Biology subjects

Marciano, G.

Publications and source records attributed to Marciano, G..

5 recordsLinked to original sources

bioRxiv2026

The centromere localization domain of kinetoplastid kinetochore protein KKT2 recognizes the free N-terminus of histone H3

Kinetochores are multiprotein complexes that drive chromosome segregation in eukaryotes. Kinetoplastids, a group of early-diverging eukaryotes that lack canonical kinetochore components, have a unique set of kinetochore proteins. How their kinetochores are assembled specifically at centromeres in the absence of a centromere-specific histone H3 variant CENP-A remains unknown. Here, we demonstrate that the centromere localization (CL) domain of KKT2 has similarities to a ZZ domain. An invariant aspartate present in histone H3-binding ZZ domains is conserved in the KKT2 CL domain. Using nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that Trypanosoma brucei KKT2 CL binds the N-terminus of histone H3. Strikingly, even mono-methylation of the N-terminal amino group of histone H3 abolishes the binding. Our study raises a possibility that centromere-specific kinetochore assembly in T. brucei is ensured by abundant N-terminal methylations of histone H3 in non-centromeric regions.

cell biology↗

bioRxiv2026

Structural basis for alternative 3' splice site selection in the human spliceosome active center

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/741260v1_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@279388org.highwire.dtl.DTLVardef@46d636org.highwire.dtl.DTLVardef@997672org.highwire.dtl.DTLVardef@12f40ee_HPS_FORMAT_FIGEXP M_FIG C_FIG Key findingsO_LICryo-EM reveals SDE2 as a novel factor stabilizing the spliceosome active center C_LIO_LISDE2 {Delta}N structures visualize a stalled C* state with impaired docking-factor engagement. C_LIO_LISDE2, Prp18, and FAM32A read a cis-code to stabilize weaker proximal 3'-ss. C_LIO_LISDE2 {Delta}N destabilizes 3'-ss docking and rescues BRCA1 and CFTR mis-splicing in vivo. C_LI Accurate alternative splicing requires discrimination between adjacent 3' splice sites (3'-ss) during catalysis and is disrupted by pathogenic AG-gain mutations that create competing 3'-ss. Here, we present cryo-EM structures of human spliceosomes assembled on native-sequence pre-mRNAs, revealing how the catalytic core controls alternative 3'-ss selection. SDE2 is a previously unrecognized active-center component that promotes a docking-competent spliceosome conformation. Machine learning, in vivo transcriptomics, and in vitro biochemistry show how SDE2 cooperates with FAM32A and Prp18 to act as readers of a cis-regulatory code that governs 3'-ss selection during catalysis. These factors promote weaker, proximal site use by counteracting an intrinsic distal bias generated by active-site interactions with the distal-site -4 nucleotide. Structural or genetic perturbation of these exon-ligation factors destabilizes proximal 3'-ss docking and restores canonical splicing in disease-relevant CFTR and BRCA1 AG-gain alleles. Our work establishes the spliceosome active center as a tunable regulatory hub for alternative splicing.

molecular biology↗

bioRxiv2022

Divergent polo boxes in KKT2 and KKT3 initiate the kinetochore assembly cascade in Trypanosoma brucei

Chromosome segregation requires assembly of the macromolecular kinetochore complex onto centromeric DNA. While most eukaryotes have canonical kinetochore proteins that are widely conserved among eukaryotes, evolutionarily divergent kinetoplastids have a unique set of kinetochore proteins. Little is known about the mechanism of kinetochore assembly in kinetoplastids. In this study, we have characterized two homologous kinetoplastid kinetochore proteins, KKT2 and KKT3, that constitutively localize at centromeres and promote the recruitment of other kinetochore proteins. KKT2 and KKT3 have three domains that are highly conserved among kinetoplastids: an N-terminal kinase domain of unknown function, the centromere localization domain in the middle, and the C-terminal domain that has weak similarity to polo boxes of Polo-like kinases. We show that the kinase activity of KKT2 is essential for accurate chromosome segregation, while that of KKT3 is dispensable for cell growth in the procyclic form of Trypanosoma brucei. Crystal structures of their divergent polo boxes reveal differences between KKT2 and KKT3. We also show that the divergent polo boxes of KKT3 are sufficient to recruit KKT2 in T. brucei. Furthermore, we identify the C-terminal domain of KKT1 as a direct interaction partner for the divergent polo boxes of KKT2, but not KKT3. KKT6 is found to interact with KKT1. These results show that KKT2 and KKT3 recruit downstream kinetochore proteins by direct protein-protein interactions using their divergent polo boxes.

cell biology↗

bioRxiv2020

Structural characterisation of KKT4, an unconventional microtubule-binding kinetochore protein

The kinetochore is the macromolecular protein machinery that drives chromosome segregation by interacting with spindle microtubules. Unlike most other eukaryotes that have canonical kinetochore proteins, a group of evolutionarily divergent eukaryotes called kinetoplastids (such as Trypanosoma brucei) have a unique set of kinetochore proteins. To date, KKT4 is the only kinetoplastid kinetochore protein that is known to bind microtubules. Here we use X-ray crystallography, NMR spectroscopy, and crosslinking mass spectrometry to characterise the structure and dynamics of KKT4. We show that its microtubule-binding domain consists of a coiled-coil structure followed by a positively charged disordered tail. The crystal structure of the C-terminal BRCT domain of KKT4 reveals that it is likely a phosphorylation-dependent protein-protein interaction domain. The BRCT domain interacts with the N-terminal region of the KKT4 microtubule-binding domain and with a phosphopeptide derived from KKT8. Finally, we show that KKT4 binds DNA with high affinity. Taken together, these results provide the first structural insights into the unconventional kinetoplastid kinetochore protein KKT4.

cell biology↗

bioRxiv2019

Unconventional kinetochore kinases KKT2 and KKT3 have a unique zinc finger that promotes their kinetochore localization

Chromosome segregation in eukaryotes is driven by the kinetochore, the macromolecular protein complex that assembles onto centromeric DNA and binds spindle microtubules. Cells must tightly control the number and position of kinetochores so that all chromosomes assemble a single kinetochore. A central player in this process is the centromere-specific histone H3 variant CENP-A, which localizes constitutively at centromeres and promotes kinetochore assembly. However, CENP-A is absent from several eukaryotic lineages including kinetoplastids, a group of evolutionarily divergent eukaryotes that have an unconventional set of kinetochore proteins. There are six proteins that localize constitutively at centromeres in the kinetoplastid parasite Trypanosoma brucei, among which two homologous protein kinases (KKT2 and KKT3) have limited similarity to polo-like kinases. In addition to the N-terminal kinase domain and the C-terminal divergent polo boxes, KKT2 and KKT3 have a central domain of unknown function as well as putative DNA-binding motifs. Here we show that KKT2 and KKT3 are important for the localization of several kinetochore proteins and that their central domains are sufficient for centromere localization in T. brucei. Crystal structures of the KKT2 central domain from two divergent kinetoplastids reveal a unique zinc-binding domain (termed the CL domain for centromere localization), which promotes its kinetochore localization in T. brucei. Mutations in the equivalent domain in KKT3 abolish its kinetochore localization and function. Our work shows that the unique central domains play a critical role in mediating the centromere localization of KKT2 and KKT3.

cell biology↗