KETCH1 imports HYL1 to nucleus for miRNA biogenesisin ArabidopsisZhonghui Zhanga,b,1, Xinwei Guoa,c,1, Chunxiao Gea,1, Zeyang Maa, Mengqiu Jianga, Tianhong Lic, Hisashi Koiwad,Seong Wook Yange, and Xiuren Zhanga,2

aDepartment of Biochemistry and Biophysics, Institute for Plant Genomics and Biotechnology, Texas A&M University, College Station, TX 77843;bGuangdong Provincial Key Laboratory of Biotechnology for Plant Development, School of Life Science, South China Normal University, Guangzhou510631, China; cCollege of Horticulture, China Agricultural University, Beijing 100193, China; dDepartment of Horticultural Sciences, Texas A&M University,College Station, TX 77843; and eDepartment of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Republicof Korea

Edited by Xuemei Chen, University of California, Riverside, CA, and approved March 9, 2017 (received for review December 2, 2016)

MicroRNA (miRNA) is processed from primary transcripts with hairpinstructures (pri-miRNAs) by microprocessors in the nucleus. Howcytoplasmic-borne microprocessor components are transported intothe nucleus to fulfill their functions remains poorly understood. Here,we report KETCH1 (karyopherin enabling the transport of thecytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein,a core component of microprocessor in Arabidopsis and functionalcounterpart of DGCR8/Pasha in animals. Null mutation of ketch1 isembryonic-lethal, whereas knockdown mutation of ketch1 causedmorphological defects, reminiscent of mutants in the miRNA path-way. ketch1 knockdown mutation also substantially reduced miRNAaccumulation, but did not alter nuclear-cytoplasmic shuttling ofmiRNAs. Rather, the mutation significantly reduced nuclear portionof HYL1 protein and correspondingly compromised the pri-miRNAprocessing in the nucleus. We propose that KETCH1 transportsHYL1 from the cytoplasm to the nucleus to constitute functionalmicroprocessor in Arabidopsis. This study provides insight into thelargely unknown nuclear-cytoplasmic trafficking process of miRNAbiogenesis components through eukaryotes.

KETCH1 | importin β | HYL1 | miRNA | pri-miRNA processing

MicroRNAs (miRNAs) are a class of noncoding RNAs, 21–24 nt in length, that play vital roles in diverse biological

processes. miRNA biogenesis starts with the transcription of longprimary miRNAs (pri-miRNAs) that contain hairpin-like fold-back. In animals, pri-miRNAs are initially processed to generateprecursor miRNAs (premiRNAs) in the nucleus by a micropro-cessor that minimally comprises of an RNase III enzyme, Drosha,and two molecules of DGCR8/Pasha (1). PremiRNAs areexported to the cytoplasm and further processed by anotherRNase III enzyme, Dicer, to release 21–22 bp miRNA/* duplexes(2, 3). In Arabidopsis, DCL1, one of four Dicer-like enzymes,fulfills consecutive cuts of pri-miRNAs to produce miRNA/*s (4,5); and the entire processes occur in the nucleus and are facilitatedby DCL1 cofactors, including hyponastic leaves 1 (HYL1), Serrate(SE), and other accessory partners (4–8). Once generated,miRNA duplexes are exported to the cytoplasm and loaded intoArgonaute (AGO)-containing RNA-induced silencing complexes(RISCs) to repress the expression of their targeted genes (4, 9).Although the biogenesis and action of mode of miRNAs have

been studied well, how the microprocessor components are trans-located from their cytoplasmic birthplaces to functional niches inthe nucleus has been poorly defined. Nuclear-cytoplasmic traf-ficking of biological macromolecules through the nuclear porecomplex entails transport receptors, namely karyopherin proteins,some of which also referred to importins and exportins. Importinsload their cargos in the cytoplasm and release them in the nucleusupon binding with Ran-GTPase, whereas exportins, coupled withRan-GTPase, associate with the cargos in the nucleus and dis-charge the cargos in the cytoplasm upon hydrolysis of GTP to GDP(10). Several importin β-family members are implicated in themiRNA pathway. Exportin-5 mediates the nuclear export of

