [v/v] 0.5 mM DTT, 2 mMEDTA, 13 cocktail protease inhibitor [Roche], and 40 units/mL RiboLockRNaseInhibitor [ThermoScientific]). Thesamplesweremixedwell, incubatedwith rotation at 4°C for 20 min, and centrifuged at 15,000 rpm for 30 min at4°C. The supernatant was filtered through a 0.22-mm membrane syringe.Agarose beads (100 mL; Promega) were washed twice with NT2 buffer(200 mM NaCl, 50 mM HEPES, pH 7.5, 2 mM EDTA, 0.05% Nonidet P-40,0.5 mM DTT, 40 units/mL RiboLock RNase Inhibitor, and 13 cocktail pro-tease inhibitor), added to the sample lysate, and incubated for 1 h at 4°C forprehybridization.Thesupernatantwascollectedasapreclearedsample.The

980 The Plant Cell

OD260 of the precleared sample was measured by diluting 1 mL lysate in500 mL ammonium acetate buffer. The total absorbance units were equal to500-fold OD260 multiplied by the total volume in milliliters. Turbo DNase(2000 units; Thermo Scientific) and 1000 units of RNase T1 (Thermo Sci-entific) were added per 25 absorbance units of the sample to partially digestthe sample at 22°C for 15min, and the samples were then placed on ice for2 min. Anti-Flag M2 magnetic beads (100 mL; Sigma-Aldrich) were pre-washed (43) with 600 mL NT2 buffer. Col-0 and ECT2pro:ECT2-Flag/ect2-1samples were incubated with the washed beads separately and rotatedcontinuously at 4°C for 4 h. The beadswere collected andwashed five timeswith 1 mL of ice-cold NT2 buffer and rotated continuously at 4°C for 5 mineach time.Thesamplewas resuspended into 400mLNT2buffer, followedbythe addition of 10 units/mLRNase T1. The samplewas incubated at 22°C for15min todigest theRNA toa size of;30nucleotides.After incubation on icefor5min,thebeadswerewashed(43)with500mLofhigh-saltbuffer (500mMKCl, 50 mM HEPES, pH 7.5, 0.05% Nonidet P-40 [v/v], 0.5 mM DTT,200units/mLRNase inhibitor, and13cocktail protease inhibitor). Thebeadswere resuspended into 400 mL 13T4 PNK buffer prior to the next step.

(3) End repair. After adding 5 mL of T4 PNK (NEB), the reaction wasincubatedat37°C for1h. Following theadditionof 4mLATP (NEB) and5mLT4 PNK, the sample was incubated for an additional 30 min. The beadswere washed (33) with 800 mL of 13 T4 PNK buffer.

(4)ProteaseKdigestion.Thebeadswere resuspended into200mLof13protease K buffer (50 mM Tris-HCl, pH 7.5, 75 mMNaCl, 6 mM EDTA, and1% [w/v] SDS), followed by the addition of 2 mg/mL protease K (NEB) andincubation at 50°C for 30 min.

(5) RNA recovery. The RNA was recovered by phenol/chloroform ex-traction followed by ethanol precipitation.

(6) Library construction and sequencing. The concentration of the re-covered RNA was measured using a Qubit kit (Thermo Scientific). Fiftynanograms of RNA was used to generate the library using an NEB NextMultiplex Small RNA Library Prep Set for Illumina (NEB).

(7) After quality control and quantification, the libraries were sequencedon the Illumina HiSeq 2500 SR50 platform.

EMSA

EMSAwas performed following a previously reportedmethod (Wang et al.,2014) with minor modifications. (1) To assay the binding affinity of GST-ECT2 and GST-ECT2m, the following fluorescently labeled RNA oligo-nucleotideswereused:59-FAM-UCUUUUGUXAGACUUGUACUCUUUA-39,where X indicates either an A or an A with m6Amodification. The RNA probeconcentrationwas4nmol.TheconcentrationofGST-ECT2andGST-ECT2mranged from 0 to 2000 nM. (2) For the motif recognition assay, [g-32P]-radiolabeledRNAprobeswereused;RNAwithdifferentmotifswassynthesizedwith the sequence 59-AUGGGCCGUUCAUCUGCUAAAA(GGXCU/UGUXA/CUXUG)GCUUUUGGGGCUUGU-39,whereXindicateseitheranAoranAwithm6A modification. The final concentrations of GST-ECT2 used for EMSAranged from 10 to 2000 nM for the UGUA and CUAUG oligonucleotides andfrom 10 to 500 nM for the GGACU oligonucleotides.

