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.

Crystal structure of the Arabidopsis SPIRAL2 C-terminal domain reveals a p80-Katanin-like domain.

Epidermal cells of dark-grown plant seedlings reorient their cortical microtubule arrays in response to blue light from a net lateral orientation to a net longitudinal orientation with respect to the long axis of cells. The molecular mechanism underlying this microtubule array reorientation involves katanin, a microtubule severing enzyme, and a plant-specific microtubule associated protein called SPIRAL2. Katanin preferentially severs longitudinal microtubules, generating seeds that amplify the longitudinal array. Upon severing, SPIRAL2 binds nascent microtubule minus ends and limits their dynamics, thereby stabilizing the longitudinal array while the lateral array undergoes net depolymerization. To date, no experimental structural information is available for SPIRAL2 to help inform its mechanism. To gain insight into SPIRAL2 structure and function, we determined a 1.8 Å resolution crystal structure of the Arabidopsis thaliana SPIRAL2 C-terminal domain. The domain is composed of seven core α-helices, arranged in an α-solenoid. Amino-acid sequence conservation maps primarily to one face of the domain involving helices α1, α3, α5, and an extended loop, the α6-α7 loop. The domain fold is similar to, yet structurally distinct from the C-terminal domain of Ge-1 (an mRNA decapping complex factor involved in P-body localization) and, surprisingly, the C-terminal domain of the katanin p80 regulatory subunit. The katanin p80 C-terminal domain heterodimerizes with the MIT domain of the katanin p60 catalytic subunit, and in metazoans, binds the microtubule minus-end factors CAMSAP3 and ASPM. Structural analysis predicts that SPIRAL2 does not engage katanin p60 in a mode homologous to katanin p80. The SPIRAL2 structure highlights an interesting evolutionary convergence of domain architecture and microtubule minus-end localization between SPIRAL2 and katanin complexes, and establishes a foundation upon which structure-function analysis can be conducted to elucidate the role of this domain in the regulation of plant microtubule arrays.

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network.

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of α-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster, and then examining the microtubule networks in the neuromuscular junction and muscle cells, we accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

The katanin A-subunits KATNA1 and KATNAL1 act co-operatively in mammalian meiosis and spermiogenesis to achieve male fertility.

Katanins, a class of microtubule-severing enzymes, are potent M-phase regulators in oocytes and somatic cells. How the complex and evolutionarily crucial, male mammalian meiotic spindle is sculpted remains unknown. Here, using multiple single and double gene knockout mice, we reveal that the canonical katanin A-subunit KATNA1 and its close paralogue KATNAL1 together execute multiple aspects of meiosis. We show KATNA1 and KATNAL1 collectively regulate the male meiotic spindle, cytokinesis and midbody abscission, in addition to diverse spermatid remodelling events, including Golgi organisation, and acrosome and manchette formation. We also define KATNAL1-specific roles in sperm flagellum development, manchette regulation and sperm-epithelial disengagement. Finally, using proteomic approaches, we define the KATNA1, KATNAL1 and KATNB1 mammalian testis interactome, which includes a network of cytoskeletal and vesicle trafficking proteins. Collectively, we reveal that the presence of multiple katanin A-subunit paralogs in mammalian spermatogenesis allows for 'customised cutting' via neofunctionalisation and protective buffering via gene redundancy.

The Scaffold Protein KATNIP Enhances CILK1 Control of Primary Cilia.

The primary cilium functions as a cellular sensory organelle and signaling antenna that detects and transduces extracellular signals. Mutations in the human gene CILK1 (ciliogenesis associated kinase 1) cause abnormal cilia elongation and faulty Hedgehog signaling, associated with developmental disorders and epilepsy. CILK1 is a protein kinase that requires dual phosphorylation of its TDY motif for activation and its extended C-terminal intrinsically disordered region (IDR) mediates targeting to the basal body and substrate recognition. Proteomics previously identified katanin-interacting protein (KATNIP), also known as KIAA0556, as a CILK1 interacting partner. In this study we discovered that CILK1 colocalizes with KATNIP at the basal body and the CILK1 IDR is sufficient to mediate binding to KATNIP. Deletion analysis of KATNIP shows one of three domains of unknown function (DUF) is required for association with CILK1. KATNIP binding with CILK1 drastically elevated CILK1 protein levels and TDY phosphorylation in cells. This resulted in a profound increase in phosphorylation of known CILK1 substrates and suppression of cilia length. Thus, KATNIP functions as a regulatory subunit of CILK1 that potentiates its actions. This advances our understanding of the molecular basis of control of primary cilia.