premiRNAs in mammalians (11, 12). Importin-8 facilitates therecruitment of AGO2-containing RISC to target mRNAs to pro-mote efficient and specific gene silencing in the cytoplasm, whereasthe protein can also transport AGO2 and AGO2 partners, GWproteins and miRNAs, into the nucleus to balance levels of cyto-plasmic gene-silencing effectors (13–15). Arabidopsis encodes18 importin β-proteins, among which few have also been reportedto function in the miRNA pathway. Loss-of-function of HASTY(HST), an ortholog of human Exportin-5 in plants (16), decreasesthe accumulation of most of tested miRNAs, implying its criticalrole in transporting miRNAs and miRNA pathway components.Supersensitive to ABA and drought 2 (SAD2)/enhanced miRNAactivity 1 (EMA1) appears to negatively impact loading of miR-NAs into AGO1-centered RISC (17). Recently, Transportin1(TRN1) has been identified as a positive regulator in miRNAloading through a suppressor screening of ema1 (18). The bonafide cargos for these importin β-proteins remain to be identifiedin Arabidopsis.Here, we identified karyopherin enabling the transport of the

cytoplasmic HYL1 (KETCH1, meaning a sailboat for HYL1), amember of the importin β-family, as a partner of HYL1. Wefound that a knockdown mutant of ketch1 through an artificialmiRNA (amiR-KETCH1) displayed developmental abnormality,

Significance

Microprocessor components are transported into the nucleusto process primary miRNAs (pri-miRNAs). Hyponastic leaves 1(HYL1) is a core component of microprocessor in Arabidopsis.This study identifies an importin β-protein, KETCH1, as a part-ner of HYL1 protein. KETCH1 knockdown mutation causes de-velopmental defect, reminiscent of mutants in the miRNApathway. The mutation also reduces miRNA abundance butdoes not affect its nuclear-cytoplasmic distribution. Rather,KETCH1 knockdown mutation decreases the accumulation ofHYL1 protein in the nucleus and compromises the HYL1-mediatedpri-miRNA processing, supporting that KETCH1 specifically trans-ports HYL1 into the nucleus to facilitate miRNA production. Giventhat HYL1 and importin β-proteins are all conserved in variousorganisms, this study sheds light on the poorly understoodnuclear-cytoplasmic trafficking of microprocessor componentsthrough eukaryotes.

Author contributions: Z.Z., C.G., and X.Z. designed research; Z.Z., X.G., C.G., Z.M., M.J., andX.Z. performed research; T.L., H.K., and S.W.Y. contributed new reagents/analytic tools;Z.Z., X.G., C.G., Z.M., M.J., T.L., H.K., and S.W.Y. analyzed data; and Z.Z. and X.Z. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1Z.Z., X.G., and C.G. contributed equally to this work.2To whom correspondence should be addressed. Email: xiuren.zhang@tamu.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1619755114/-/DCSupplemental.

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characteristic of the mutants with the miRNA defect. The hypo-morphic mutants also exhibited significantly reduced accumulationof miRNAs, but no change in nuclear-cytoplasmic distribution. Incontrast, the mutation caused less accumulation of nuclear-localizedHYL1 and compromised pri-miRNA processing in the nucleus. Weconclude that KETCH1 imports HYL1 protein from the cytoplasmto nucleus to participate in miRNA biogenesis. This study shed lighton the poorly understood nuclear-cytoplasmic trafficking mecha-nism of microprocessor components in eukaryotes.

ResultsIdentification of KETCH1 as an HYL1 Partner. To identify new compo-nents in microprocessor in plants, we generated stable transgeniclines overexpressing Flag-4Myc (FM) –tagged HYL1 (Fig. S1A). Weisolated the HYL1-containing complex through two-step immuno-precipitation (IP) and resolved the complex on SDS/PAGE gradientgels (Fig. 1A). We selected a few distinct bands clearly visible inHYL1 IP for mass spectrometric analysis. From one band corre-sponding to ∼130 kDa, 15 unique peptides matched specifically toEmbryo Defective 2734 (EMB2734, At5g19820) were recovered fromHYL1 IP but absent in the control IP using Col-0 plants (Fig. 1Aand Fig. S1B). EMB2734 is a member of Karyopherin/Importin βfamily in Arabidopsis (Fig. S2), and we renamed the gene KETCH1.To examine whether KETCH1 is a bona fide partner of HYL1,

