Dottorato in GENETICA E BIOLOGIA MOLECOLARE

Titolo della tesi: Nuclear transport factors, nucleoporins and SUMOylation in mitotic cells

Protein conjugation with SUMO (Small Ubiquitin-like MOdifier) peptides, or SUMOylation, modulates protein interactions and localization in many cellular dynamic processes. In particular, growing evidence implicate protein SUMOylation in control of mitosis. Indeed, several factors acting in SUMOylation and deSUMOylation reside at kinetochores. Among them, RANBP2 is a large nucleoporin with SUMO E3 ligase and SUMO stabilizing activity. It is therefore of interest to identify mitotic targets of RANBP2-dependent SUMOylation. Here, by combining Proximity Ligation Assay (PLA) and functional assays, I have identified a key role of RANBP2 in control of the microtubule-regulatory factor NuSAP1 (Nucleolar and spindle-associated protein 1) via temporally and spatially regulated SUMOylation. First, I demonstrated that NuSAP1 is actually SUMOylated in HeLa mitotic cells. Furthermore, I showed that RANBP2 modulates the stability of a NuSAP1 fraction at microtubule plus ends, and needed to stabilize the interactions of chromosomes with the spindle microtubules. To assess directly the effect of NUSAP1 SUMOylation, I mutagenized two putative SUMO consensus sites (K44 and K316, in the DNA-binding and microtubule-binding domain of NuSAP1, respectively). I generated stable inducible cell lines expressing either wild-type NuSAP1 or SUMO mutants. I found that NuSAP1K 316R mutant in the microtubule-binding domain is indeed impaired in microtubule regulatory functions. Together these results indicate that RANBP2-mediated SUMOylation regulates the localization and function of NuSAP1, with implications in the mechanisms regulating chromosome segregation. Loss of this regulation may contribute to originate genetic instability in daughter cells.