P150 Subunit Research Articles

Elf1 promotes transcription-coupled repair in yeast by using its C-terminal domain to bind TFIIH

Transcription coupled-nucleotide excision repair (TC-NER) removes DNA lesions that block RNA polymerase II (Pol II) transcription. A key step in TC-NER is the recruitment of the TFIIH complex, which initiates DNA unwinding and damage verification; however, the mechanism by which TFIIH is recruited during TC-NER, particularly in yeast, remains unclear. Here, we show that the C-terminal domain (CTD) of elongation factor-1 (Elf1) plays a critical role in TC-NER in yeast by binding TFIIH. Analysis of genome-wide repair of UV-induced cyclobutane pyrimidine dimers (CPDs) using CPD-seq indicates that the Elf1 CTD in yeast is required for efficient TC-NER. We show that the Elf1 CTD binds to the pleckstrin homology (PH) domain of the p62 subunit of TFIIH in vitro, and identify a putative TFIIH-interaction region (TIR) in the Elf1 CTD that is important for PH binding and TC-NER. The Elf1 TIR shows functional, structural, and sequence similarities to a conserved TIR in the mammalian UV sensitivity syndrome A (UVSSA) protein, which recruits TFIIH during TC-NER in mammalian cells. These findings suggest that the Elf1 CTD acts as a functional counterpart to mammalian UVSSA in TC-NER by recruiting TFIIH in response to Pol II stalling at DNA lesions.

Molecular mechanism of dynein-dynactin complex assembly by LIS1.

Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.

Microtubule-binding domains in Katanin p80 subunit are essential for severing activity in C. elegans.

Microtubule-severing enzymes (MSEs), such as Katanin, Spastin, and Fidgetin play essential roles in cell division and neurogenesis. They damage the microtubule (MT) lattice, which can either destroy or amplify the MT cytoskeleton, depending on the cellular context. However, little is known about how they interact with their substrates. We have identified the microtubule-binding domains (MTBD) required for Katanin function in C. elegans. Katanin is a heterohexamer of dimers containing a catalytic subunit p60 and a regulatory subunit p80, both of which are essential for female meiotic spindle assembly. Here, we report that p80-like(MEI-2) dictates Katanin binding to MTs via two MTBDs composed of basic patches. Substituting these patches reduces Katanin binding to MTs, compromising its function in female meiotic-spindle assembly. Structural alignments of p80-like(MEI-2) with p80s from different species revealed that the MTBDs are evolutionarily conserved, even if the specific amino acids involved vary. Our findings highlight the critical importance of the regulatory subunit (p80) in providing MT binding to the Katanin complex.

Structural insights into histone binding and nucleosome assembly by chromatin assembly factor-1.

Chromatin inheritance entails de novo nucleosome assembly after DNA replication by chromatin assembly factor-1 (CAF-1). Yet direct knowledge about CAF-1's histone binding mode and nucleosome assembly process is lacking. In this work, we report the crystal structure of human CAF-1 in the absence of histones and the cryo-electron microscopy structure of CAF-1 in complex with histones H3 and H4. One histone H3-H4 heterodimer is bound by one CAF-1 complex mainly through the p60 subunit and the acidic domain of the p150 subunit. We also observed a dimeric CAF-1-H3-H4 supercomplex in which two H3-H4 heterodimers are poised for tetramer assembly and discovered that CAF-1 facilitates right-handed DNA wrapping of H3-H4 tetramers. These findings signify the involvement of DNA in H3-H4 tetramer formation and suggest a right-handed nucleosome precursor in chromatin replication.

A potential posttranscriptional regulator for p60-katanin: miR-124-3p.