we initially conducted yeast-two hybrid (Y2H) assays. Interestingly,KETCH1 strongly interacted with Arabidopsis Ran-GTPase,RAN1, but surprisingly not with HYL1, in the Y2H assays (Fig.S3A). However, Western blot analysis of yeast cotransfected withHYL1 and KETCH1 plasmids showed that these two proteins couldnot be coexpressed in a single colony, implying their potential in-terplay in vivo (Fig. S3B). We next carried out a split luciferasecomplementation assay (LCI) (Fig. S3C). In our LCI assays,HYL1 indeed displayed LUC complementation with KETCH1(Fig. S3C), as did AGO1 with the positive control, Cucumbermosaic virus-encoded 2b protein (CMV 2b) (19), implying thatKETCH1 interacts with HYL1 in plants. Next, we validated theHYL1–KETCH1 interaction in vivo by FRET assay, using HYL1-CFP as a donor and YFP-KETCH1 as an acceptor (Fig. 1B). Wecoexpressed both HYL1-CFP and YFP-KETCH1 in Nicotinabenthamiana cells. Using confocal microscopy, under the laser inthe wavelengths specific for CFP/YFP excision, respectively, weobserved that both HYL1-CFP and YFP-KETCH1 were pre-dominantly localized in the nucleus, but also distributed in cytoplasmthrough the corresponding CFP/YFP channels (Fig. 1B). Consis-tent with this finding, KETCH1 protein is also localized in bothnucleus and cytoplasm in the stable transgenic plants (Fig. S3D).Under the laser in the wavelength specific for CFP excision, we

could observe the strong FRET signals in the nucleus and residueamount in some spots near the nuclear envelope, suggesting thattranslocation of the KETCH1–HYL1 complex may happen nearthe nuclear envelope through KETCH1–HYL1 interaction. Wedid not detect obvious FRET signals in any discrete foci, likeD-bodies, where endogenous HYL1 is typically located (20),suggesting that the HYL1–KETCH1 interaction is dynamic andHYL1 is discharged before its entrance into D-bodies. Alterna-tively, overaccumulation of HYL1 and KETCH1 proteins in theinfiltrated cells might mask the signal from the D-bodies. We alsoconducted co-IP experiments with ketch1-2; PKETCH1-FM-KETCH1 complementation lines using an anti-Flag antibody anddetected co-IP products with antibodies specifically recognizingendogenous proteins. Again, we observed the specific KETCH1–HYL1 interactions in Arabidopsis, but not control proteins, such asactin, and validated this interaction was RNA-independent (Fig.1C). Finally, an in vitro pull-down assay indicated that recombi-nant His-SUMO-KETCH1, but not the control protein STING(21), could physically interact with HYL1 (Fig. S3E). Taking thesedata together, we concluded that KETCH1 is a bona fide partnerof HYL1 protein in vivo.

Loss-of-Function Mutation of ketch1 Causes Developmental Defect.To study biological function of the KETCH1 gene, we genotypeda T-DNA insertion line SALK_050129/emb2734-2, which isrenamed as ketch1-2 here (Fig. S4A). ketch1-2 (+/−) heterozygotesyielded ∼25% abortive seeds in their siliques; and the embryonicdefect of ketch1-2 could be fully rescued by transgene expressingPKETCH1-FM-KETCH1 (Fig. 2A and Fig. S4 A–C). This observa-tion was consistent with a previous report that null mutation of thisgene is embryonic-lethal (22). We next generated knockdowntransgenic lines of ketch1 by expressing artificial miRNA con-structs specifically targeting KETCH1 (amiR-KETCH1) (Fig.S4D). Approximately 50% (∼60 of 118) of 35S-amiR-KETCH1primary transformants exhibited developmental abnormalities withvarying severity (Fig. 2B). The most severe transgenic lines hadspoon-shaped cotyledons and narrow and strongly upward curledleaves, and they died soon after emergence of a few pairs of trueleaves. Lines with less-severe defect also phenocopied hypomor-phic ago1 mutants (Fig. 2B), and these plants survived and werepropagated to higher generations for various assays below. Simi-larly, we have also generated amiR-KETCH1 transgenic plantscontrolled by an XVE-inducible promoter (23). Again, theseplants phenocopied the mutants in the miRNA pathway once theinducer β-estradiol was applied (Fig. 2C).Analyses of small RNA (sRNA) blots using the T2 transgenic

