Principy jaderné organizace

Basello, Davide Alessandro

Cajal bodies (CBs) are dynamic, membraneless nuclear compartments involve in the biogenesis and maturation of small nuclear ribonucleoproteins (snRNPs), with the protein coilin serving as their primary costituent. This thesis investigates the molecular mechanisms underlying CB assembly and maintenance, focusing on the roles of coilin oligomerization, RNA interactions, and coilin-snRNP binding. Through the generation and analysis of CRISPR/Cas9-mediated coilin knockout cell lines and a series of coilin mutants, including a previusly unknown mutation (K496E) present in a widly use coilin plasmid, this work reveals that both coilin self-interaction (mediated by the N-terminus) and its association with snRNPs (via the C-terminus) are indispensable for CB formation. Advance microscopy techniques show that CBs are structurally heterogeneous and possess a selective diffusion barrier, with coilin exchange between CBs and nucleoplasm being tightly regulated. Crucially, RNA is shown to be essential for coilin oligomerization and CB integrity: RNase microinjection leads to rapid CB disassembly, and RNA depletion impairs coilin self- association. Detailed mutational analyses demonstrate that conserved residues and loops in the Tudor domain are required for snRNP binding and CB formation, while coilin...

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