Katanin is a microtubule severing protein belonging to the ATPase family and consists of two subunits; p60-katanin synthesized by the KATNA1 gene and p80-katanin synthesized by the KATNB1 gene. Microtubule severing is one of the mechanisms that allow the reorganization of microtubules depending on cellular needs. While this reorganization of microtubules is associated with mitosis in dividing cells, it primarily takes part in the formation of structures such as axons and dendrites in nondividing mature neurons. Therefore, it is extremely important in neuronal branching. p60 and p80 katanin subunits coexist in the cell. While p60-katanin is responsible for cutting microtubules with its ATPase function, p80-katanin is responsible for the regulation of p60-katanin and its localization in the centrosome. Although katanin has vital functions in the cell, there are no known posttranscriptional regulators of it. MicroRNAs (miRNAs) are a group of small noncoding ribonucleotides that have been found to have important roles in regulating gene expression posttranscriptionally. Despite being important in gene regulation, so far no microRNA has been experimentally associated with katanin regulation. In this study, the effects of miR-124-3p, which we detected as a result of bioinformatics analysis to have the potential to bind to the p60 katanin mRNA, were investigated. For this aim, in this study, SH-SY5Y neuroblastoma cells were transfected with pre-miR-124-3p mimics and pre-mir miRNA precursor as a negative control, and the effect of this transfection on p60-katanin expression was measured at both RNA and protein levels by quantitative real-time PCR (qRT-PCR) and western blotting, respectively. The results of this study showed for the first time that miR-124-3p, which was predicted to bind p60-katanin mRNA by bioinformatic analysis, may regulate the expression of the KATNA1 gene. The data obtained within the scope of this study will make important contributions in order to better understand the regulation of the expression of p60-katanin which as well will have an incontrovertible impact on the understanding of the importance of cytoskeletal reorganization in both mitotic and postmitotic cells.

Structural polymorphism of the PH domain in TFIIH.

The general transcription factor TFIIH is a multi-subunit complex involved in transcription, DNA repair, and cell cycle in eukaryotes. In the human p62 subunit and the budding yeast Saccharomyces cerevisiae Tfb1 subunit of TFIIH, the pleckstrin homology (PH) domain (hPH/scPH) recruits TFIIH to transcription-start and DNA-damage sites by interacting with an acidic intrinsically disordered region in transcription and repair factors. Whereas metazoan PH domains are highly conserved and adopt a similar structure, fungal PH domains are divergent and only the scPH structure is available. Here, we have determined the structure of the PH domain from Tfb1 of fission yeast Schizosaccharomyces pombe (spPH) by NMR. spPH holds an architecture, including the core and external backbone structures, that is closer to hPH than to scPH despite having higher amino acid sequence identity to scPH. In addition, the predicted target-binding site of spPH shares more amino acid similarity with scPH, but spPH contains several key residues identified in hPH as required for specific binding. Using chemical shift perturbation, we have identified binding modes of spPH to spTfa1, a homologue of hTFIIEα, and to spRhp41, a homologue of the repair factors hXPC and scRad4. Both spTfa1 and spRhp41 bind to a similar but distinct surface of spPH by modes that differ from those of target proteins binding to hPH and scPH, revealing that the PH domain of TFIIH interacts with its target proteins in a polymorphic manner in Metazoa, and budding and fission yeasts.

Structural characterization of transcription-coupled repair protein UVSSA and its interaction with TFIIH protein

UV-stimulated scaffold protein A (UVSSA) is a key protein in the Transcription-Coupled Nucleotide Excision Repair (TC-NER) pathway. UVSSA, an intrinsically disordered protein, interacts with multiple members of the pathway, tethering them into the complex. Several studies have reported that UVSSA recruits Transcription Factor IIH (TFIIH) via direct interaction, following which CSB is degraded and the lesion recognition TC-NER complex dissociates from the damage site to facilitate the DNA repair. Structural insights into these events remain largely unknown. Herein, we have investigated the interaction of human UVSSA with the Pleckstrin-Homology-domain of p62 subunit of TFIIH (p62-PHD) using biophysical techniques. We observed that UVSSA forms a stable complex with the p62-PHD in vitro. Small-angle scattering measurements using X-rays and neutrons revealed a significant change in pair-distance distribution function for UVSSA662/p62-PHD complex compared to UVSSA alone. Additionally, a significant decrease was observed in the radius of gyration of the complex. Our findings suggest that TFIIH binding to UVSSA causes significant conformational changes in UVSSA. We hypothesize that these conformational changes play an important role in the dissociation of the lesion recognition TC-NER complex.