seedlings showed that amiR-KETCH1 were processed and

Fig. 1. KETCH1 is a partner of HYL1 in Arabidopsis. (A) Silver-stained SDS/PAGE of purified HYL1 complexes for proteomics analysis. (B and C) SpecificHYL1-KETCH1 interaction was confirmed in N. benthamiana by FRET (B) and inArabidopsis by co-IP assays (C). In B: CFP, donor HYL1-CFP fluorescence; nFRET,normalized FRET fluorescence; YFP, acceptor YFP-KETCH1 fluorescence. Thecolor bars indicate the scale of the signal strength or the distance between thedonor and the acceptor (DCFP/YFP). In C, actin is a control. (Scale bars, 10 μm.)

Fig. 2. ketch1 knockdown mutant has developmental defects in Arabi-dopsis. (A) The representative seed-set phenotypes in the siliques of Col-0,ketch1-2 heterozygote, and two individual KETCH1 complementation lines.The red arrows indicate abortive seeds. (B) Morphological phenotypes ofketch1 and other miRNA pathway mutants at the 7-d stage. (C) Morpho-logical phenotypes of XVE-amiR-KETCH1 transgenic plants induced with (+)or without (−) 25 μM β-estradiol (β-ES). (Scale bars, 0.4 cm.)

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accumulated to a moderate level (Fig. S4D). Correspondingly,qRT-PCR assays showed that levels of KETCH1 transcript de-creased approximately ∼80–90% compared with wild-type plants(Fig. S4D). Reexamination of the T2 amiR-KETCH1 linesrevealed that the amiR-KETCH1 adult plants typically exhibitedshorter statues and serrated rosette leaves (Fig. S4E). Comparedwith Col-0 plants, the rosette leaves of adult plants readily dis-played yellow and necrosis symptoms, suggestive of acceleratedleaf senescence (Fig. S4 E and F). Taken together, these datashow that knockdown of KETCH1 transcripts clearly impactedgrowth and development in Arabidopsis.

ketch1 Mutation Decreases miRNA Accumulation. Physical interactionof KETCH1 with HYL1 and morphological abnormality of theamiR-KETCH1mutants prompted us to examine whether KETCH1functions in the miRNA pathway. sRNA blot analysis showed thataccumulation of all tested miRNAs was significantly decreased inthe amiR-KETCH1 mutants compared with Col-0 plants (Fig. 3Aand Fig. S5). However, the down-regulation of mature miRNAs inthe amiR-KETCH1 mutants was to a less extent relative to hyl1-2and se-2 null mutants (Fig. 3A and Fig. S5). The scenario was likelybecause of the fact that the amiR-KETCH1 knockdown lines wereweak alleles. Of note, sad2mutants did not display obvious changesin the steady-state level of miRNAs (17), suggesting that KETCH1functions differently from SAD2 in the miRNA pathway.To examine whether miRNA targets are deregulated in the

amiR-KETCH1mutants, qRT-PCR was conducted assays for PHV,MYB33, and targets of miR159 and miR166, respectively. We ob-served that the steady-state levels of these transcripts were clearlyup-regulated in the amiR-KETCH1 mutants compared with Col-0plants, although to a less extent than the up-regulation in hyl1-2and se-2 (Fig. 3B). Of note, ORE1, the target of miR164 (24), wasenhanced in the amiR-KETCH1 mutants but not in hyl1 and se-2mutants. Taken together, all these results indicated that KETCH1is a positive regulator in the miRNA-mediated gene silencingin Arabidopsis.

ketch1 Mutation Elevates Expression Levels of Few Core Componentsin the miRNA Pathways. Up to date, more than a dozen of geneshave been reported to affect miRNA biogenesis and turnoverin vivo (4, 7). Down-regulation of miRNA accumulation in theketch1mutant could result from altered expression of componentsin the miRNA metabolism. To test this theory, we conductedRNA gel blots and qRT-PCR assays for some key components.These results showed that the transcript levels of most of thetested components were essentially not altered in the ketch1 mu-tant, whereas SDN1, the component engaged in miRNA degra-dation (25), was in fact down-regulated (Fig. 3 C and D). Notably,the amount of some core effectors exemplified by HYL1, SE, andAGO1 was even enhanced in the amiR-KETCH1 mutants relativeto Col-0 plants (Fig. 3 D and E). AGO1 and SE are targeted bymiR168 and miR863-3p, respectively, and their up-regulation waslikely because of the reduced levels of the miRNAs in ketch1mutants (Fig. 3A). Thus, our results indicated that impact ofKETCH1 on miRNA expression was not through repressing ex-pression levels of the key components in the miRNA micropro-cessor nor through up-regulating expression of the establishedcomponents tested in the miRNA decay pathway.Because KETCH1 belongs to the importin β-family as SAD2, we