Preparation of Nuclear Extracts from Arabidopsis Seedlings

Nuclear fractions were extracted from 14-d-old Col-0 Arabidopsis seed-lings as follows: Protoplasts were isolated using a previously reportedmethod (Zhai et al., 2009) and separated into the cytoplasmic and nuclearfractions using a nuclear extraction kit (Abcam).

Splint Ligation to Generate a Site-Specific [g-32P]-RadiolabeledRNA Substrate

Splint ligation was performed to generate site-specific radiolabeled RNAsubstrates for in vitro methylation assays using a previously reported

method (Moore and Query, 2000). Briefly, the 59 end of a donor RNA oligowas phosphorylated with [g-32P]ATP in a 5-mL reaction containing 0.9 mL[g-32P]ATP (10mCi/mL), 0.5mL 10PNKbuffer, 1mL 20mMdonor RNA, 1mLT4 PNK (NEB), and 1.6 mL diethyl pyrocarbonate (DEPC) water. The re-action was incubated at 37°C for 2 h and heated at 75°C for 15 min toinactivate the kinase. Next, 1.5 mL of 10 ligation buffer, 1 mL of 20 mMacceptor, 1 mL of 20 mMDNA bridge, and 3.5 mL DEPCwater were added,and the reaction was heated at 75°C for 2 min. The samples were cooledslowly to room temperature. Onemicroliter of 10mM cold ATP and 2 mL ofT4 DNA ligase (NEB) were then added to the samples, which were in-cubated for an additional 4 h at 30°C. The DNA bridge was digested with1 mL of RQ1 RNase-free DNase (Promega) at 37°C for 30 min. Finally, thesamples were incubated at 75°C for 20 min to inactivate the DNase. Theresulting site-specific radiolabeledRNAsubstrateswere used in the in vitromethylation assays.

In Vitro Methylation Assay

Each 50-mL methylation reaction contained 10 mL 53 reaction buffer(75 mMHEPES, pH 7.5, 20%glycerol, 250 mMKCl, 250mMNaCl, 5 mMMgCl2, 2.5 mM DTT, and 100 mM ATP), 5 mL 32 mM SAM (NEB), 1 mLRNase inhibitor (40units/mL), 15mLnuclear extract (1.1mg/mL), 1mLsite-specific radiolabeled RNA substrates, and 16.5 mL DEPC water. Thereaction was incubated at 37°C for various times ranging from 5 min to1 h. The reactions were quenched at 75°C for 20 min. Ten microliters ofmethylationmixture (as well as a 59 radiolabeledm6A control RNA [9-merRNAoligowith am6Aat the 59 terminus]) was added to the samples, alongwith 1 mL Nuclease P1 (Wako) and 1 mL 0.1 M NH4Ac. The samples weredigested at 42°C for 2 h. After the digestion, 0.5-mL sample aliquots wereloaded on cellulose TLC plates (Merck) and separated for 28 h in a so-lutionof isopropanol:HCl:water (70:15:15 [v/v]) . Theplatewasair-dried ina fume hood at room temperature, and radioactive signal detection andquantification were performed with a Typhoon FLA 9500 phosphor im-ager (GE Healthcare).

In Vitro RIP-LC/MS/MS

In vitro pull-down assays were performed as previously reported (Wanget al., 2014), with the following modifications: RNA was extracted from14-d-oldArabidopsis seedlings, and0.2mgofmRNAwasusedas the Inputsample. mRNA (0.8 mg) and GST-ECT2 (final concentration 500 nM) werediluted in 200mL binding buffer (150mMNaCl, 0.1%Nonidet P-40, 10mMTris-HCl, pH 7.4, 40 units/mL RNase inhibitor, and 0.5 mM DTT). Theprotein-RNA solution wasmixed and rotated at 4°C for 2 h. Tenmicrolitersof GST-affinity magnetic beads (Pierce) were resuspended in 50 mL ofbinding buffer after being washed four times with 200mL of binding buffer.The solutionwascombinedwith thebeads and rotated for an additional 2 hat 4°C. The aqueous phase was collected by ethanol precipitation and thepelletwasdissolved in50mLRNase-freewater. The recovered fractionwassaved as the flow-through sample. The beads were washed three timeswith200mLofbindingbuffer, and500mLofTRIzol reagentwas thenadded.The purified RNA was saved as the ECT2-bound sample. LC-MS/MS wasused to measure the level of m6A in the input, flow-through, and ECT2-bound samples.

