HYPONASTIC LEAVES1 Research Articles

Forkhead-associated domain 2 links light signal to miRNA biogenesis

MicroRNAs (miRNAs) are one of the most important regulators of gene expression. Their biogenesis starts with the transcription of primary miRNA transcripts (pri-miRNAs). In plants, the RNase III enzyme DICER-LIKER 1 (DCL1), together with its partner, the double-stranded RNA-binding protein HYPONASTIC LEAVES 1 (HYL1), and the zinc-finger protein SERRATE (SE), process the imperfect miRNA-residing stem loop of pri-miRNAs to release the miRNA/miRNA∗ duplexes in the nucleus, which are then methylated by HUA1 ENHANCER 1 (Rogers and Chen, 2013; Song et al., 2019).

DEAD-BOX RNA HELICASE 27 regulates microRNA biogenesis, zygote division, and stem cell homeostasis.

After double fertilization, zygotic embryogenesis initiates a new life cycle, and stem cell homeostasis in the shoot apical meristem (SAM) and root apical meristem (RAM) allows plants to produce new tissues and organs continuously. Here, we report that mutations in DEAD-BOX RNA HELICASE 27 (RH27) affect zygote division and stem cell homeostasis in Arabidopsis (Arabidopsis thaliana). The strong mutant allele rh27-1 caused a zygote-lethal phenotype, while the weak mutant allele rh27-2 led to minor defects in embryogenesis and severely compromised stem cell homeostasis in the SAM and RAM. RH27 is expressed in embryos from the zygote stage, and in both the SAM and RAM, and RH27 is a nucleus-localized protein. The expression levels of genes related to stem cell homeostasis were elevated in rh27-2 plants, alongside down-regulation of their regulatory microRNAs (miRNAs). Further analyses of rh27-2 plants revealed reduced levels of a large subset of miRNAs and their pri-miRNAs in shoot apices and root tips. In addition, biochemical studies showed that RH27 associates with pri-miRNAs and interacts with miRNA-biogenesis components, including DAWDLE, HYPONASTIC LEAVES 1, and SERRATE. Therefore, we propose that RH27 is a component of the microprocessor complex and is critical for zygote division and stem cell homeostasis.

MRNA adenosine methylase (MTA) deposits m6A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana

In Arabidopsis thaliana, the METTL3 homolog, mRNA adenosine methylase (MTA) introduces N6-methyladenosine (m6A) into various coding and noncoding RNAs of the plant transcriptome. Here, we show that an MTA-deficient mutant (mta) has decreased levels of microRNAs (miRNAs) but accumulates primary miRNA transcripts (pri-miRNAs). Moreover, pri-miRNAs are methylated by MTA, and RNA structure probing analysis reveals a decrease in secondary structure within stem-loop regions of these transcripts in mta mutant plants. We demonstrate interaction between MTA and both RNA Polymerase II and TOUGH (TGH), a plant protein needed for early steps of miRNA biogenesis. Both MTA and TGH are necessary for efficient colocalization of the Microprocessor components Dicer-like 1 (DCL1) and Hyponastic Leaves 1 (HYL1) with RNA Polymerase II. We propose that secondary structure of miRNA precursors induced by their MTA-dependent m6A methylation status, together with direct interactions between MTA and TGH, influence the recruitment of Microprocessor to plant pri-miRNAs. Therefore, the lack of MTA in mta mutant plants disturbs pri-miRNA processing and leads to the decrease in miRNA accumulation. Furthermore, our findings reveal that reduced miR393b levels likely contributes to the impaired auxin response phenotypes of mta mutant plants.

Strontium stress disrupts miRNA biogenesis by reducing HYL1 protein levels in Arabidopsis