wondered whether KETCH1 acted as SAD2 to impact the loadingof miRNAs into the AGO1 protein. To test this theory, weimmunoprecipitated AGO1 protein from planta and recoveredmiRNAs from the complexes. Despite the level of AGO1 proteinbeing enhanced in the amiR-KETCH1 mutants compared withCol-0 plants, the miRNA amount recovered from AGO1 com-plexes was slightly decreased in the mutants vs. Col-0 plants (Fig.3F). The reduced amount was likely because of the fact thatmiRNA input was lesser and AGO1 protein was more in themutants compared with Col-0 plants, rather than because ofcompromised loading of miRNAs into AGO1 effectors in theketch1 mutant, because unlike SAD2, KETCH1 did not have di-rect interaction with AGO1 (Fig. S6).

Fig. 3. ketch1 mutation reduces miRNA accumulation but not through impacting key components in the miRNA pathways. (A) sRNA blot analyses of theindicated miRNAs in the indicated mutants. U6 is a loading control. Additional replicates are shown in Fig. S5. (B and C) qRT-PCR analysis of selected miRNAtargets (B) and the mRNAs of selected genes in the miRNA pathway (C) in the indicated mutants. EF1A is an internal control. The asterisk (*) indicates thesignificance between mutants and Col-0 control (*P < 0.05) in B and C. (D) RNA blot analyses for AGO1, HYL1, and SE transcripts. rRNAs are the loadingcontrol. (E) Western blot analyses of AGO1, HYL1, and SE proteins in the ketch1 mutants using the indicated antibodies. Actin is a control. (F) The amount ofthe tested miRNAs loaded into RISC in the ketch1 mutants.

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KETCH1 Impacts the Nuclear-Cytoplasmic Distribution of HYL1 Proteinbut Not miRNAs. We next hypothesized that KETCH1 might beinvolved in the translocation of miRNA/*s from microprocessorsbetween the nucleus and cytoplasm. To test this hypothesis, weperformed the nuclear-cytoplasmic fractionation of cell extractsfrom Col-0 and amiR-KETCH1 mutants, and extracted totalRNA from the nuclear and cytoplasmic fractions for RNA gelblots. sRNA blot analysis showed that distribution patterns of alltested miRNAs were approximately the same in the mutants andCol-0 plants (Fig. S7), indicating that miRNAs are unlikely to bethe cargos of KETCH1 protein. In line with this result, weimmunoprecipated KETCH1 and did not recover miRNA fromthe KETCH1 protein (Fig. S6).Importin proteins could also transport protein cargos to the

nucleus (10). HYL1 has been known to be predominantly lo-calized and function in the nucleus (26, 27); however, a recentstudy reported that a substantial amount of HYL1 is present incytosol (28). This observation was reproducible in our hands(Fig. 4A). Because KETCH1 directly interacted with HYL1, wenext investigated whether KETCH1 is involved in nuclear importof HYL1 protein. Western blot for the nuclear and cytoplasmicfractions of proteins were performed with anti-HYL1 antibody.

We quantified nuclear and cytoplasmic signals of pixel intensitiesof tested and control proteins and normalized ratios of nuclear vs.cytoplasmic HYL1 based on the signals of Histone 3 (H3) andRubisco (Rbsc) controls. Upon ketch1 knockdown, the ratio ofnucleus-localized HYL1 relative to the cytoplasmic HYL1 wassignificantly decreased (Fig. 4 A and B). In contrast, lower distri-bution of HYL1 in the nucleus was not observed in the loss-of-function mutants ofHST and SAD2 that are supposed to transportsome components in the miRNA pathway in Arabidopsis (Fig. S8A and B). Furthermore, we examined a few other critical com-ponents in the miRNA pathway, and did not observe any signifi-cant change of nuclear-cytoplasmic distribution of the componentsin the ketch1 mutant (Fig. 4 A and B). Thus, these components—different from HYL1—are not the cargos of KETCH1.To further validate whether KETCH1 imported HYL1 protein,