Reduced levels of ALS gene DCTN1 induce motor defects in Drosophila

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neuromuscular disease that has a strong genetic component. Deleterious variants in the DCTN1 gene are known to be a cause of ALS in diverse populations. DCTN1 encodes the p150 subunit of the molecular motor dynactin which is a key player in the bidirectional transport of cargos within cells. Whether DCTN1 mutations lead to the disease through either a gain or loss of function mechanism remains unresolved. Moreover, the contribution of non-neuronal cell types, especially muscle tissue, to ALS phenotypes in DCTN1 carriers is unknown. Here we show that gene silencing of Dctn1, the Drosophila main orthologue of DCTN1, either in neurons or muscles is sufficient to cause climbing and flight defects in adult flies. We also identify Dred, a protein with high homology to Drosophila Dctn1 and human DCTN1, that on loss of function also leads to motoric impairments. A global reduction of Dctn1 induced a significant reduction in the mobility of larvae and neuromuscular junction (NMJ) deficits prior to death at the pupal stage. RNA-seq and transcriptome profiling revealed splicing alterations in genes required for synapse organisation and function, which may explain the observed motor dysfunction and synaptic defects downstream of Dctn1 ablation. Our findings support the possibility that loss of DCTN1 function can lead to ALS and underscore an important requirement for DCTN1 in muscle in addition to neurons.

Dynamic structures of intrinsically disordered proteins related to the general transcription factor TFIIH, nucleosomes, and histone chaperones.

Advances in structural analysis by cryogenic electron microscopy (cryo-EM) and X-ray crystallography have revealed the tertiary structures of various chromatin-related proteins, including transcription factors, RNA polymerases, nucleosomes, and histone chaperones; however, the dynamic structures of intrinsically disordered regions (IDRs) in these proteins remain elusive. Recent studies using nuclear magnetic resonance (NMR), together with molecular dynamics (MD) simulations, are beginning to reveal dynamic structures of the general transcription factor TFIIH complexed with target proteins including the general transcription factor TFIIE, the tumor suppressor p53, the cell cycle protein DP1, the DNA repair factors XPC and UVSSA, and three RNA polymerases, in addition to the dynamics of histone tails in nucleosomes and histone chaperones. In complexes of TFIIH, the PH domain of the p62 subunit binds to an acidic string formed by the IDR in TFIIE, p53, XPC, UVSSA, DP1, and the RPB6 subunit of three RNA polymerases by a common interaction mode, namely extended string-like binding of the IDR on the positively charged surface of the PH domain. In the nucleosome, the dynamic conformations of the N-tails of histones H2A and H2B are correlated, while the dynamic conformations of the N-tails of H3 and H4 form a histone tail network dependent on their modifications and linker DNA. The acidic IDRs of the histone chaperones of FACT and NAP1 play important roles in regulating the accessibility to histone proteins in the nucleosome.

Unorthodox PCNA Binding by Chromatin Assembly Factor 1.

The eukaryotic DNA replication fork is a hub of enzymes that continuously act to synthesize DNA, propagate DNA methylation and other epigenetic marks, perform quality control, repair nascent DNA, and package this DNA into chromatin. Many of the enzymes involved in these spatiotemporally correlated processes perform their functions by binding to proliferating cell nuclear antigen (PCNA). A long-standing question has been how the plethora of PCNA-binding enzymes exert their activities without interfering with each other. As a first step towards deciphering this complex regulation, we studied how Chromatin Assembly Factor 1 (CAF-1) binds to PCNA. We demonstrate that CAF-1 binds to PCNA in a heretofore uncharacterized manner that depends upon a cation-pi (π) interaction. An arginine residue, conserved among CAF-1 homologs but absent from other PCNA-binding proteins, inserts into the hydrophobic pocket normally occupied by proteins that contain canonical PCNA interaction peptides (PIPs). Mutation of this arginine disrupts the ability of CAF-1 to bind PCNA and to assemble chromatin. The PIP of the CAF-1 p150 subunit resides at the extreme C-terminus of an apparent long α-helix (119 amino acids) that has been reported to bind DNA. The length of that helix and the presence of a PIP at the C-terminus are evolutionarily conserved among numerous species, ranging from yeast to humans. This arrangement of a very long DNA-binding coiled-coil that terminates in PIPs may serve to coordinate DNA and PCNA binding by CAF-1.