Strontium (Sr) is an emerging environmental pollutant that has become a major global concern after the nuclear accident at the Fukushima Daiichi Nuclear Power Plant in 2011. Although many studies have demonstrated the harmful effects of Sr on plant growth and development at the physiological level, knowledge regarding how plants sense and respond to Sr stress at the molecular level is limited. Recent studies have suggested that microRNAs (miRNAs) function as key regulators of plant growth and development as well as in the responses of plants to environmental stresses, including salinity, drought, cold, nutrient starvation, and heavy metals. In this study, we examined the global expression profile of miRNAs under Sr stress using small RNA sequencing analysis in Arabidopsis to better understand the molecular basis of plant responses to Sr stress. To identify specific Sr-responsive miRNAs, we performed comparative miRNA expression profiling analysis using control, CaCl2-, and SrCl2-treated seedlings. Compared to the control treatment, the expressions of most miRNAs were considerably decreased in the Sr-treated seedlings. However, under Sr stress, the expressions of primary miRNAs (pri-miRNAs) and their target genes were significantly increased; the protein levels of HYPONASTIC LEAVES 1 (HYL1), one of the core components of the microprocessor complex, were strongly reduced despite the increased HYL1 mRNA expression. In addition, hyl1-2 mutant plants were shown to be more sensitive to Sr stress than wild-type plants. Collectively, our results strongly suggested that Sr stress may be associated with the disruption of miRNA biogenesis by reducing the protein level of HYL1, which is required to maintain proper growth and development for plants. Our findings further indicated that some miRNAs may play important roles in plant responses to Sr stress.

Hyponastic Leaves 1 protects pri-miRNAs from nuclear exosome attack

Biogenesis of plant microRNAs (miRNAs) takes place in nuclear dicing bodies (D-bodies), where the ribonulease III-type enzyme Dicer-like 1 (DCL1) processes primary transcripts of miRNAs (pri-miRNAs) into miRNA/miRNA* (*, passenger strand) duplexes from either base-to-loop or loop-to-base directions. Hyponastic Leaves 1 (HYL1), a double-stranded RNA-binding protein, is crucial for efficient and accurate processing. However, whether HYL1 has additional function remains unknown. Here, we report that HYL1 plays a noncanonical role in protecting pri-miRNAs from nuclear exosome attack in addition to ensuring processing. Loss of functions in SOP1 or HEN2, two cofactors of the nucleoplasmic exosome, significantly suppressed the morphological phenotypes of hyl1-2 Remarkably, mature miRNAs generated from loop-to-base processing were partially but preferentially restored in the hyl1 sop1 and hyl1 hen2 double mutants. Accordingly, loop-to-base-processed pri-miRNAs accumulated to higher levels in double mutants. In addition, dysfunction of HEN2, but not of SOP1, in hyl1-2 resulted in overaccumulation of many base-to-loop-processed pri-miRNAs, with most of their respective miRNAs unaffected. In summary, our findings reveal an antagonistic action of exosome in pri-miRNA biogenesis and uncover dual roles of HYL1 in stabilizing and processing of pri-miRNAs.

The Intrinsically Disordered Protein CARP9 Bridges HYL1 to AGO1 in the Nucleus to Promote MicroRNA Activity.

In plants, small RNAs are loaded into ARGONAUTE (AGO) proteins to fulfill their regulatory functions. MicroRNAs (miRNAs), one of the most abundant classes of endogenous small RNAs, are preferentially loaded into AGO1. Such loading, long believed to happen exclusively in the cytoplasm, was recently proposed to also occur in the nucleus. Here, we identified CONSTITUTIVE ALTERATIONS IN THE SMALL RNAS PATHWAYS9 (CARP9), a nuclear-localized, intrinsically disordered protein, as a factor promoting miRNA activity in Arabidopsis (Arabidopsis thaliana). Mutations in the CARP9-encoding gene led to a mild reduction of miRNAs levels, impaired gene silencing, and characteristic morphological defects, including young leaf serration and altered flowering time. Intriguingly, we found that CARP9 was able to interact with HYPONASTIC LEAVES1 (HYL1), but not with other proteins of the miRNA biogenesis machinery. In the same way, CARP9 appeared to interact with mature miRNA, but not with primary miRNA, positioning it after miRNA processing in the miRNA pathway. CARP9 was also able to interact with AGO1, promoting its interaction with HYL1 to facilitate miRNA loading in AGO1. Plants deficient in CARP9 displayed reduced levels of AGO1-loaded miRNAs, partial retention of miRNA in the nucleus, and reduced levels of AGO1. Collectively, our data suggest that CARP9 might modulate HYL1-AGO1 cross talk, acting as a scaffold for the formation of a nuclear post-primary miRNA-processing complex that includes at least HYL1, AGO1, and HEAT SHOCK PROTEIN 90. In such a complex, CARP9 stabilizes AGO1 and mature miRNAs, allowing the proper loading of miRNAs in the effector complex.

HUA ENHANCER1 Mediates Ovule Development.