we expressed HYL1-CFP in the protoplasts prepared from a dif-ferent genetic background, including Col-0, amiR-KETCH1, andsad2-2. A recent study indicates that cytoplasmic partitioning ofCOP1, a well-known RING-type E3 ligase, could protect cyto-plasmic HYL1 from the degradation by certain unknown prote-ases under light conditions (28). However, cytoplasmic HYL1would be easily degraded under dark conditions and MG132treatment could increase its cytoplasmic localization (28). To fa-cilitate the observation of HYL1 localization in the cytoplasm, wekept the transformed protoplasts under light conditions beforesampling for confocal imaging. In the transformed protoplast ofCol-0, HYL1-CFP was localized in both the cytoplasm and nu-cleus, and the signal was clearly different from the fluorescencebackground in untransformed cells (Fig. 4C and Fig. S8C). Simi-larly, the subcellular localization of HYL1-CFP in the protoplastof the sad2-2 mutant was not changed, suggesting that SAD2 maynot be involved in HYL1 translocation. In contrast, in the trans-formed protoplasts of two individual ketch1 knock-down lines, thenuclear localization of HYL1-CFP was largely attenuated (Fig.4C). In parallel, we did transformation experiments of SE-CFPin the protoplasts of these genetic backgrounds; SE-CFPs wereall predominantly localized in nucleus, suggesting that bothKETCH1 and SAD2 are not responsible for SE translocationbetween the cytoplasm and the nucleus (Fig. 4C). In summary, weprovided clear evidences that KETCH1 is an importin specific fornuclear import of HYL1 protein in Arabidopsis.

ketch1 Mutation Compromises pri-miRNA Processing. If KETCH1functions as a specific importin for HYL1, one prediction of thismodel is that KETCH1 and HYL1 should be in the same geneticpathway. To test this prediction, we crossed the 35S-amiR-KETCH1 lines (#41) with several mutants in the miRNA path-way, including hyl1-2, se-2, sad2-2, and hst-16. Clearly, theketch1 hyl1-2 double mutant resembles the hyl1-2 single mutantboth in morphology and in miRNA defect (Fig. S9). In contrast,the ketch1 se-2 double mutant has more severe phenotypescompared with either single mutant, suggesting that SE has ad-ditional function beside a role in the miRNA pathway. Consis-tent with this notion is that the hyl1-1 se-1 double mutant isembryonically lethal (29). Similarly, in ketch1 sad2-2 and ketch1hst-16 double mutants, additional phenotypes were also ob-served, suggesting that KETCH1 acts in the genetic pathwaydifferent from both SAD2 and HST.Another prediction for the model above is that the pri-miRNA

processing would be compromised in the ketch1 mutant. To testthis prediction, we performed an RNA gel blot to measure thelevels of a few selected pri-miRNAs exemplified by pri-miR159,pri-miR164, and pri-miR165. MIR159, MIR164, and MIR165 arethree founding members and each contains two to three loci inArabidopsis. Previous reports show thatMIR159a and -b, MIR164b,and MIR165a are predominant contributors to accumulation oftheir mature miRNAs (30–32). Excitingly, the levels of pri-miR159a, pri-miR164b, and pri-miR165a were indeed enhanced

Fig. 4. ketch1 mutation decreases the nuclear-localized HYL1 protein.(A) Western blot analysis of the indicated core components of miRNApathway in total extraction (T), the cytoplasm (C), and nucleus (N) fractionsin the ketch1 and ran1 mutants. Rubisco (Rbsc) stained with Ponceau S andHistone 3 detected by anti-H3 serve as a loading control for cytoplasmic andnuclear proteins, respectively. Endogenous proteins were detected with theindicated antibodies, respectively. The asterisk (*) indicates the nonspecificcross-reaction. (B) Quantification of the nuclear-cytoplasmic distribution ofthe tested components in miRNA pathway. The nuclear-cytoplasmic distri-bution (N/C) ratio of tested proteins in Col-0 was arbitrarily designated as1.0, and the ones in mutants were normalized to that of Col-0. (C) Repre-sentative subcellular localizations of HYL1-CFP or SE-CFP in the protoplastsof the indicated lines. Greater than 50 individual transformed protoplasts foreach were checked by confocal microscopy. (Scale bars, 10 μm.)

