KETCH1 Imports HYL1 to Nucleus for Mirna Biogenesis in Arabidopsis

KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis

Zhonghui 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, Guangzhou 510631, 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, Republic of 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 hairpin premiRNAs in mammalians (11, 12). Importin-8 facilitates the structures (pri-miRNAs) by microprocessors in the nucleus. How recruitment of AGO2-containing RISC to target mRNAs to pro- cytoplasmic-borne microprocessor components are transported into mote efficient and specific gene silencing in the cytoplasm, whereas the nucleus to fulfill their functions remains poorly understood. Here, the protein can also transport AGO2 and AGO2 partners, GW we report KETCH1 (karyopherin enabling the transport of the proteins and miRNAs, into the nucleus to balance levels of cyto- cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, plasmic gene-silencing effectors (13–15). Arabidopsis encodes a core component of microprocessor in Arabidopsis and functional 18 importin β-proteins, among which few have also been reported counterpart of DGCR8/Pasha in animals. Null mutation of ketch1 is to function in the miRNA pathway. Loss-of-function of HASTY embryonic-lethal, whereas knockdown mutation of ketch1 caused (HST), an ortholog of human Exportin-5 in plants (16), decreases morphological defects, reminiscent of mutants in the miRNA path- the accumulation of most of tested miRNAs, implying its critical way. ketch1 knockdown mutation also substantially reduced miRNA role in transporting miRNAs and miRNA pathway components. accumulation, but did not alter nuclear-cytoplasmic shuttling of Supersensitive to ABA and drought 2 (SAD2)/enhanced miRNA miRNAs. Rather, the mutation significantly reduced nuclear portion

activity 1 (EMA1) appears to negatively impact loading of miR- PLANT BIOLOGY of HYL1 protein and correspondingly compromised the pri-miRNA NAs into AGO1-centered RISC (17). Recently, Transportin1 processing in the nucleus. We propose that KETCH1 transports (TRN1) has been identified as a positive regulator in miRNA HYL1 from the cytoplasm to the nucleus to constitute functional loading through a suppressor screening of ema1 (18). The bona microprocessor in Arabidopsis. This study provides insight into the fide cargos for these importin β-proteins remain to be identified largely unknown nuclear-cytoplasmic trafficking process of miRNA in Arabidopsis. biogenesis components through eukaryotes. Here, we identified karyopherin enabling the transport of the cytoplasmic HYL1 (KETCH1, meaning a sailboat for HYL1), a KETCH1 | importin β | HYL1 | miRNA | pri-miRNA processing member of the importin β-family, as a partner of HYL1. We found that a knockdown mutant of ketch1 through an artificial icroRNAs (miRNAs) are a class of noncoding RNAs, 21– miRNA (amiR-KETCH1) displayed developmental abnormality, M24 nt in length, that play vital roles in diverse biological processes. miRNA biogenesis starts with the transcription of long Significance primary miRNAs (pri-miRNAs) that contain hairpin-like fold- back. In animals, pri-miRNAs are initially processed to generate Microprocessor components are transported into the nucleus precursor miRNAs (premiRNAs) in the nucleus by a micropro- to process primary miRNAs (pri-miRNAs). Hyponastic leaves 1 cessor that minimally comprises of an RNase III enzyme, Drosha, (HYL1) is a core component of microprocessor in Arabidopsis. and two molecules of DGCR8/Pasha (1). PremiRNAs are This study identifies an importin β-protein, KETCH1, as a part- exported to the cytoplasm and further processed by another KETCH1 – ner of HYL1 protein. knockdown mutation causes de- RNase III enzyme, Dicer, to release 21 22 bp miRNA/* duplexes velopmental defect, reminiscent of mutants in the miRNA (2, 3). In Arabidopsis, DCL1, one of four Dicer-like enzymes, pathway. The mutation also reduces miRNA abundance but fulfills consecutive cuts of pri-miRNAs to produce miRNA/*s (4, does not