Ovules are female reproductive organs of angiosperms, containing sporophytic integuments and gametophytic embryo sacs. After fertilization, embryo sacs develop into embryos and endosperm whereas integuments into seed coat. Ovule development is regulated by transcription factors (TF) whose expression is often controlled by microRNAs. Mutations of Arabidopsis DICER-LIKE 1 (DCL1), a microRNA processing protein, caused defective ovule development and reduced female fertility. However, it was not clear whether other microRNA processing proteins participate in this process and how defective ovule development influenced female fertility. We report that mutations of HUA ENHANCER1 (HEN1) and HYPONASTIC LEAVES 1 (HYL1) interfered with integument growth. The sporophytic defect caused abnormal embryo sac development and inability of mutant ovules to attract pollen tubes, leading to reduced female fertility. We show that the role of HEN1 in integument growth is cell-autonomous. Although AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 were ectopically expressed in mutant ovules, consistent with the reduction of microRNA167 in hen1, introducing arf6;arf8 did not suppress ovule defects of hen1, suggesting the involvement of more microRNAs in this process. Results presented indicate that the microRNA processing machinery is critical for ovule development and seed production through multiple microRNAs and their targets.

Arabidopsis KETCH1 Is Critical for the Nuclear Accumulation of Ribosomal Proteins and Gametogenesis.

Male and female gametophytes are generated from micro- or megaspore mother cells through consecutive meiotic and mitotic cell divisions. Defects in these divisions often result in gametophytic lethality. Gametophytic lethality was also reported when genes encoding ribosome-related proteins were mutated. Although numerous ribosomal proteins (RPs) have been identified in plants based on homology with their yeast and metazoan counterparts, how RPs are regulated, e.g., through dynamic subcellular targeting, is unknown. We report here that an Arabidopsis (Arabidopsis thaliana) importin β, KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1), is critical for gametogenesis. Karyopherins are molecular chaperones mediating nucleocytoplasmic protein transport. However, the role of KETCH1 during gametogenesis is independent of HYPONASTIC LEAVES 1 (HYL1), a previously reported KETCH1 cargo. Instead, KETCH1 interacts with several RPs and is critical for the nuclear accumulation of RPL27a, whose mutations caused similar gametophytic defects. We further showed that knocking down KETCH1 caused reduced ribosome biogenesis and translational capacity, which may trigger the arrest of mitotic cell cycle progression and lead to gametophytic lethality.

BcpLH organizes a specific subset of microRNAs to form a leafy head in Chinese cabbage (Brassica rapa ssp. pekinensis)

HYL1 (HYPONASTIC LEAVES 1) in Arabidopsis thaliana encodes a double-stranded RNA-binding protein needed for proper miRNA maturation, and its null mutant hyl1 shows a typical leaf-incurvature phenotype. In Chinese cabbage, BcpLH (Brassica rapa ssp. pekinensis LEAFY HEADS), a close homolog of HYL1, is differentially expressed in juvenile leaves, which are flat, and in adult leaves, which display extreme incurvature. BcpLH lacks protein–protein interaction domains and is much shorter than HYL1. To test whether BcpLH is associated with defects in microRNA (miRNA) biogenesis and leaf flatness, we enhanced and repressed the activity of BcpLH by transgenics and investigated BcpLH-dependent miRNAs and plant morphology. BcpLH promoted miRNA biogenesis by the proper processing of primary miRNAs. BcpLH downregulation via antisense decreased a specific subset of miRNAs and increased the activities of their target genes, causing upward curvature of rosette leaves and early leaf incurvature, concurrent with the enlargement, earliness, and round-to-oval shape transition of leafy heads. Moreover, BcpLH-dependent miRNAs in Chinese cabbage are not the same as HYL1-dependent miRNAs in Arabidopsis. We suggest that BcpLH controls a specific subset of miRNAs in Chinese cabbage and coordinates the direction, extent, and timing of leaf curvature during head formation in Brassica rapa.

Light Triggers the miRNA-Biogenetic Inconsistency for De-etiolated Seedling Survivability in Arabidopsis thaliana

The shift of dark-grown seedlings into light causes enormous transcriptome changes followed by a dramatic developmental transition. Here, we show that microRNA (miRNA) biogenesis also undergoes regulatory changes during de-etiolation. Etiolated seedlings maintain low levels of primary miRNAs (pri-miRNAs) and miRNA processing core proteins, such as Dicer-like 1, SERRATE, and HYPONASTIC LEAVES 1, whereas during de-etiolation both pri-miRNAs and the processing components accumulate to high levels. However, the levels of most miRNAs do not notably increase in response to light. To reconcile this inconsistency, we demonstrated that an unknown suppressor decreases miRNA-processing activity and light-induced SMALL RNA DEGRADING NUCLEASE 1 shortens the half-life of several miRNAs in de-etiolated seedlings. Taken together, these data suggest a novel mechanism, miRNA-biogenetic inconsistency, which accounts for the intricacy of miRNA biogenesis during de-etiolation. This mechanism is essential for the survival of de-etiolated seedlings after long-term skotomorphogenesis and their optimal adaptation to ever-changing light conditions.