LC-MS/MS for m6A Quantification

RNA (100 ng) was digested with 1 unit of Nuclease P1 in 50 mL of buffercontaining 10% 0.1 M ammonium acetate (pH 5.3) at 42°C for more than3 h, followed by the addition of 1 unit of shrimp alkaline phosphatase (NEB)and 10% Cutsmart buffer. The mixture was incubated at 37°C for anadditional 3 h. The sampleswere then centrifuged at 15,000 rpm for 30minand the aqueous phase was injected into an LC-MS/MS system.

m6A Read by ECT2 Affects Trichome Morphology 981

Nucleosides were separated using a UPLC pump (Shimadzu) witha ZORBAX SB-Aq column (Agilent) and analyzed by MS/MS using a TripleQuad 5500 mass spectrometer (AB SCIEX) running in positive ion modeand the multiple reaction monitoring feature. MS parameters were opti-mized for m6A detection. Nucleosides were quantified using the nucleo-side-to-base ion mass transitions of m/z 268.0 to 136.0 (A), m/z 282.0 to150.1 (m6A),m/z244.0 to112.0 (C),m/z284.0 to 152.0 (G), andm/z245.0 to113.1 (U). Standard curves were generated using a concentration series ofpure commercial nucleosides (Sigma-Aldrich) analyzed using the samemethod. Concentrations of nucleosides and m6A/A ratio in samples werecalculated by fitting the signal intensities to the standard curves (Wanget al., 2014; Duan et al., 2017).

RNA-Seq

Fourteen-day-oldCol-0andect2-1seedlingswerecollectedandground inliquid nitrogen. Total RNA was extracted by adding TRIzol to the groundsamples. mRNA was then isolated using Dynabead oligos (dT)25 (ThermoScientific). One hundred nanograms of the extracted mRNA was used asthe template for library construction using a NEBNext Ultra RNA LibraryPrep Kit for Illumina kit (NEB).

In Vivo FA-RIP-LC/MS/MS and in Vivo FA-RIP-qPCR

For in vivo FA-RIP, most of the procedures were same as those used forthe FA-CLIP method, but without the RNase T1 digestion step. Theresulting input and IP RNA were separated into two parts: one part wassubjected to LC-MS/MS to measure the m6A level and the other wasreverse transcribed into cDNA and analyzed via qPCR to measure theenrichment levels. AT2G07689 (encoding NADH-Ubiquinone/plasto-quinone [complex I] protein) was used as an internal control, since (1)AT2G07689 mRNA did not show any obvious m6A peak from m6A-seqdata; (2) AT2G07689mRNAwas not enriched by ECT2 from the FA-CLIPdata; (3) AT2G07689 showed relatively invariant expression levels betweenCol-0andect2plants;and(4)AT2G07689isconsideredtobeahousekeepinggene. Samples were performed in 2 biological replicates 3 3 technicalreplicates.

m6A-IP-qPCR

m6A-IP-qPCR was performed as previously reported (Duan et al., 2017).Briefly, 14-d-old wild-type seedlings were used for the IP assays. Fivemicrograms of mRNA was used for each m6A-IP sample. mRNA wasfragmented into ;200-nucleotide molecules with RNA fragmentation re-agents (NEB) and incubatedwith5mgm6Aantibody (SynapticSystems) for4 h at 4°C. The m6A-containing fragments were immunoprecipitated withpreblockedProtein ADynabeads (ThermoScientific) and elutedwith 7mMm6A nucleoside-containing solution. After ethanol precipitation, the m6A-bound fraction RNA, as well as input mRNA, were subjected to reversetranscription and qPCR assays using AT2G07689 as the internal controlgene. Samples were performed in 2 biological replicates 3 3 technicalreplicates.