affect its nuclear-cytoplasmic distribution. Rather, 5); and the entire processes occur in the nucleus and are facilitated KETCH1 knockdown mutation decreases the accumulation of by DCL1 cofactors, including hyponastic leaves 1 (HYL1), Serrate – HYL1 protein in the nucleus and compromises the HYL1-mediated (SE), and other accessory partners (4 8). Once generated, pri-miRNA processing, supporting that KETCH1 specifically trans- miRNA duplexes are exported to the cytoplasm and loaded into ports HYL1 into the nucleus to facilitate miRNA production. Given Argonaute (AGO)-containing RNA-induced silencing complexes that HYL1 and importin β-proteins are all conserved in various (RISCs) to repress the expression of their targeted genes (4, 9). organisms, this study sheds light on the poorly understood Although the biogenesis and action of mode of miRNAs have nuclear-cytoplasmic trafficking of microprocessor components been studied well, how the microprocessor components are trans- through eukaryotes. located from their cytoplasmic birthplaces to functional niches in

the nucleus has been poorly defined. Nuclear-cytoplasmic traf- Author contributions: Z.Z., C.G., and X.Z. designed research; Z.Z., X.G., C.G., Z.M., M.J., and ficking of biological macromolecules through the nuclear pore X.Z. performed research; T.L., H.K., and S.W.Y. contributed new reagents/analytic tools; complex entails transport receptors, namely karyopherin proteins, 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. wrote some of which also referred to importins and exportins. Importins the paper. load their cargos in the cytoplasm and release them in the nucleus The authors declare no conflict of interest. upon binding with Ran-GTPase, whereas exportins, coupled with This article is a PNAS Direct Submission. Ran-GTPase, associate with the cargos in the nucleus and dis- 1Z.Z., X.G., and C.G. contributed equally to this work. charge the cargos in the cytoplasm upon hydrolysis of GTP to GDP 2To whom correspondence should be addressed. Email: [email protected]. β (10). Several importin -family members are implicated in the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. miRNA pathway. Exportin-5 mediates the nuclear export of 1073/pnas.1619755114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1619755114 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 could observe the strong FRET signals in the nucleus and residue amount in some spots near the nuclear envelope, suggesting that translocation of the KETCH1–HYL1 complex may happen near the nuclear envelope through KETCH1–HYL1 interaction. We did not detect obvious FRET signals in any discrete foci, like D-bodies, where endogenous HYL1 is typically located (20), suggesting that the HYL1–KETCH1 interaction is dynamic and HYL1 is discharged before its entrance into D-bodies. Alterna- tively, overaccumulation of HYL1 and KETCH1 proteins in the infiltrated cells might mask the signal from the D-bodies. We also conducted co-IP experiments with ketch1-2; PKETCH1-FM- KETCH1 complementation lines using an anti-Flag antibody and Fig. 1. KETCH1 is a partner of HYL1 in Arabidopsis.(A) Silver-stained SDS/ detected co-IP products with antibodies specifically recognizing PAGE of purified HYL1 complexes for proteomics analysis. (B and C) Specific endogenous proteins. Again, we observed the specific KETCH1– HYL1-KETCH1 interaction was confirmed in N. benthamiana by FRET (B)andin HYL1 interactions in Arabidopsis, but not control proteins, such as Arabidopsis by co-IP assays (C). In B: CFP, donor HYL1-CFP fluorescence; nFRET, normalized FRET fluorescence; YFP, acceptor YFP-KETCH1 fluorescence. The actin, and validated this interaction was RNA-independent (Fig. color bars indicate the scale of the signal strength or the distance between the 1C). Finally, an in vitro pull-down assay indicated that recombi-