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.

A Tale of 12 Tails: Katanin Severing Activity Affected by Carboxy-Terminal Tail Sequences.

In cells, microtubule location, length, and dynamics are regulated by a host of microtubule-associated proteins and enzymes that read where to bind and act based on the microtubule "tubulin code," which is predominantly encoded in the tubulin carboxy-terminal tail (CTT). Katanin is a highly conserved AAA ATPase enzyme that binds to the tubulin CTTs to remove dimers and sever microtubules. We have previously demonstrated that short CTT peptides are able to inhibit katanin severing. Here, we examine the effects of CTT sequences on this inhibition activity. Specifically, we examine CTT sequences found in nature, alpha1A (TUBA1A), detyrosinated alpha1A, Δ2 alpha1A, beta5 (TUBB/TUBB5), beta2a (TUBB2A), beta3 (TUBB3), and beta4b (TUBB4b). We find that these natural CTTs have distinct abilities to inhibit, most noticeably beta3 CTT cannot inhibit katanin. Two non-native CTT tail constructs are also unable to inhibit, despite having 94% sequence identity with alpha1 or beta5 sequences. Surprisingly, we demonstrate that poly-E and poly-D peptides are capable of inhibiting katanin significantly. An analysis of the hydrophobicity of the CTT constructs indicates that more hydrophobic polypeptides are less inhibitory than more polar polypeptides. These experiments not only demonstrate inhibition, but also likely interaction and targeting of katanin to these various CTTs when they are part of a polymerized microtubule filament.

Crystal structure of the C-terminal domain of the plant-specific microtubule-associated protein Spiral2.

Plant cells form microtubule arrays, called `cortical microtubules', beneath the plasma membrane which are critical for cell-wall organization and directional cell growth. Cortical microtubules are nucleated independently of centrosomes. Spiral2 is a land-plant-specific microtubule minus-end-targeting protein that stabilizes the minus ends by inhibiting depolymerization of the filament. Spiral2 possesses an N-terminal microtubule-binding domain and a conserved C-terminal domain whose function is unknown. In this study, the crystal structure of the conserved C-terminal domain of Spiral2 was determined using the single-wavelength anomalous dispersion method. Refinement of the model to a resolution of 2.2 Šrevealed a helix-turn-helix fold with seven α-helices. The protein crystallized as a dimer, but SEC-MALS analysis showed the protein to be monomeric. A structural homology search revealed that the protein has similarity to the C-terminal domain of the katanin regulatory subunit p80. The structure presented here suggests that the C-terminal domain of Spiral2 represents a new class of microtubule dynamics modulator across the kingdom.

New insights into the functions and regulations of MAP215/MOR1 and katanin, two conserved microtubule-associated proteins in Arabidopsis.

Plant microtubules (MTs) form highly dynamic and distinct arrays throughout the cell cycle and are essential for cell and organ morphogenesis. A plethora of microtubule associated-proteins (MAPs), both conserved and plant-specific, ensure the dynamic response of MTs to internal and external cues. The MAP215 family MT polymerase/nucleation factor and the MT severing enzyme katanin are among the most conserved MAPs in eukaryotes. Recent studies have revealed unexpected functional and physical interactions between MICROTUBULE ORGANIZATION 1 (MOR1), the Arabidopsis homolog of MAP215, and KATANIN 1 (KTN1), the catalytic subunit of katanin. In this minireview, we provide a short overview on current understanding of the functions and regulations of MOR1 and katanin in cell morphogenesis and plant growth and development.

IQ67 DOMAIN protein 21 is critical for indentation formation in pavement cell morphogenesis.

In plants, cortical microtubules anchor to the plasma membrane in arrays and play important roles in cell shape. However, the molecular mechanism of microtubule binding proteins, which connect the plasma membrane and cortical microtubules in cell morphology remains largely unknown. Here, we report that a plasma membrane and microtubule dual-localized IQ67 domain protein, IQD21, is critical for cotyledon pavement cell (PC) morphogenesis in Arabidopsis. iqd21 mutation caused increased indentation width, decreased lobe length, and similar lobe number of PCs, whereas IQD21 overexpression had a different effect on cotyledon PC shape. Weak overexpression led to increased lobe number, decreased indentation width, and similar lobe length, while moderate or great overexpression resulted in decreased lobe number, indentation width, and lobe length of PCs. Live-cell observations revealed that IQD21 accumulation at indentation regions correlates with lobe initiation and outgrowth during PC development. Cell biological and genetic approaches revealed that IQD21 promotes transfacial microtubules anchoring to the plasma membrane via its polybasic sites and bundling at the indentation regions in both periclinal and anticlinal walls. IQD21 controls cortical microtubule organization mainly through promoting Katanin 1-mediated microtubule severing during PC interdigitation. These findings provide the genetic evidence that transfacial microtubule arrays play a determinant role in lobe formation, and the insight into the molecular mechanism of IQD21 in transfacial microtubule organization at indentations and puzzle-shaped PC development.