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in the ketch1 mutant compared with the amount in Col-0 plants(Fig. 5 A–D). However, the enhancement was lesser in the mutantcompared with the amount in hyl1-2 and se-2 mutants. Oneplausible reason is that the ketch1 knockdown line by the artificialmiRNAs still produced residual amount of KETCH1 protein,allowing partial translocation of the functional HYL1 into nucleus(Fig. 5E). Importantly, the ketch1 mutation also increased levelsof premiRNAs, in particular premiR165a, compared with the onein wild-type (Fig. 5 A–D). The overaccumulation of some pre-miRNAs in the ketch1 mutant suggested that processing of thepremiRNAs might be more sensitive to the level of HYL1 proteinthan the event with the pri-miRNA species in nucleus. Takingthese data together, we show that the ketch1 mutation would leadto compromised processing of pri-miRNAs and premiRNAs,resulting from reduced import of HYL1 to nucleus.

DiscussionIn this study we reported KETCH1 as a new player with themicroprocessor machinery. We propose that KETCH1 trans-ports HYL1 from the cytoplasm to the nucleus to participate inpri-miRNA processing. Several pieces of evidences supportedthis notion: (i) KETCH1 physically associated with HYL1, andthus has capability to recognize and transport HYL1 protein(Fig. 1); (ii) ketch1 mutation caused developmental defect,reminiscent of mutants in the miRNA pathway (Fig. 2 B and C);

(iii) ketch1 mutation significantly decreased miRNA levels (Fig.3A); (iv) ketch1 mutation did not repress expression of corecomponents of microprocessor and effectors of RISC in themiRNA pathway nor enhance established negative regulators inthe miRNA decay (Fig. 3 C–E); (v) KETCH1 protein did nottransport miRNAs (Fig. S7); (vii) ketch1 mutation reduced nu-clear distribution of HYL1 (Fig. 4); and finally but importantly,(vii) ketch1 mutation compromised pri-miRNA processing, a keyfunction of HYL1 protein in vivo (Fig. 5).To our best knowledge, KETCH1 is an importin protein that

has been identified to be specifically engaged in transporting themicroprocessor components throughput eukaryotes. In mammals,importin 8 protein has been reported to account for importingnumerous components in the miRNA pathway, such as miRNA,AGO, and TNRC6 proteins, but not for microprocessor proteins,such as Drosha, DGCR8/Pasha, and Dicer protein (13–15). In ourstudy, KETCH1 is very unlikely to be responsible for trafficking ofRISC components, as we did not see any obvious changes ofnuclear-cytoplasmic distributions of RISC components (Fig. 4 Aand B and Fig. S7). Thus, KETCH1 and importin 8 select differentcargos in the miRNA pathway for trafficking. Phylogenetic anal-ysis of importin β-members in Arabidopsis and mammals suggestthat KETCH1 is genetically close to importin 5 (IPO5) in human,and whether IPO5 in human functions similar to KETCH1 totransport the DGCR8–HYL1 counterpart in mammals is un-known (Fig. S2). How the target specificity of importin β-membersevolves in different organisms would be an interesting question inthe future.In plants, genetic functions of numerous importin β-members

have been reported. Among the members, HST and TRN1 arepositive regulators, whereas SAD2 is a negative regulator in in themiRNA pathway (16–18). The common feature for hst, sad2, trn1,and ketch1 herein is that none of these mutations impacts nuclear-cytoplasmic distribution of miRNAs. However, the different thingis that KETCH1 transports HYL1, whereas the bona fide cargosin the miRNA pathway for HST, SAD2, and TRN1 remain tobe validated. Although TRN1 has been shown to interact withAGO1 in a RNA-independent manner, TRN1 dysfunction doesnot impact the nuclear-cytoplasmic distribution of AGO1 (18).Our preliminary results did not reveal any significant changes ofcomponents in microprocessor machinery and RISC complexes inthe hst-16 and sad2-2 mutants (Fig. S8 A and B). Thus, our resultssuggest that KETCH1 functions distinctly from these threemembers (Fig. 5E).It has not escaped our attention that the null allele of the ketch1