donor and the acceptor (DCFP/YFP). In C, actin is a control. (Scale bars, 10 μm.) nant His-SUMO-KETCH1, but not the control protein STING (21), could physically interact with HYL1 (Fig. S3E). Taking these data together, we concluded that KETCH1 is a bona fide partner characteristic of the mutants with the miRNA defect. The hypo- of HYL1 protein in vivo. morphic mutants also exhibited significantly reduced accumulation of miRNAs, but no change in nuclear-cytoplasmic distribution. In Loss-of-Function Mutation of ketch1 Causes Developmental Defect. contrast, the mutation caused less accumulation of nuclear-localized To study biological function of the KETCH1 gene, we genotyped HYL1 and compromised pri-miRNA processing in the nucleus. We a T-DNA insertion line SALK_050129/emb2734-2,whichis conclude that KETCH1 imports HYL1 protein from the cytoplasm renamed as ketch1-2 here (Fig. S4A). ketch1-2 (+/−) heterozygotes to nucleus to participate in miRNA biogenesis. This study shed light yielded ∼25% abortive seeds in their siliques; and the embryonic on the poorly understood nuclear-cytoplasmic trafficking mecha- defect of ketch1-2 could be fully rescued by transgene expressing – nism of microprocessor components in eukaryotes. PKETCH1-FM-KETCH1 (Fig. 2A and Fig. S4 A C). This observation was consistent with a previous report that null mutation of this Results gene is embryonic-lethal (22). We next generated knockdown Identification of KETCH1 as an HYL1 Partner. To identify new compo- transgenic lines of ketch1 by expressing artificial miRNA con- nents in microprocessor in plants, we generated stable transgenic structs specifically targeting KETCH1 (amiR-KETCH1)(Fig. ∼ lines overexpressing Flag-4Myc (FM) –tagged HYL1 (Fig. S1A). We S4D). Approximately 50% ( 60 of 118) of 35S-amiR-KETCH1 isolated the HYL1-containing complex through two-step immuno- primary transformants exhibited developmental abnormalities with precipitation (IP) and resolved the complex on SDS/PAGE gradient varying severity (Fig. 2B). The most severe transgenic lines had gels (Fig. 1A). We selected a few distinct bands clearly visible in spoon-shaped cotyledons and narrow and strongly upward curled HYL1 IP for mass spectrometric analysis. From one band corre- leaves, and they died soon after emergence of a few pairs of true sponding to ∼130 kDa, 15 unique peptides matched specifically to leaves. Lines with less-severe defect also phenocopied hypomor- Embryo Defective 2734 (EMB2734, At5g19820) were recovered from phic ago1 mutants (Fig. 2B), and these plants survived and were propagated to higher generations for various assays below. Simi- HYL1 IP but absent in the control IP using Col-0 plants (Fig. 1A larly, we have also generated amiR-KETCH1 transgenic plants and Fig. S1B). EMB2734 is a member of Karyopherin/Importin β controlled by an XVE-inducible promoter (23). Again, these family in Arabidopsis (Fig. S2), and we renamed the gene KETCH1. plants phenocopied the mutants in the miRNA pathway once the To examine whether KETCH1 is a bona fide partner of HYL1, inducer β-estradiol was applied (Fig. 2C). we initially conducted yeast-two hybrid (Y2H) assays. Interestingly, Analyses of small RNA (sRNA) blots using the T2 transgenic KETCH1 strongly interacted with Arabidopsis Ran-GTPase, seedlings showed that amiR-KETCH1 were processed and RAN1, but surprisingly not with HYL1, in the Y2H assays (Fig. S3A). However, Western blot analysis of yeast cotransfected with HYL1 and KETCH1 plasmids showed that these two proteins could not be coexpressed in a single colony, implying their potential in- terplay in vivo (Fig. S3B). We next carried out a split luciferase complementation assay (LCI) (Fig. S3C). In our LCI assays, HYL1 indeed displayed LUC complementation with KETCH1 (Fig. S3C), as did AGO1 with the positive control, Cucumber mosaic virus-encoded 2b protein (CMV 2b) (19), implying that KETCH1 interacts with HYL1 in plants. Next, we validated the HYL1–KETCH1interactioninvivobyFRETassay,usingHYL1- CFP as a donor and YFP-KETCH1 as an acceptor (Fig. 1B). We coexpressed both HYL1-CFP and YFP-KETCH1 in Nicotina benthamiana cells. Using confocal microscopy, under the laser in the wavelengths specific for CFP/YFP excision, respectively, we observed that both HYL1-CFP and YFP-KETCH1 were pre- Fig. 2. ketch1 knockdown mutant has developmental defects in Arabi- dominantly localized in the nucleus, but also distributed in cytoplasm dopsis.