Modification of the 4Fe-4S Cluster Charge Transport Pathway Alters RNA Synthesis by Yeast DNA Primase.

DNA synthesis during replication begins with the generation of an ∼10-nucleotide primer by DNA primase. Primase contains a redox-active 4Fe-4S cluster in the C-terminal domain of the p58 subunit (p58C). The redox state of this 4Fe-4S cluster can be modulated via the transport of charge through the protein and the DNA substrate (redox switching); changes in the redox state of the cluster alter the ability of p58C to associate with its substrate. The efficiency of redox switching in p58C can be altered by mutating tyrosine residues that bridge the 4Fe-4S cluster and the nucleic acid binding site. Here, we report the effects of mutating bridging tyrosines to phenylalanines in yeast p58C. High-resolution crystal structures show that these mutations, even with six tyrosines simultaneously mutated, do not perturb the three-dimensional structure of the protein. In contrast, measurements of the electrochemical properties on DNA-modified electrodes of p58C containing multiple tyrosine to phenylalanine mutations reveal deficiencies in their ability to engage in DNA charge transport. Significantly, this loss of electrochemical activity correlates with decreased primase activity. While single-site mutants showed modest decreases in activity compared to that of the wild-type primase, the protein containing six mutations exhibited a 10-fold or greater decrease. Thus, many possible tyrosine-mediated pathways for charge transport in yeast p58C exist, but inhibiting these pathways together diminishes the ability of yeast primase to generate primers. These results support a model in which redox switching is essential for primase activity.

MOR1/MAP215 acts synergistically with katanin to control cell division and anisotropic cell elongation in Arabidopsis.

The MAP215 family of microtubule (MT) polymerase/nucleation factors and the MT severing enzyme katanin are widely conserved MT-associated proteins (MAPs) across the plant and animal kingdoms. However, how these two essential MAPs coordinate to regulate plant MT dynamics and development remains unknown. Here, we identified novel hypomorphic alleles of MICROTUBULE ORGANIZATION 1 (MOR1), encoding the Arabidopsis thaliana homolog of MAP215, in genetic screens for mutants oversensitive to the MT-destabilizing drug propyzamide. Live imaging in planta revealed that MOR1-green fluorescent protein predominantly tracks the plus-ends of cortical MTs (cMTs) in interphase cells and labels preprophase band, spindle and phragmoplast MT arrays in dividing cells. Remarkably, MOR1 and KATANIN 1 (KTN1), the p60 subunit of Arabidopsis katanin, act synergistically to control the proper formation of plant-specific MT arrays, and consequently, cell division and anisotropic cell expansion. Moreover, MOR1 physically interacts with KTN1 and promotes KTN1-mediated severing of cMTs. Our work establishes the Arabidopsis MOR1-KTN1 interaction as a central functional node dictating MT dynamics and plant growth and development.

Biochemical and Structural Insights into the Winged Helix Domain of P150, the Largest Subunit of the Chromatin Assembly Factor 1.

The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features.

A mutation in <i>CsKTN1</i> for the Katanin p60 protein results in miniature plant in cucumber, <i>Cucumis sativus</i> L.