Brassinosteroid signals cooperate with katanin-mediated microtubule severing to control stamen filament elongation.

Proper stamen filament elongation is essential for pollination and plant reproduction. Plant hormones are extensively involved in every stage of stamen development; however, the cellular mechanisms by which phytohormone signals couple with microtubule dynamics to control filament elongation remain unclear. Here, we screened a series of Arabidopsis thaliana mutants showing different microtubule defects and revealed that only those unable to sever microtubules, lue1 and ktn80.1234, displayed differential floral organ elongation with less elongated stamen filaments. Prompted by short stamen filaments and severe decrease in KTN1 and KTN80s expression in qui-2 lacking five BZR1-family transcription factors (BFTFs), we investigated the crosstalk between microtubule severing and brassinosteroid (BR) signaling. The BFTFs transcriptionally activate katanin-encoding genes, and the microtubule-severing frequency was severely reduced in qui-2. Taken together, our findings reveal how BRs can regulate cytoskeletal dynamics to coordinate the proper development of reproductive organs.

Microtubule Severing Enzymes Oligomerization and Allostery: A Tale of Two Domains.

Severing proteins are nanomachines from the AAA+ (ATPases associated with various cellular activities) superfamily whose function is to remodel the largest cellular filaments, microtubules. The standard AAA+ machines adopt hexameric ring structures for functional reasons, while being primarily monomeric in the absence of the nucleotide. Both major severing proteins, katanin and spastin, are believed to follow this trend. However, studies proposed that they populate lower-order oligomers in the presence of cofactors, which are functionally relevant. Our simulations show that the preferred oligomeric assembly is dependent on the binding partners and on the type of severing protein. Essential dynamics analysis predicts that the stability of an oligomer is dependent on the strength of the interface between the helical bundle domain (HBD) of a monomer and the convex face of the nucleotide binding domain (NBD) of a neighboring monomer. Hot spots analysis found that the region consisting of the HBD tip and the C-terminal (CT) helix is the only common element between the allosteric networks responding to nucleotide, substrate, and intermonomer binding. Clustering analysis indicates the existence of multiple pathways for the transition between the secondary structure of the HBD tip in monomers and the structure(s) it adopts in oligomers.

Combinatorial and antagonistic effects of tubulin glutamylation and glycylation on katanin microtubule severing.

Microtubules have spatiotemporally complex posttranslational modification patterns. How cells interpret this tubulin modification code is largely unknown. We show that C. elegans katanin, a microtubule severing AAA ATPase mutated in microcephaly and critical for cell division, axonal elongation, and cilia biogenesis, responds precisely, differentially, and combinatorially to three chemically distinct tubulin modifications-glycylation, glutamylation, and tyrosination-but is insensitive to acetylation. Glutamylation and glycylation are antagonistic rheostats with glycylation protecting microtubules from severing. Katanin exhibits graded and divergent responses to glutamylation on the α- and ß-tubulin tails, and these act combinatorially. The katanin hexamer central pore constrains the polyglutamate chain patterns on ß-tails recognized productively. Elements distal to the katanin AAA core sense α-tubulin tyrosination, and detyrosination downregulates severing. The multivalent microtubule recognition that enables katanin to read multiple tubulin modification inputs explains in vivo observations and illustrates how effectors can integrate tubulin code signals to produce diverse functional outcomes.

Microtubule-severing enzymes.

Stephanie Sarbanes et al. discuss microtubule-severing enzymes, highlighting their shared structure and mechanism and the diversity of processes in which they participate.

Opto-katanin, an optogenetic tool for localized, microtubule disassembly.