(A) The representative seed-set phenotypes in the siliques of Col-0, ketch1-2 heterozygote, and two individual KETCH1 complementation lines. through the corresponding CFP/YFP channels (Fig. 1B). Consis- The red arrows indicate abortive seeds. (B) Morphological phenotypes of tent with this finding, KETCH1 protein is also localized in both ketch1 and other miRNA pathway mutants at the 7-d stage. (C) Morpho- nucleus and cytoplasm in the stable transgenic plants (Fig. S3D). logical phenotypes of XVE-amiR-KETCH1 transgenic plants induced with (+) Under the laser in the wavelength specific for CFP excision, we or without (−)25μM β-estradiol (β-ES). (Scale bars, 0.4 cm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1619755114 Zhang et al. Downloaded by guest on September 25, 2021 accumulated to a moderate level (Fig. S4D). Correspondingly, ketch1 Mutation Elevates Expression Levels of Few Core Components qRT-PCR assays showed that levels of KETCH1 transcript de- in the miRNA Pathways. Up to date, more than a dozen of genes creased approximately ∼80–90% compared with wild-type plants have been reported to affect miRNA biogenesis and turnover (Fig. S4D). Reexamination of the T2 amiR-KETCH1 lines in vivo (4, 7). Down-regulation of miRNA accumulation in the revealed that the amiR-KETCH1 adult plants typically exhibited ketch1 mutant could result from altered expression of components shorter statues and serrated rosette leaves (Fig. S4E). Compared in the miRNA metabolism. To test this theory, we conducted with Col-0 plants, the rosette leaves of adult plants readily dis- RNA gel blots and qRT-PCR assays for some key components. played yellow and necrosis symptoms, suggestive of accelerated These results showed that the transcript levels of most of the leaf senescence (Fig. S4 E and F). Taken together, these data tested components were essentially not altered in the ketch1 mu- show that knockdown of KETCH1 transcripts clearly impacted tant, whereas SDN1, the component engaged in miRNA degra- growth and development in Arabidopsis. dation (25), was in fact down-regulated (Fig. 3 C and D). Notably, the amount of some core effectors exemplified by HYL1, SE, and ketch1 Mutation Decreases miRNA Accumulation. Physical interaction AGO1 was even enhanced in the amiR-KETCH1 mutants relative of KETCH1 with HYL1 and morphological abnormality of the to Col-0 plants (Fig. 3 D and E). AGO1 and SE are targeted by amiR-KETCH1 mutants prompted us to examine whether KETCH1 miR168 and miR863-3p, respectively, and their up-regulation was functions in the miRNA pathway. sRNA blot analysis showed that likely because of the reduced levels of the miRNAs in ketch1 accumulation of all tested miRNAs was significantly decreased in mutants (Fig. 3A). Thus, our results indicated that impact of the amiR-KETCH1 mutants compared with Col-0 plants (Fig. 3A and Fig. S5). However, the down-regulation of mature miRNAs in KETCH1 on miRNA expression was not through repressing ex- the amiR-KETCH1 mutants was to a less extent relative to hyl1-2 pression levels of the key components in the miRNA micropro- and se-2 null mutants (Fig. 3A and Fig. S5). The scenario was likely cessor nor through up-regulating expression of the established components tested in the miRNA decay pathway. because of the fact that the amiR-KETCH1 knockdown lines were β weak alleles. Of note, sad2 mutants did not display obvious changes Because KETCH1 belongs to the importin -family as SAD2,we in the steady-state level of miRNAs (17), suggesting that KETCH1 wondered whether KETCH1 acted as SAD2 to impact the loading functions differently from SAD2 in the miRNA pathway. of miRNAs into the AGO1 protein. To test this theory, we To examine whether miRNA targets are deregulated in the immunoprecipitated AGO1 protein from planta and recovered