Katanin, a microtubule shearing protein, plays an important role in plant architecture formation. However, little is known about its mechanisms in regulating plant architecture in cucumber. In the present study, through EMS mutagenesis, we identified a novel <em>micro-plant</em> (<em>mp</em>) mutant in the North China type inbred line CCMC that may be of value for cucumber breeding. The size and number of stem cells were altered in the <em>mp</em> mutant. Through bulked segregant analysis (BSA) sequencing approach combined with genetic mapping, the <em>mp</em> locus was delimited to an interval of 130.9-kb. Multiple lines of evidence suggested that the <em>mp</em> mutation is due to a single nucleotide polymorphism in <em>Csa7G435510</em> that is predicted to encode the Katanin p60 subunit protein (CsKTN1). The expression levels of <em>CsKTN1</em> decreased significantly in all tissues except the tendril of <em>mp</em> mutant. Subcellular localization showed that both wild-type and mutant CsKTN1 proteins were located in cell membrane, cytoplasm and nucleus of tobacco leaf cells. The mutant protein lost part of its ability to bind and shear microtubule <em>in vitro</em>. These findings provide new insight into the regulatory function of microtubule-shearing protein, Katanin p60, in plant architecture of cucumber.

CHAF1b, chromatin assembly factor-1 subunit b, is essential for mouse preimplantation embryos

Chromatin assembly factor-1, subunit b (CHAF1b), the p60 subunit of the chromatin-assembly factor-1 (CAF-1) complex, is an evolutionarily conserved protein that has been implicated in various biological processes. Although a variety of functions have been attributed to CHAF1b, its function in preimplantation embryos remains obscure. In this study, we showed that CHAF1b knockdown did not affect the blastocyst rate, but resulted in a low blastocyst hatching rate, outgrowth failure in vitro, and embryonic lethality after implantation in vivo. Notably, CHAF1b depletion increased apoptosis and caused down-regulated expression of key regulators of cell fate specification, including Oct4, Cdx2, Sox2, and Nanog. Further analysis revealed that CHAF1b mediated the replacement of H3.3 with H3.1/3.2, which was associated with decreased repressive histone marks (H3K9me2/3 and H3K27me2/3) and increased active histone marks (H3K4me2/3). Moreover, RNA-sequencing analysis revealed that CHAF1b depletion resulted in the differential expression of 1508 genes, including epigenetic modifications genes, multiple lineage-specific genes, and several genes encoding apoptosis proteins. In addition, assay for transposase-accessible chromatin-sequencing analysis demonstrated that silencing CHAF1b altered the chromatin accessibility of lineage-specific genes and epigenetic modifications genes. Taken together, these data imply that CHAF1b plays significant roles in preimplantation embryos, probably by regulating epigenetic modifications and lineage specification.

Cesium based phasing of macromolecules: a general easy to use approach for solving the phase problem

Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a “quick-soak”-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.

CHAF1A Blocks Neuronal Differentiation and Promotes Neuroblastoma Oncogenesis via Metabolic Reprogramming.

Neuroblastoma (NB) arises from oncogenic disruption of neural crest (NC) differentiation. Treatment with retinoic acid (RA) to induce differentiation has improved survival in some NB patients, but not all patients respond, and most NBs eventually develop resistance to RA. Loss of the chromatin modifier chromatin assembly factor 1 subunit p150 (CHAF1A) promotes NB cell differentiation; however, the mechanism by which CHAF1A drives NB oncogenesis has remained unexplored. This study shows that CHAF1A gain‐of‐function supports cell malignancy, blocks neuronal differentiation in three models (zebrafish NC, human NC, and human NB), and promotes NB oncogenesis. Mechanistically, CHAF1A upregulates polyamine metabolism, which blocks neuronal differentiation and promotes cell cycle progression. Targeting polyamine synthesis promotes NB differentiation and enhances the anti‐tumor activity of RA. The authors' results provide insight into the mechanisms that drive NB oncogenesis and suggest a rapidly translatable therapeutic approach (DFMO plus RA) to enhance the clinical efficacy of differentiation therapy in NB patients.