mRNA Stability Measurements

An mRNA stability measurement assay was performed as previously de-scribed (Duan et al., 2017) withminor modification. Briefly, 7-d-old wild-typeandect2-1Arabidopsis seedlingsgrownon1/2MSmediumwere transferredto 10-cm Petri dishes containing 10 mL 1/2 MS liquid medium. After 30 minincubation, 0.2mMactinomycinDwas added to thebuffer. The tissueswerecollected at 30 min after the transcription inhibitor was added; thesesamples are referred to as 0 h samples. The 3, 6, and 8 h samples werecollected and immediately frozen in liquid nitrogen. The tissues were

stored at 280°C or subjected to total RNA extraction. cDNAs weregenerated with SuperScript IV reverse transcriptase (Thermo Scientific)using the oligo d(T) primer. mRNA levels were quantified by RT-qPCRwith gene-specific qPCR primers (Supplemental Table 1). 18S RNA wasused as the internal control, and the primers for 18S were designed aspreviously reported (Cao et al., 2016). Samples were performed in 2 bi-ological replicates 3 3 technical replicates.

Sequencing Data Processing

RNA-Seq Data Processing

After adapter trimming with Cutadapt (Martin, 2012), the reads weremapped to TAIR10 (Lamesch et al., 2012) using TopHat (Trapnell et al.,2009), and RPKM values were calculated using Cufflink (v2.2.1) (Trapnellet al., 2010). The differentially expressed genes between ect2-1 and thewild typewere definedbased on a cutoff criterion of FPKM fold change$ 2and P value < 0.05. Gene annotations were downloaded from the Ensemblplants database (TAIR10 release 31).

FA-CLIP Data Analysis

After adapter trimming with Cutadapt, the reads were mapped to TAIR10using Bowtie2 (Langmead and Salzberg, 2012). ECT2 binding peaks(termed FA-CLIP peaks) were defined as overlapping peaks of FA-CLIPmutation peaks and FA-CLIP enrichment peaks.

Mutation Peaks

MutationpeakswerecalculatedusingPARalyzer v1.1withdefault settings,except that amutationpeakcontainedat least twomutationsites insteadofonly one mutation site and all types of mutations were taken into accountrather than only T-to-C conversion (Corcoran et al., 2011). The mutation-based ECT2 binding peaks (termed FA-CLIP mutation peaks) were ob-tained by subtracting themutation peaks inMock from themutation peaksin FA-CLIP-ECT2.

PARalyzer is commonly used togenerate a high-resolutionmapof high-confidence RNA-protein interaction sites from CLIP deep-sequencingdatabased on patterns of nucleotide mutations coupled with read density.The parameters used are as follows: bandwidth = 3; minimum_read_count_per_group = 10; minimum_read_count_per_cluster = 5; minimum_read_count_for_KDE = 5; minimum_cluster_size = 10; minimum_conversion_locations_for_cluster = 2; minimum_conversion_count_for_cluster = 1; minimum_read_count_for_cluster_inclusion = 5; minimum_read_length = 13; minimum_number_of_non_conversion_mismatches = 1;and additional_nucleotides_beyond_signal = 5.

Enrichment-Based Peak

Peak calling was performed as previously reported using the same criteriawith enrichment fold (FA-CLIP-ECT2 [or Mock]/Input)$ 2 and FDR < 0.01,and theonlydifferencewas that thealigned readswerenot extendeddue tothe small average fragments size (Ma et al., 2017). IP enrichment-basedECT2 binding peaks (termed FA-CLIP enrichment peak) were obtained bysubtracting the enrichment peaks in Mock (Mock versus Input) from theenrichment peaks in FA-CLIP-ECT2 (FA-CLIP-ECT2 versus Input).

Gene annotationswere downloaded from the Ensembl plants database(TAIR10 release 31). HOMER (Heinz et al., 2010) was used for motifidentification. DAVID (Huang et al., 2007) was used to performed GOanalysis of the ECT2-targeted genes that were identified in the FA-CLIPanalysis and the 197differentially expressedgenes identified in themRNA-seq analysis. The top 40 GO items for the ECT2-targeted genes wereselected for visualization using the interactive graph function of REVIGO(Supek et al., 2011).