Alternative use of miRNA-biogenesis co-factors in plants at low temperatures.

Plants use molecular mechanisms to sense temperatures, trigger quick adaptive responses and thereby cope with environmental changes. MicroRNAs (miRNAs) are key regulators of plant development under such conditions. The catalytic action of DICER LIKE 1 (DCL1), in conjunction with HYPONASTIC LEAVES 1 (HYL1) and SERRATE (SE), produces miRNAs from double-stranded RNAs. As plants lack a stable internal temperature to which enzymatic reactions could be optimized during evolution, reactions such as miRNA processing have to be adjusted to fluctuating environmental temperatures. Here, we report that with decreasing ambient temperature, the plant miRNA biogenesis machinery becomes more robust, producing miRNAs even in the absence of the key DCL1 co-factors HYL1 and SE. This reduces the morphological and reproductive defects of se and hyl1 mutants, restoring seed production. Using small RNA-sequencing and bioinformatics analyses, we have identified specific miRNAs that become HYL1/SE independent for their production in response to temperature decrease. We found that the secondary structure of primary miRNAs is key for this temperature recovery. This finding may have evolutionary implications as a potential adaptation-driving mechanism to a changing climate.

The PROTEIN PHOSPHATASE4 Complex Promotes Transcription and Processing of Primary microRNAs in Arabidopsis.

PROTEIN PHOSPHATASE4 (PP4) is a highly conserved Ser/Thr protein phosphatase found in yeast, plants, and animals. The composition and functions of PP4 in plants are poorly understood. Here, we uncovered the complexity of PP4 composition and function in Arabidopsis (Arabidopsis thaliana) and identified the composition of one form of PP4 containing the regulatory subunit PP4R3A. We show that PP4R3A, together with one of two redundant catalytic subunit genes, PROTEIN PHOSPHATASE X (PPX)1 and PPX2, promotes the biogenesis of microRNAs (miRNAs). PP4R3A is a chromatin-associated protein that interacts with RNA polymerase II and recruits it to the promoters of miRNA-encoding (MIR) genes to promote their transcription. PP4R3A likely also promotes the cotranscriptional processing of miRNA precursors, because it recruits the microprocessor component HYPONASTIC LEAVES1 to MIR genes and to nuclear dicing bodies. Finally, we show that hundreds of introns exhibit splicing defects in pp4r3a mutants. Together, this study reveals roles for Arabidopsis PP4 in transcription and nuclear RNA metabolism.

Detection of MicroRNA Processing Intermediates Through RNA Ligation Approaches.

MicroRNAs (miRNA) are small RNAs of 20-22nt that regulate diverse biological pathways through the modulation of gene expression. miRNAs recognize target RNAs by base complementarity and guide them to degradation or translational arrest. They are transcribed as longer precursors with extensive secondary structures. In plants, these precursors are processed by a complex harboring DICER-LIKE1 (DCL1), which cuts on the precursor stem region to release the mature miRNA together with the miRNA*. In both plants and animals, the miRNA precursors contain spatial clues that determine the position of the miRNA along their sequences. DCL1 is assisted by several proteins, such as the double-stranded RNA binding protein, HYPONASTIC LEAVES1 (HYL1), and the zinc finger protein SERRATE (SE). The precise biogenesis of miRNAs is of utter importance since it determines the exact nucleotide sequence of the mature small RNAs and therefore the identity of the target genes. miRNA processing itself can be regulated and therefore can determine the final small RNA levels and activity. Here, we describe methods to analyze miRNA processing intermediates in plants. These approaches can be used in wild-type or mutant plants, as well as in plants grown under different conditions, allowing a molecular characterization of the miRNA biogenesis from the RNA precursor perspective.

