The protein-protein interaction network of KNL2 in plants
Cell Biology
Final Report Abstract
KNL2 is a centromere-associated protein that serves as a licensing factor for CENH3 deposition and kinetochore assembly. In Arabidopsis, αKNL2 features an N-terminal SANTA domain and a C-terminal CENPC-k motif. To investigate its molecular function and regulation, we identified interaction partners using yeast two-hybrid (Y2H) screening and affinity purification coupled with mass spectrometry (AP-MS). Screens with full-length, N-terminal, and C-terminal fragments of αKNL2 revealed overlapping interactors. To determine whether plant KNL2 forms complexes with proteins from conserved pathways across species, we conducted gene ontology and pathway enrichment analyses of AP-MS data from Arabidopsis and C. elegans. These analyses revealed conserved enrichment in posttranslational modification pathways, particularly ubiquitination and SUMOylation. Guided by these results, we selected candidate proteins including ubiquitin-conjugating enzymes (UBC19, UBC20), E3 ligase components (APC2, APC10, CDC20), and SUMO-related proteins (SUMO3, ULP1D), and confirmed their interaction with αKNL2 using BiFC, Y2H, and co-immunoprecipitation assays. Functional analyses showed that αKNL2 is polyubiquitinated and degraded by the 26S proteasome. Treatment with proteasome inhibitors significantly stabilized αKNL2 levels in planta. Deletion or mutation of predicted degrons (D-box1 motif) and ubiquitination sites resulted in degradation-resistant αKNL2 variants that mis-regulated cell cycle progression. Transgenic plants expressing these forms exhibited reduced root growth and mitotic defects, such as metaphase misalignment and anaphase bridges, underscoring the developmental impact of disrupted αKNL2 turnover. SUMOylation was also critical, regulating αKNL2 localization and its interaction with centromeric chromatin. Mutations in conserved SUMOylation sites and SIM motif impaired centromeric targeting and interaction with SUMO3, ULP1D, and CENH3. Further, transcriptome profiling identified a natural antisense transcript (NDC80as) from the NDC80 locus, encoding a 115-amino acid protein with coiled-coil and DNA-binding motifs. This protein localized to the nucleus and periphery, and its overexpression in Arabidopsis caused developmental defects. These findings suggest that, alongside post-translational control, transcriptional regulation via NATs also contributes to kinetochore function. Overall, our study reveals a multilayered regulatory system integrating protein modifications and antisense transcription to control centromere and kinetochore dynamics.
Publications
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A CENH3 mutation promotes meiotic exit and restores fertility in SMG7-deficient Arabidopsis. PLOS Genetics, 17(9), e1009779.
Capitao, Claudio; Tanasa, Sorin; Fulnecek, Jaroslav; Raxwal, Vivek K.; Akimcheva, Svetlana; Bulankova, Petra; Mikulkova, Pavlina; Crhak Khaitova, Lucie; Kalidass, Manikandan; Lermontova, Inna; Mittelsten Scheid, Ortrun & Riha, Karel
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High temperature increases centromere-mediated genome elimination frequency and enhances haploid induction in Arabidopsis. Plant Communications, 4(3), 100507.
Ahmadli, Ulkar; Kalidass, Manikandan; Khaitova, Lucie Crhak; Fuchs, Joerg; Cuacos, Maria; Demidov, Dmitri; Zuo, Sheng; Pecinkova, Jana; Mascher, Martin; Ingouff, Mathieu; Heckmann, Stefan; Houben, Andreas; Riha, Karel & Lermontova, Inna
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The role of centromeric repeats and transcripts in kinetochore assembly and function. The Plant Journal, 118(4), 982-996.
Ramakrishnan Chandra, Jothipriya; Kalidass, Manikandan; Demidov, Dmitri; Dabravolski, Siarhei A. & Lermontova, Inna
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Centromeric localization of αKNL2 and CENP-C proteins in plants depends on their centromere-targeting domain and DNA-binding regions. Nucleic Acids Research, 53(4).
Yalagapati, Surya Prakash; Ahmadli, Ulkar; Sinha, Aditya; Kalidass, Manikandan; Dabravolski, Siarhei; Zuo, Sheng; Yadala, Ramakrishna; Rutten, Twan; Talbert, Paul; Berr, Alexandre & Lermontova, Inna
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Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis. eLife, 12.
Crhak Khaitova, Lucie; Mikulkova, Pavlina; Pecinkova, Jana; Kalidass, Manikandan; Heckmann, Stefan; Lermontova, Inna & Riha, Karel
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Ubiquitin-dependent proteolysis of KNL2 driven by APC/CCDC20 is critical for centromere integrity and mitotic fidelity. The Plant Cell, 37(7).
Kalidass, Manikandan; Jarubula, Venkata Ganesh; Ratnikava, Maryia; Chandra, Jothipriya Ramakrishnan; Le Goff, Samuel; Probst, Aline V.; Esposito, Silvia; Grasser, Klaus D.; Bruckmann, Astrid; Gagneux, Jérôme F.; Prosée, Reinier F.; Rutten, Twan; Schubert, Veit; Demidov, Dmitri; Lechner, Esther; Steiner, Florian A.; Genschik, Pascal & Lermontova, Inna