Phylogeny and structural peculiarities of the EB proteins of diatoms

The end-binding proteins are a family of microtubule-associated proteins; this family belongs to plus-end-tracking proteins (+TIPs) that regulate microtubule growth and stabilisation. Although the genes encoding EB proteins are found in all eukaryotic genomes, most studies of them have centred on one or another taxonomic group, without a broad comparative analysis. Here, we present a first phylogenetic analysis and a comparative analysis of domain structures of diatom EB proteins in comparison with other phyla of Chromista, red and green algae, as well as model organisms A. thaliana and H. sapiens. Phylogenetically, diatom EB proteins are separated into six clades, generally corresponding to the phylogeny of their respective organisms. The domain structure of this family is highly variable, but the CH and EBH domains responsible for binding tubulin and other MAPs are mostly conserved. Homologous modelling of the F. cylindrus EB protein shows that conserved motifs of the CH domain are positioned on the protein surface, which is necessary for their functioning. We hypothesise that high variance of the diatom C-terminal domain is caused by previously unknown interactions with a CAP-GLY motif of dynactin subunit p150. Our findings contribute to wider possibilities for further investigations of the cytoskeleton in diatoms.

Three human RNA polymerases interact with TFIIH via a common RPB6 subunit.

In eukaryotes, three RNA polymerases (RNAPs) play essential roles in the synthesis of various types of RNA: namely, RNAPI for rRNA; RNAPII for mRNA and most snRNAs; and RNAPIII for tRNA and other small RNAs. All three RNAPs possess a short flexible tail derived from their common subunit RPB6. However, the function of this shared N-terminal tail (NTT) is not clear. Here we show that NTT interacts with the PH domain (PH-D) of the p62 subunit of the general transcription/repair factor TFIIH, and present the structures of RPB6 unbound and bound to PH-D by nuclear magnetic resonance (NMR). Using available cryo-EM structures, we modelled the activated elongation complex of RNAPII bound to TFIIH. We also provide evidence that the recruitment of TFIIH to transcription sites through the p62–RPB6 interaction is a common mechanism for transcription-coupled nucleotide excision repair (TC-NER) of RNAPI- and RNAPII-transcribed genes. Moreover, point mutations in the RPB6 NTT cause a significant reduction in transcription of RNAPI-, RNAPII- and RNAPIII-transcribed genes. These and other results show that the p62–RPB6 interaction plays multiple roles in transcription, TC-NER, and cell proliferation, suggesting that TFIIH is engaged in all RNAP systems.

CsKTN1 for a katanin p60 subunit is associated with the regulation of fruit elongation in cucumber (Cucumis sativus L.).

We identified a short fruit3 (sf3) mutant in cucumber. Map-based cloning revealed that CsKTN1 gene encodes a katanin p60 subunit, which is associated with the regulation of fruit elongation. Fruit length is an important horticultural trait for both fruit yield and quality of cucumber (Cucumis sativus L.). Knowledge on the molecular regulation of fruit elongation in cucumber is very limited. In this study, we identified and characterized a cucumber short fruit3 (sf3) mutant. Histological examination indicated that the shorter fruit in the mutant was due to reduced cell numbers. Genetic analysis revealed that the phenotype of the sf3 mutant was controlled by a single gene with semi-dominant inheritance. By map-based cloning and Arabidopsis genetic transformation, we showed that Sf3 was a homolog of KTN1 (CsKTN1) encoding a katanin p60 subunit. A non-synonymous mutation in the fifth exon of CsKTN1 resulted in an amino acid substitution from Serine in the wild type to Phenylalanine in the sf3 mutant. CsKTN1 expressed in all tissues of both the wild type and the sf3 mutant. However, there was no significant difference in CsKTN1 expression levels between the wild type and the sf3 mutant. The hormone quantitation and RNA-seq analysis suggested that auxin and gibberellin contents are decreased in sf3 by changing the expression levels of genes related with auxin and gibberellin metabolism and signaling. This work helps understand the function of the katanin and the molecular mechanisms of fruit growth regulation in cucumber.