A Quick HYL1-Dependent Reactivation of MicroRNA Production Is Required for a Proper Developmental Response after Extended Periods of Light Deprivation

Light is the most influential environmental stimulus for plant growth. In response to deficient light, plants reprogram their development to adjust their growth in search for a light source. A fine reprogramming of gene expression orchestrates this adaptive trait.Here we show that plants alter microRNA (miRNA) biogenesis in response to light transition. When plants suffer an unusual extended period of light deprivation, the miRNA biogenesis factor HYPONASTIC LEAVES 1 (HYL1) is degraded but an inactive pool of phosphorylated protein remains stable inside the nucleus. Degradation of HYL1 leads to the release of gene silencing, triggering a proper response to dark and shade. Upon light restoration, a quick dephosphorylation of HYL1 leads to the reactivation of miRNA biogenesis and a switch toward a developmental program that maximizes the light uptake. Our findings define a unique and fast regulatory mechanism controlling the plant silencing machinery during plant light response.

Regulation of Plant Microprocessor Function in Shaping microRNA Landscape.

MicroRNAs are small molecules (∼21 nucleotides long) that are key regulators of gene expression. They originate from long stem–loop RNAs as a product of cleavage by a protein complex called Microprocessor. The core components of the plant Microprocessor are the RNase type III enzyme Dicer-Like 1 (DCL1), the zinc finger protein Serrate (SE), and the double-stranded RNA binding protein Hyponastic Leaves 1 (HYL1). Microprocessor assembly and its processing of microRNA precursors have been reported to occur in discrete nuclear bodies called Dicing bodies. The accessibility of and modifications to Microprocessor components affect microRNA levels and may have dramatic consequences in plant development. Currently, numerous lines of evidence indicate that plant Microprocessor activity is tightly regulated. The cellular localization of HYL1 is dependent on a specific KETCH1 importin, and the E3 ubiquitin ligase COP1 indirectly protects HYL1 from degradation in a light-dependent manner. Furthermore, proper localization of HYL1 in Dicing bodies is regulated by MOS2. On the other hand, the Dicing body localization of DCL1 is regulated by NOT2b, which also interacts with SE in the nucleus. Post-translational modifications are substantial factors that contribute to protein functional diversity and provide a fine-tuning system for the regulation of protein activity. The phosphorylation status of HYL1 is crucial for its activity/stability and is a result of the interplay between kinases (MPK3 and SnRK2) and phosphatases (CPL1 and PP4). Additionally, MPK3 and SnRK2 are known to phosphorylate SE. Several other proteins (e.g., TGH, CDF2, SIC, and RCF3) that interact with Microprocessor have been found to influence its RNA-binding and processing activities. In this minireview, recent findings on the various modes of Microprocessor activity regulation are discussed.

The Arabidopsis MOS4-Associated Complex Promotes MicroRNA Biogenesis and Precursor Messenger RNA Splicing.

In Arabidopsis thaliana, the MOS4-ASSOCIATED COMPLEX (MAC) is required for defense and development. The evolutionarily conserved, putative RNA helicase MAC7 is a component of the Arabidopsis MAC, and the human MAC7 homolog, Aquarius, is implicated in pre-mRNA splicing. Here, we show that mac7-1, a partial loss-of-function mutant in MAC7, and two other MAC subunit mutants, mac3a mac3b and prl1 prl2 (pleiotropic regulatory locus), exhibit reduced microRNA (miRNA) levels, indicating that MAC promotes miRNA biogenesis. The mac7-1 mutant shows reduced primary miRNA (pri-miRNA) levels without affecting miRNA gene (MIR) promoter activity or the half-life of pri-miRNA transcripts. As a nuclear protein, MAC7 is not concentrated in dicing bodies, but it affects the localization of HYPONASTIC LEAVES1 (HYL1), a key protein in pri-miRNA processing, to dicing bodies. Immunoprecipitation of HYL1 retrieved 11 known MAC subunits, including MAC7, indicating association between HYL1 and MAC. We propose that MAC7 links MIR transcription to pri-miRNA processing. RNA-seq analysis showed that downregulated genes in MAC subunit mutants are mostly involved in plant defense and stimulus responses, confirming a role of MAC in biotic and abiotic stress responses. We also discovered global intron retention defects in mutants in three subunits of MAC, thus linking MAC function to splicing in Arabidopsis.