982 The Plant Cell

Statistical Analysis

Statisticalanalysiswasperformedbyone-wayANOVAfollowedbyLSDpost-hoc tests usingSPSS20.0 (IBM) (seeSupplemental File 1 forANOVA tables).

Accession Numbers

Sequence data in this study can be found under the following accessionnumbers: ECT1, AT3G03950; ECT2, AT3g13460; ECT3 At5g61020; ECT4,At1g55500; ECT5, At3g13060; ECT6, At3g17330; ECT7, At1g48110; ECT8,AT1G79270; ECT9, At1g27960; ECT10, AT5G58190; ECT11, At1g09810;ECT12,At4g11970;CPSF,At1g30460;TTG1,AT5G24520; ITB1,AT2G38440;DIS2,AT1G30825; andNADH-Ubiquinone/plastoquinone (Complex I) protein,AT2G07689.All rawhigh-throughputsequencingdatahavebeensubmitted toGene Expression Omnibus under accession number GSE108119.

Supplemental Data

Supplemental Figure 1. Relative expression levels of YTH domainfamily genes in Arabidopsis seedlings.

Supplemental Figure 2. ect2 mutants and transgenic lines.

Supplemental Figure 3. Sequence alignment of YTH domain familyproteins in Arabidopsis and human.

Supplemental Figure 4. Binding affinity of GST-ECT2 and GST-ECT2m to m6A methylated RNA probe.

Supplemental Figure 5. Subcellular localization of ECT2 based ontransient expression of 35Spro:ECT2-eGFP in Nicotiana benthamiana.

Supplemental Figure 6. ECT2 binding peaks identified by FA-CLIP.

Supplemental Figure 7. ECT2 binding motif identified by HOMER.

Supplemental Figure 8. ECT2 is involved in 39 UTR processing andmRNA stability.

Supplemental Figure 9. GO analysis of ECT2-related transcripts.

Supplemental Table 1. Primers and oligonucleotide probes used inthis study.

Supplemental Data Set 1. FA-CLIP binding sites of ECT2.

Supplemental Data Set 2. FA-CLIP peaks of ECT2 overlapping withthose identified by m6A-seq.

Supplemental Data Set 3. RNA-seq of wild-type Col-0 and ect2-1.

Supplemental Data Set 4. Downregulated genes in ect2-1 comparedwith the wild type revealed by RNA-seq.

Supplemental Data Set 5. Upregulated genes in ect2-1 comparedwith the wild type revealed by RNA-seq.

Supplemental File 1. ANOVA tables.

ACKNOWLEDGMENTS

We thank W. Qian and H. Guo for providing vectors, X. Hao and Y. Liufor helping with the cryo-SEM imaging, and S. Huang for helpingwith the radiolabeling assay. This work was supported by theNational Basic Research Program of China (nos. 2017YFA0505201,MOST2016YFC0900302, and 2014CB964900) and the National NaturalScience Foundation of China (nos. 21432002, 21372022, and 21210003).

AUTHOR CONTRIBUTIONS

G.J. conceived the project. L.-H.W. performed the experiments withthe help of P.S., Y.W., Z.L., Q.T., Q.Y., Y.X., X.Z., and H.-C.D. G.J. and

L.-H.W. designed the experiments, interpreted the results, and wrote themanuscript.

Received December 4, 2017; revised April 17, 2018; accepted April 30,2018; published April 30, 2018.

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m6A Read by ECT2 Affects Trichome Morphology 985

DOI 10.1105/tpc.17.00934; originally published online April 30, 2018; 2018;30;968-985Plant Cell

Hong-Chao Duan and Guifang JiaLian-Huan Wei, Peizhe Song, Ye Wang, Zhike Lu, Qian Tang, Qiong Yu, Yu Xiao, Xiao Zhang,

ArabidopsisA Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in6The m

 This information is current as of June 23, 2020

 

Supplemental Data /content/suppl/2018/04/30/tpc.17.00934.DC1.html /content/suppl/2018/05/02/tpc.17.00934.DC2.html

References /content/30/5/968.full.html#ref-list-1

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