amiR-KETCH1 mutants, qRT-PCR was conducted assays for PHV, miRNAs from the complexes. Despite the level of AGO1 protein PLANT BIOLOGY MYB33, and targets of miR159 and miR166, respectively. We ob- being enhanced in the amiR-KETCH1 mutants compared with served that the steady-state levels of these transcripts were clearly Col-0 plants, the miRNA amount recovered from AGO1 com- up-regulated in the amiR-KETCH1 mutants compared with Col-0 plexes was slightly decreased in the mutants vs. Col-0 plants (Fig. plants, although to a less extent than the up-regulation in hyl1-2 3F). The reduced amount was likely because of the fact that and se-2 (Fig. 3B). Of note, ORE1, the target of miR164 (24), was miRNA input was lesser and AGO1 protein was more in the enhanced in the amiR-KETCH1 mutants but not in hyl1 and se-2 mutants compared with Col-0 plants, rather than because of mutants. Taken together, all these results indicated that KETCH1 compromised loading of miRNAs into AGO1 effectors in the is a positive regulator in the miRNA-mediated gene silencing ketch1 mutant, because unlike SAD2, KETCH1 did not have di- in Arabidopsis. 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 the indicated 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 miRNA targets (B) and the mRNAs of selected genes in the miRNA pathway (C) in the indicated mutants. EF1A is an internal control. The asterisk (*) indicates the significance between mutants and Col-0 control (*P < 0.05) in B and C.(D) RNA blot analyses for AGO1, HYL1,andSE transcripts. rRNAs are the loading control. (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 of the tested miRNAs loaded into RISC in the ketch1 mutants.

Zhang et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 KETCH1 Impacts the Nuclear-Cytoplasmic Distribution of HYL1 Protein We quantified nuclear and cytoplasmic signals of pixel intensities but Not miRNAs. We next hypothesized that KETCH1 might be of tested and control proteins and normalized ratios of nuclear vs. involved in the translocation of miRNA/*s from microprocessors cytoplasmic HYL1 based on the signals of Histone 3 (H3) and between the nucleus and cytoplasm. To test this hypothesis, we Rubisco (Rbsc) controls. Upon ketch1 knockdown, the ratio of performed the nuclear-cytoplasmic fractionation of cell extracts nucleus-localized HYL1 relative to the cytoplasmic HYL1 was from Col-0 and amiR-KETCH1 mutants, and extracted total significantly decreased (Fig. 4 A and B). In contrast, lower distri- RNA from the nuclear and cytoplasmic fractions for RNA gel bution of HYL1 in the nucleus was not observed in the loss-of- blots. sRNA blot analysis showed that distribution patterns of all function mutants of HST and SAD2 that are supposed to transport tested miRNAs were approximately the same in the mutants and some components in the miRNA pathway in Arabidopsis (Fig. S8 Col-0 plants (Fig. S7), indicating that miRNAs are unlikely to be A and B). Furthermore, we examined a few other critical com- the cargos of KETCH1 protein. In line with this result, we ponents in the miRNA pathway, and did not observe any signifi- immunoprecipated KETCH1 and did not recover miRNA from cant change of nuclear-cytoplasmic distribution of the components in the ketch1 mutant (Fig. 4 A and B). Thus, these components— the KETCH1 protein (Fig. S6). different from HYL1—are not the cargos of KETCH1. Importin proteins could also transport protein cargos to the To further validate whether KETCH1 imported HYL1 protein, nucleus (10). HYL1 has been known to be predominantly lo- we expressed HYL1-CFP in the protoplasts prepared from a dif- calized and function in the nucleus (26, 27); however, a recent ferent genetic background, including Col-0, amiR-KETCH1,and study reported that a substantial amount of HYL1 is present in sad2-2. A recent study indicates that cytoplasmic partitioning of cytosol (28). This observation was reproducible in our hands COP1, a well-known RING-type E3 ligase, could protect cyto- (Fig. 4A). Because KETCH1 directly interacted with HYL1, we plasmic HYL1 from the degradation by certain unknown