Arabidopsis thaliana miRNAs promote embryo pattern formation beginning in the zygote

miRNAs are essential regulators of cell identity, yet their role in early embryo development in plants remains largely unexplored. To determine the earliest stage at which miRNAs act to promote pattern formation in embryogenesis, we examined a series of mutant alleles in the Arabidopsis thaliana miRNA biogenesis enzymes DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1). Cellular and patterning defects were observed in dcl1, se and hyl1 embryos from the zygote through the globular stage of embryogenesis. To identify miRNAs that are expressed in early embryogenesis, we sequenced mRNAs from globular stage Columbia wild type (wt) and se-1 embryos, and identified transcripts potentially corresponding to 100 miRNA precursors. Considering genome location and transcript increase between wt and se-1, 39 of these MIRNAs are predicted to be bona fide early embryo miRNAs. Among these are conserved miRNAs such as miR156, miR159, miR160, miR161, miR164, miR165, miR166, miR167, miR168, miR171, miR319, miR390 and miR394, as well as miRNAs whose function has never been characterized. Our analysis demonstrates that miRNAs promote pattern formation beginning in the zygote, and provides a comprehensive dataset for functional studies of individual miRNAs in Arabidopsis embryogenesis.

The Protein Phosphatase 4 and SMEK1 Complex Dephosphorylates HYL1 to Promote miRNA Biogenesis by Antagonizing the MAPK Cascade in Arabidopsis

Phosphorylation plays an essential role in microRNA (miRNA) processing by regulating co-factors of the miRNA biogenesis machinery. HYL1 (Hyponastic Leaves 1), a core co-factor in plant miRNA biogenesis, is a short-lived phosphoprotein. However, the precise balance and regulatory mechanism of the stability and phosphorylation of HYL1 remain unclear. Here, we show that a highly conserved PP4 (Protein Phosphatase 4) and SMEK1 (Suppressor ofMEK 1) complex dephosphorylates HYL1 to promote miRNA biogenesis, by antagonizing the MAPK cascade in Arabidopsis. The smek1 mutants exhibit defective miRNA biogenesis due to accelerated degradation of HYL1. SMEK1 stabilizes HYL1 in adual manner: SMEK1, as a suppressor, inhibits MAPK activation to attenuate HYL1 phosphorylation; SMEK1 assembles a functional PP4 to target HYL1 for dephosphorylation. Moreover, the protein level of SMEK1 is increased in response to abscisic acid. Our results provide insights into the delicate balance between a protein kinase and a phosphatase during miRNA biogenesis.

The SnRK2 kinases modulate miRNA accumulation in Arabidopsis.

MicroRNAs (miRNAs) regulate gene expression and play critical roles in growth and development as well as stress responses in eukaryotes. miRNA biogenesis in plants requires a processing complex that consists of the core components DICER-LIKE 1 (DCL1), SERRATE (SE) and HYPONASTIC LEAVES (HYL1). Here we show that inactivation of functionally redundant members of the SnRK2 kinases, which are the core components of abscisic acid (ABA) and osmotic stress signaling pathways, leads to reduction in miRNA accumulation under stress conditions. Further analysis revealed that the steady state level of HYL1 protein in plants under osmotic stress is dependent on the SnRK2 kinases. Additionally, our results suggest that the SnRK2 kinases physically associate with the miRNA processing components SE and HYL1 and can phosphorylate these proteins in vitro. These findings reveal an important role for the SnRK2 kinases in the regulation of miRNA accumulation and establish a mechanism by which ABA and osmotic stress signaling is linked to miRNA biogenesis.

KETCH1 imports HYL1 to nucleus for miRNA biogenesis in Arabidopsis

MicroRNA (miRNA) is processed from primary transcripts with hairpin structures (pri-miRNAs) by microprocessors in the nucleus. How cytoplasmic-borne microprocessor components are transported into the nucleus to fulfill their functions remains poorly understood. Here, we report KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1) as a partner of hyponastic leaves 1 (HYL1) protein, a core component of microprocessor in Arabidopsis and functional counterpart of DGCR8/Pasha in animals. Null mutation of ketch1 is embryonic-lethal, whereas knockdown mutation of ketch1 caused morphological defects, reminiscent of mutants in the miRNA pathway. ketch1 knockdown mutation also substantially reduced miRNA accumulation, but did not alter nuclear-cytoplasmic shuttling of miRNAs. Rather, the mutation significantly reduced nuclear portion of HYL1 protein and correspondingly compromised the pri-miRNA processing in the nucleus. We propose that KETCH1 transports HYL1 from the cytoplasm to the nucleus to constitute functional microprocessor in Arabidopsis This study provides insight into the largely unknown nuclear-cytoplasmic trafficking process of miRNA biogenesis components through eukaryotes.