Biology subjects

van Rooijen, L. E.

Publications and source records attributed to van Rooijen, L. E..

4 recordsLinked to original sources

bioRxiv2025

Unconventional components complement the cryptic kinetochore of the ciliate Tetrahymena thermophila

The accurate segregation of chromosomes is mediated by kinetochores, multi-protein structures that connect centromeric chromatin to the dynamic microtubules of the spindle apparatus. Comparative genomics surveys predict a complex kinetochore in the last eukaryotic common ancestor (LECA) and recurrent loss or replacement of its subcomplexes across the eukaryotic tree of life. Understanding kinetochore composition and organization in diverse lineages can reveal the trajectories of kinetochore evolution in eukaryotes and aid in dissecting the function of each subcomplex. Tetrahymena thermophila is a unicellular eukaryote of the phylum Ciliophora with a largely elusive kinetochore composition. Here, we leverage proximity proteomics coupled to deep homology detection approaches to identify 16 kinetochore proteins in T. thermophila, dubbed KiTTs (Kinetochore of Tetrahymena thermophila 1-16). We find that nine KiTTs (3-9 + 15-16) are cryptic orthologs of conventional kinetochore proteins that previously remained undetected due to extensive sequence divergence. Four KiTTs (10-13) are not orthologous to known subunits and therefore represent unconventional kinetochore proteins. Super-resolution imaging places three of these novel proteins (KiTT10/11/13) at the inner kinetochore, whereas the fourth (KiTT12) localizes near the MIS12 complex at the outer kinetochore. RNAi-mediated depletion of KiTT12 reduces levels of the outer kinetochore protein KiTT1NDC80 and causes chromosome segregation errors, showcasing a bona fide role at the kinetochore. Our work reveals a unique kinetochore composition in a ciliate, providing new insights into the evolution of an essential cellular protein machine.

cell biology↗

bioRxiv2023

Distinct functions for the paralogous RBM41 and U11/U12-65K proteins in the minor spliceosome

In this work, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of the U11/U12-65K protein, a known unique component of the minor spliceosome that functions during the early steps of minor intron recognition as a component of the U11/U12 di-snRNP. We show that both proteins use their highly similar C-terminal RRMs to bind to 3-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with the U11/U12-65K protein, which uses the N-terminal region to interact with U11 snRNP during the intron recognition step. Finally, we show that while RBM41 knockout cells are viable, they show alterations in the splicing of U12-type introns, particularly differential U12-type 3 splice site usage. Together, our results highlight the role 3-terminal stem-loop of U12 snRNA as a dynamic binding platform for the paralogous U11/U12-65K and RBM41 proteins, which function at distinct stages of minor spliceosome assembly/disassembly cycle.

molecular biology↗

bioRxiv2022

Increased sampling and intra-complex homologies favor vertical over horizontal inheritance of the Dam1 complex

Kinetochores connect chromosomes to spindle microtubules to ensure their correct segregation during cell division. Kinetochores of human and yeast are largely homologous, their ability to track depolymerizing microtubules however is carried out by the non-homologous complexes Ska1-C and Dam1-C, respectively. We previously reported the unique anti-correlating phylogenetic profiles of Dam1-C and Ska-C found amongst a wide variety of eukaryotes. Based on these profiles and the limited presence of Dam1-C, we speculated that horizontal gene transfer (HGT) could have played a role in the evolutionary history of Dam1-C. Here, we present expanded analyses of Dam1-c evolution, using additional genome as well as transcriptome sequences and recently acquired 3D structure data. This analysis revealed a wider and more complete presence of Dam1-C in Cryptista, Rhizaria, Ichthyosporea, CRuMs, and Colponemidia. The fungal Dam1-C cryo-EM structure supports earlier hypothesized intra-complex homologies, which enables the reconstruction of rooted and unrooted phylogenies. The rooted tree of concatenated Dam1-C subunits is statistically consistent with the species tree of eukaryotes, suggesting that Dam1-C is ancient, and that the present-day phylogenetic distribution is best explained by multiple, independent losses and no HGT was involved. Furthermore, we investigated the ancient origin of Dam1-C via profile-profile searches. Homology among eight out of the ten Dam1-C subunits suggested that the complex largely evolved from a single multimerizing subunit that diversified into an octameric core via stepwise duplication and sub-functionalization of the subunits before the origin of the Last Common Ancestor. SignificanceThe Dam1 complex has a crucial role in cell division yet its distribution across species is very patchy. To resolve the evolutionary origin of this peculiar distribution, we used the recently acquired 3D structure to obtain a rooted phylogeny. This study makes an important step in discovering the evolutionary history of the Dam1 complex, by determining that Dam1-C was part of the Last eukaryotic Common Ancestor and arose via stepwise duplications during the transition from prokaryotes to eukaryotes.

evolutionary biology↗

bioRxiv2020

Chromosomal instability by mutations in a novel specificity factor of the minor spliceosome

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ~100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing with a specific impact on the chromosome segregation process, and show how defects herein can cause constitutional aneuploidy.

molecular biology↗