prote- next investigated whether KETCH1 is involved in nuclear import ases under light conditions (28). However, cytoplasmic HYL1 of HYL1 protein. Western blot for the nuclear and cytoplasmic would be easily degraded under dark conditions and MG132 fractions of proteins were performed with anti-HYL1 antibody. treatment could increase its cytoplasmic localization (28). To facilitate the observation of HYL1 localization in the cytoplasm, we kept the transformed protoplasts under light conditions before sampling for confocal imaging. In the transformed protoplast of Col-0, HYL1-CFP was localized in both the cytoplasm and nucleus, and the signal was clearly different from the fluorescence background in untransformed cells (Fig. 4C and Fig. S8C). Simi- larly, the subcellular localization of HYL1-CFP in the protoplast of the sad2-2 mutant was not changed, suggesting that SAD2 may not be involved in HYL1 translocation. In contrast, in the transformed protoplasts of two individual ketch1 knock-down lines, the nuclear localization of HYL1-CFP was largely attenuated (Fig. 4C). In parallel, we did transformation experiments of SE-CFP in the protoplasts of these genetic backgrounds; SE-CFPs were all predominantly localized in nucleus, suggesting that both KETCH1 and SAD2 are not responsible for SE translocation between the cytoplasm and the nucleus (Fig. 4C). In summary, we provided clear evidences that KETCH1 is an importin specific for nuclear import of HYL1 protein in Arabidopsis.

ketch1 Mutation Compromises pri-miRNA Processing. If KETCH1 functions as a specific importin for HYL1, one prediction of this model is that KETCH1 and HYL1 should be in the same genetic pathway. To test this prediction, we crossed the 35S-amiR- KETCH1 lines (#41) with several mutants in the miRNA pathway, including hyl1-2, se-2, sad2-2, and hst-16. Clearly, the ketch1 hyl1-2 double mutant resembles the hyl1-2 single mutant both in morphology and in miRNA defect (Fig. S9). In contrast, the ketch1 se-2 double mutant has more severe phenotypes compared with either single mutant, suggesting that SE has additional function beside a role in the miRNA pathway. Consis- tent with this notion is that the hyl1-1 se-1 double mutant is embryonically lethal (29). Similarly, in ketch1 sad2-2 and ketch1 Fig. 4. ketch1 mutation decreases the nuclear-localized HYL1 protein. hst-16 double mutants, additional phenotypes were also ob- (A) Western blot analysis of the indicated core components of miRNA served, suggesting that KETCH1 acts in the genetic pathway pathway in total extraction (T), the cytoplasm (C), and nucleus (N) fractions different from both SAD2 and HST. in the ketch1 and ran1 mutants. Rubisco (Rbsc) stained with Ponceau S and Another prediction for the model above is that the pri-miRNA Histone 3 detected by anti-H3 serve as a loading control for cytoplasmic and processing would be compromised in the ketch1 mutant. To test nuclear proteins, respectively. Endogenous proteins were detected with the this prediction, we performed an RNA gel blot to measure the indicated antibodies, respectively. The asterisk (*) indicates the nonspecific levels of a few selected pri-miRNAs exemplified by pri-miR159, cross-reaction. (B) Quantification of the nuclear-cytoplasmic distribution of pri-miR164, and pri-miR165. MIR159, MIR164,andMIR165 are the tested components in miRNA pathway. The nuclear-cytoplasmic distribution (N/C) ratio of tested proteins in Col-0 was arbitrarily designated as three founding members and each contains two to three loci in 1.0, and the ones in mutants were normalized to that of Col-0. (C) Repre- Arabidopsis. Previous reports show that MIR159a and -b, MIR164b, sentative subcellular localizations of HYL1-CFP or SE-CFP in the protoplasts and MIR165a are predominant contributors to accumulation of of the indicated lines. Greater than 50 individual transformed protoplasts for their mature miRNAs (30–32). Excitingly, the levels of pri- each were checked by confocal microscopy. (Scale bars, 10 μm.) miR159a, pri-miR164b, and pri-miR165a were indeed enhanced