mutant is embryotic-lethal, whereas the hypomorphic mutant ofketch1 has additional phenotypes exemplified by earlier necrosiscompared with hyl1 mutants (Fig. S4 E and F). Notably, thetranscription of ORE1, a positive regulator for leaf senescence (24,33), is up-regulated in ketch1 mutants but almost completelyabolished in both hyl1-2 and se-2 mutants (Fig. 3B). Such expres-sion patterns of ORE1 are consistent with the fact that senescenceis accelerated in ketch1 but the process is attenuated in hyl1-2 andse-2. A trial view is that hyl1-2 and se-2 have substantial effect onbroader range of miRNAs so that certain miRNAs and their fur-ther targets have cross-talk and antagonized effects in the leaf-senescence pathway. However, we favor an idea that KETCH1,like other importin β-proteins, might have multiple targets in vivo.SAD2 has been reported to be involved in the nuclear import oftranscription factor MYB4 (34), and a recent report that Y2Hscreening using TRN1 recovers a handful of potential cargos (35).In line with these reports, it’s not surprising that KETCH1 hassome other cargos in addition to HYL1. Certain cargos ofKETCH1 might play a pivotal role in embryonic developmentand be directly responsible for the embryonic defects caused bydysfunction of KETCH1. Thus, future effort in systematicidentification of additional cargos for KETCH1 would provide

Fig. 5. ketch1 mutation compromises HYL1-engaged miRNA biogenesisin vivo. (A–D) RNA blot analyses of pri-miRNAs in Col-0 and various mutantsusing probes against the indicated pri-miRNAs. Ten micrograms of totalRNAs was resolved in 1% agarose gel for A–C and in 5% urea polyacrylmidegel for D. The red open and closed rectangles show premiR159b (C) and withdifferent contrast (D). The asterisks (** and *) indicate the tested pri- andpremiRNAs, respectively. rRNAs and U6 serve as the loading control. (E) Theproposed model for KETCH1 that imports HYL1 from cytoplasm to nucleus tofacilitate miRNA biogenesis. NPC, nuclear pore complex.

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comprehensive insight into biological functions of the importinβ-member that is evolutionarily conserved in eukaryotes.

Materials and MethodsMaterials. The mutants, ketch1-2/emb2734-2/SALK_050129, hyl1-2, se-2,sad2-2/SALK_133577C, and hst-16/SALK_006481C are T-DNA insertion linesfrom the Arabidopsis Resource Center.

HYL1 Complex Isolation and Proteomic Analysis. Arabidopsis thaliana transgeniclines expressing 35S-FM-HYL1were generated and the isolation of HYL1 complexfrom plants, its resolution in SDS/PAGE, and the recovery of unique bands aswell as proteomics analysis are described in SI Materials and Methods.

DNA Construction, Assays of Protein–Protein Interaction, qRT-PCR, RNA Blot,and Western Blot Analyses. Plasmid construction, assays for protein–proteininteraction including Y2H, FRET, co-IP, and in vitro pull-down, qRT-PCR, RNAblot, and Western blot analyses are described in SI Materials and Methods.See Table S1 for a list of primers used.

Protoplast Transformation and Imaging by Confocal Microscopy. The well-expanded leaves of three-week-old Arabidopsis plants in soil were usedfor protoplast preparation and plasmid transformation as described in SIMaterials and Methods. After transformation, the transformed protoplastswere incubated in 23 °C under light conditions until sampling for the im-aging by confocal microscopy (Olympus).

Nuclear-Cytoplasmic Fractionation. The 4-wk-old Arabidopsis plants in soilwere used for nuclear-cytoplasmic fractionation performed as previouslydescribed (17). The resulting protein and RNA samples from the total extract,nuclear and cytoplasmic fractions were used for Western blot and small RNAgel blot, respectively.

ACKNOWLEDGMENTS. We thank L. Zeng and the Microscopy and ImagingCenter at Texas A&M University for imaging facilities; and B. Yu for theprecious gift of antibodies against HYL1 and SE. This work was supportedby NSF Grant CAREER MCB-1253369 and Cancer Prevention Research Insti-tute of Texas Grant RP160822 (to X.Z.). X.G. was supported by the ChineseScholarship Council.

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