Microtubules are cytoskeletal polymers that separate chromosomes during mitosis and serve as rails for intracellular transport and organelle positioning. Manipulation of microtubules is widely used in cell and developmental biology, but tools for precise subcellular spatiotemporal control of microtubules are currently lacking. Here, we describe a light-activated system for localized recruitment of the microtubule-severing enzyme katanin. This system, named opto-katanin, uses targeted illumination with blue light to induce rapid, localized, and reversible microtubule depolymerization. This tool allows precise clearing of a subcellular region of microtubules while preserving the rest of the microtubule network, demonstrating that regulation of katanin recruitment to microtubules is sufficient to control its severing activity. The tool is not toxic in the absence of blue light and can be used to disassemble both dynamic and stable microtubules in primary neurons as well as in dividing cells. We show that opto-katanin can be used to locally block vesicle transport and to clarify the dependence of organelle morphology and dynamics on microtubules. Specifically, our data indicate that microtubules are not required for the maintenance of the Golgi stacks or the tubules of the endoplasmic reticulum but are needed for the formation of new membrane tubules. Finally, we demonstrate that this tool can be applied to study the contribution of microtubules to cell mechanics by showing that microtubule bundles can exert forces constricting the nucleus.

circ-Katnal1 Enhances Inflammatory Pyroptosis in Sepsis-Induced Liver Injury through the miR-31-5p/GSDMD Axis.

Background: Sepsis is a systemic inflammatory response that can elicit organ dysfunction as well as circulatory diseases in serious cases. When inflammatory responses are especially dysregulated, severe complications can arise, including sepsis-induced liver injury. Various microRNAs along with circular (circ) RNAs are involved in inflammatory responses; nevertheless, their functions in regulating sepsis-induced liver injury remain unknown. The cecal ligation and puncture (CLP) procedure can induce liver injury as well as polymicrobial sepsis. Methods: In this study, CLP was used to induce liver injury as well as polymicrobial sepsis. Then, liver function, inflammatory cytokine expression, and hepatic histopathology were evaluated. High-throughput sequencing was employed to investigate the abnormal hepatic circRNA expression after CLP. Raw264.7 cells were utilized to simulation an in vitro sepsis inflammation model with LPS induce. The relative mRNA as well as protein levels of TNF-α, IL-1ß, and IL-6 was explored by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays. We explored functional connections among circRNAs, miR-31-5p, and gasdermin D (GSDMD) using dual-luciferase reporter assays. Western blot was employed to test GSDMD, caspase-1, and NLRP3 expression in mice and cell models. Results: Our results showed that CLP-induced sepsis promoted liver injury via increasing inflammatory pyroptosis. The abnormal expression of circ-Katnal1 played an important role in CLP-induced sepsis. Downregulating circ-Katnal1 suppressed LPS-induced inflammatory pyroptosis in Raw264.7 cells. Bioinformatics and luciferase reporter results confirmed that miR-31-5p and GSDMD were downstream targets of circ-Katnal1. Inhibiting miR-31-5p or upregulating GSDMD reversed the protective effects of silencing circ-Katnal1. Conclusion: Taken together, circ-Katnal1 enhanced inflammatory pyroptosis in sepsis-induced liver injury through the miR-31-5p/GSDMD axis.

The IPGA1-ANGUSTIFOLIA module regulates microtubule organisation and pavement cell shape in Arabidopsis.

Plant cells continuously experience mechanical stress resulting from the cell wall that bears internal turgor pressure. Cortical microtubules align with the predicted maximal tensile stress direction to guide cellulose biosynthesis and therefore results in cell wall reinforcement. We have previously identified Increased Petal Growth Anisotropy (IPGA1) as a putative microtubule-associated protein in Arabidopsis, but the function of IPGA1 remains unclear. Here, using the Arabidopsis cotyledon pavement cell as a model, we demonstrated that IPGA1 forms protein granules and interacts with ANGUSTIFOLIA (AN) to cooperatively regulate microtubule organisation in response to stress. Application of mechanical perturbations, such as cell ablation, led to microtubule reorganisation into aligned arrays in wild-type cells. This microtubule response to stress was enhanced in the IPGA1 loss-of-function mutant. Mechanical perturbations promoted the formation of IPGA1 granules on microtubules. We further showed that IPGA1 physically interacted with AN both in vitro and on microtubules. The ipga1 mutant alleles exhibited reduced interdigitated growth of pavement cells, with smooth shape. IPGA1 and AN had a genetic interaction in regulating pavement cell shape. Furthermore, IPGA1 genetically and physically interacted with the microtubule-severing enzyme KATANIN. We propose that the IPGA1-AN module regulates microtubule organisation and pavement cell shape.

Knockout of Katnal2 Leads to Autism-like Behaviors and Developmental Delay in Zebrafish.