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1619755114 Zhang et al. Downloaded by guest on September 25, 2021 in the ketch1 mutant compared with the amount in Col-0 plants (iii) ketch1 mutation significantly decreased miRNA levels (Fig. (Fig. 5 A–D). However, the enhancement was lesser in the mutant 3A); (iv) ketch1 mutation did not repress expression of core compared with the amount in hyl1-2 and se-2 mutants. One components of microprocessor and effectors of RISC in the plausible reason is that the ketch1 knockdown line by the artificial miRNA pathway nor enhance established negative regulators in miRNAs still produced residual amount of KETCH1 protein, the miRNA decay (Fig. 3 C–E); (v) KETCH1 protein did not allowing partial translocation of the functional HYL1 into nucleus transport miRNAs (Fig. S7); (vii) ketch1 mutation reduced nu- (Fig. 5E). Importantly, the ketch1 mutation also increased levels clear distribution of HYL1 (Fig. 4); and finally but importantly, of premiRNAs, in particular premiR165a, compared with the one (vii) ketch1 mutation compromised pri-miRNA processing, a key in wild-type (Fig. 5 A–D). The overaccumulation of some pre- function of HYL1 protein in vivo (Fig. 5). miRNAs in the ketch1 mutant suggested that processing of the To our best knowledge, KETCH1 is an importin protein that premiRNAs might be more sensitive to the level of HYL1 protein has been identified to be specifically engaged in transporting the than the event with the pri-miRNA species in nucleus. Taking microprocessor components throughput eukaryotes. In mammals, these data together, we show that the ketch1 mutation would lead importin 8 protein has been reported to account for importing to compromised processing of pri-miRNAs and premiRNAs, numerous components in the miRNA pathway, such as miRNA, resulting from reduced import of HYL1 to nucleus. AGO, and TNRC6 proteins, but not for microprocessor proteins, such as Drosha, DGCR8/Pasha, and Dicer protein (13–15). In our Discussion study, KETCH1 is very unlikely to be responsible for trafficking of In this study we reported KETCH1 as a new player with the RISC components, as we did not see any obvious changes of microprocessor machinery. We propose that KETCH1 trans- nuclear-cytoplasmic distributions of RISC components (Fig. 4 A ports HYL1 from the cytoplasm to the nucleus to participate in and B and Fig. S7). Thus, KETCH1 and importin 8 select different pri-miRNA processing. Several pieces of evidences supported cargos in the miRNA pathway for trafficking. Phylogenetic anal- β this notion: (i) KETCH1 physically associated with HYL1, and ysis of importin -members in Arabidopsis and mammals suggest thus has capability to recognize and transport HYL1 protein that KETCH1 is genetically close to importin 5 (IPO5) in human, (Fig. 1); (ii) ketch1 mutation caused developmental defect, and whether IPO5 in human functions similar to KETCH1 to – reminiscent of mutants in the miRNA pathway (Fig. 2 B and C); transport the DGCR8 HYL1 counterpart in mammals is unknown (Fig. S2). How the target specificity of importin β-members evolves in different organisms would be an interesting question in the future. PLANT BIOLOGY In plants, genetic functions of numerous importin β-members have been reported. Among the members, HST and TRN1 are positive regulators, whereas SAD2 is a negative regulator in in the miRNA 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 thing is that KETCH1 transports HYL1, whereas the bona fide cargos in the miRNA pathway for HST, SAD2, and TRN1 remain to be validated. Although TRN1 has been shown to interact with AGO1 in a RNA-independent manner, TRN1 dysfunction does not impact the nuclear-cytoplasmic distribution of AGO1 (18). Our preliminary results did not reveal any significant changes of components in microprocessor machinery and RISC complexes in the hst-16 and sad2-2 mutants (Fig. S8 A and B). Thus, our results suggest that KETCH1 functions distinctly from these three members (Fig. 5E). It has not escaped our attention that the null allele of the ketch1 mutant is embryotic-lethal, whereas the hypomorphic mutant of ketch1 has additional phenotypes exemplified by earlier necrosis compared with hyl1 mutants (Fig. S4 E and F). Notably, the transcription of ORE1, a positive regulator for leaf senescence (24, 33), is up-regulated in ketch1 mutants but almost completely abolished in both hyl1-2 and se-2 mutants (Fig. 3B). Such expression patterns of ORE1 are consistent with the fact that senescence is accelerated in ketch1 but the process is attenuated in hyl1-2 and se-2. A trial view is that hyl1-2 and se-2 have substantial effect on broader range of miRNAs so that certain miRNAs and their further 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 of Fig. 5. ketch1 mutation compromises HYL1-engaged miRNA biogenesis transcription factor MYB4 (34), and a recent report that Y2H in vivo. (A–D) RNA blot analyses of pri-miRNAs in Col-0 and various mutants screening using TRN1 recovers a handful of potential cargos (35). using probes against the indicated pri-miRNAs. Ten micrograms of total In line with these reports, it’s not surprising that KETCH1 has – RNAs was resolved in 1% agarose gel for A C and in 5% urea polyacrylmide some other cargos in addition to HYL1. Certain cargos of gel for D. The red open and closed rectangles show premiR159b (C) and with different contrast (D). The asterisks (** and *) indicate the tested pri- and KETCH1 might play a pivotal role in embryonic development premiRNAs, respectively. rRNAs and U6 serve as the loading control. (E) The and be directly responsible for the embryonic defects caused by proposed model for KETCH1 that imports HYL1 from cytoplasm to nucleus to dysfunction of KETCH1. Thus, future effort in systematic facilitate miRNA biogenesis. NPC, nuclear pore complex. identification of additional cargos for KETCH1 would provide

Zhang et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 comprehensive insight into biological functions of the importin Protoplast Transformation and Imaging by Confocal Microscopy. The well- β-member that is evolutionarily conserved in eukaryotes. expanded leaves of three-week-old Arabidopsis plants in soil were used for protoplast preparation and plasmid transformation as described in SI Materials and Methods Materials and Methods. After transformation, the transformed protoplasts were incubated in 23 °C under light conditions until sampling for the im- Materials. The mutants, ketch1-2/emb2734-2/SALK_050129, hyl1-2, se-2, aging by confocal microscopy (Olympus). sad2-2/SALK_133577C, and hst-16/SALK_006481C are T-DNA insertion lines from the Arabidopsis Resource Center. Nuclear-Cytoplasmic Fractionation. The 4-wk-old Arabidopsis plants in soil were used for nuclear-cytoplasmic fractionation performed as previously HYL1 Complex Isolation and Proteomic Analysis. Arabidopsis thaliana transgenic described (17). The resulting protein and RNA samples from the total extract, lines expressing 35S-FM-HYL1 were generated and the isolation of HYL1 complex nuclear and cytoplasmic fractions were used for Western blot and small RNA from plants, its resolution in SDS/PAGE, and the recovery of unique bands as gel blot, respectively. well as proteomics analysis are described in SI Materials and Methods.

ACKNOWLEDGMENTS. We thank L. Zeng and the Microscopy and Imaging DNA Construction, Assays of Protein–Protein Interaction, qRT-PCR, RNA Blot, Center at Texas A&M University for imaging facilities; and B. Yu for the – and Western Blot Analyses. Plasmid construction, assays for protein protein precious gift of antibodies against HYL1 and SE. This work was supported interaction including Y2H, FRET, co-IP, and in vitro pull-down, qRT-PCR, RNA by NSF Grant CAREER MCB-1253369 and Cancer Prevention Research Insti- blot, and Western blot analyses are described in SI Materials and Methods. tute of Texas Grant RP160822 (to X.Z.). X.G. was supported by the Chinese See Table S1 for a list of primers used. Scholarship Council.

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