KATNAL2 mutations have been associated with autism spectrum disorder (ASD) and other related neurodevelopmental disorders (NDDs) such as intellectual disability (ID) in several cohorts. KATNAL2 has been implicated in brain development, as it is required for ciliogenesis in Xenopus and is required for dendritic arborization in mice. However, a causative relationship between the disruption of Katnal2 function and behavioral defects has not been established. Here, we generated a katnal2 null allele in zebrafish using CRISPR/Cas9-mediated genome editing and carried out morphological and behavioral characterizations. We observed that katnal2-/- embryos displayed delayed embryonic development especially during the convergence and extension (CE) movement. The hatched larvae showed reduced brain size and body length. In the behavioral tests, the katnal2-/- zebrafish exhibited reduced locomotor activity both in larvae and adults; increased nocturnal waking activity in larvae; and enhanced anxiety-like behavior, impaired social interaction, and reduced social cohesion in adults. These findings indicate an important role for katnal2 in development and behavior, providing an in vivo model to study the mechanisms underlying the ASD related to KATNAL2 mutations.

SUMOylation of microtubule-cleaving enzyme KATNA1 promotes microtubule severing and neurite outgrowth.

Katanin p60 ATPase-containing subunit A1 (KATNA1) is a microtubule-cleaving enzyme that regulates the development of neural protrusions through cytoskeletal rearrangements. However, the mechanism underlying the linkage of the small ubiquitin-like modifier (SUMO) protein to KATNA1 and how this modification regulates the development of neural protrusions is unclear. Here we discovered, using mass spectrometry analysis, that SUMO-conjugating enzyme UBC9, an enzyme necessary for the SUMOylation process, was present in the KATNA1 interactome. Moreover, GST-pull down and co-immunoprecipitation assays confirmed that KATNA1 and SUMO interact. We further demonstrated using immunofluorescence experiments that KATNA1 and the SUMO2 isoform colocalized in hippocampal neurites. We also performed a bioinformatics analysis of KATNA1 protein sequences to identify three potentially conserved SUMOylation sites (K77, K157, and K330) among vertebrates. Mutation of K330, but not K77 or K157, abolished KATNA1-induced microtubule severing and decreased the level of binding observed for KATNA1 and SUMO2. Cotransfection of SUMO2 and wildtype KATNA1 in COS7 cells increased microtubule severing, whereas no effect was observed after cotransfection with the K330R KATNA1 mutant. Furthermore, in cultured hippocampal neurons, overexpression of wildtype KATNA1 significantly promoted neurite outgrowth, whereas the K330R mutant eliminated this effect. Taken together, our results demonstrate that the K330 site in KATNA1 is modified by SUMOylation and SUMOylation of KATNA1 promotes microtubule dynamics and hippocampal neurite outgrowth.

Common genetic variation in KATNAL1 non-coding regions is involved in the susceptibility to severe phenotypes of male infertility.

BACKGROUND: Previous studies in animal models evidenced that genetic mutations of KATNAL1, resulting in dysfunction of its encoded protein, lead to male infertility through disruption of microtubule remodelling and premature germ cell exfoliation. Subsequent studies in humans also suggested a possible role of KATNAL1 single-nucleotide polymorphisms in the development of male infertility as a consequence of severe spermatogenic failure. OBJECTIVES: The main objective of the present study is to evaluate the effect of the common genetic variation of KATNAL1 in a large and phenotypically well-characterised cohort of infertile men because of severe spermatogenic failure. MATERIALS AND METHODS: A total of 715 infertile men because of severe spermatogenic failure, including 210 severe oligospermia and 505 non-obstructive azoospermia patients, as well as 1058 unaffected controls were genotyped for three KATNAL1 single-nucleotide polymorphism taggers (rs2077011, rs7338931 and rs2149971). Case-control association analyses by logistic regression assuming different models and in silico functional characterisation of risk variants were conducted. RESULTS: Genetic associations were observed between the three analysed taggers and different severe spermatogenic failure groups. However, in all cases, the haplotype model (rs2077011*C | rs7338931*T | rs2149971*A) better explained the observed associations than the three risk alleles independently. This haplotype was associated with non-obstructive azoospermia (adjusted p = 4.96E-02, odds ratio = 2.97), Sertoli-cell only syndrome (adjusted p = 2.83E-02, odds ratio = 5.16) and testicular sperm extraction unsuccessful outcomes (adjusted p = 8.99E-04, odds ratio = 6.13). The in silico analyses indicated that the effect on severe spermatogenic failure predisposition could be because of an alteration of the KATNAL1 splicing pattern. CONCLUSIONS: Specific allelic combinations of KATNAL1 genetic polymorphisms may confer a risk of developing severe male infertility phenotypes by favouring the overrepresentation of a short non-functional transcript isoform in the testis.