Non-transport roles of nuclear import receptors: In need of the right balance

Nuclear import receptors ensure the recognition and transport of proteins across the nuclear envelope into the nucleus. In addition, as diverse processes as mitosis, post-translational modifications at mitotic exit, ciliogenesis, and phase separation, all share a common need for regulation by nuclear import receptors - particularly importin beta-1 and importin beta-2/transportin - independent on nuclear import. In particular, 1) nuclear import receptors regulate the mitotic spindle after nuclear envelope breakdown, 2) they shield cargoes from unscheduled ubiquitination, regulating their timely proteolysis; 3) they regulate ciliary factors, crucial to cell communications and tissue architecture during development; and 4) they prevent phase separation of toxic proteins aggregates in neurons. The balance of nuclear import receptors to cargoes is critical in all these processes, albeit in opposite directions: overexpression of import receptors, as often found in cancer, inhibits cargoes and impairs downstream processes, motivating the therapeutic design of specific inhibitors. On the contrary, elevated expression is beneficial in neuronal contexts, where nuclear import receptors are regarded as potential therapeutic tools in counteracting the formation of aggregates that may cause neurodegeneration. This paradox demonstrates the amplitude of nuclear import receptors-dependent functions in different contexts and adds complexity in considering their therapeutic implications

Importin-beta and CRM1 control a RANBP2 spatiotemporal switch essential for mitotic kinetochore function

Protein conjugation with small ubiquitin-related modifier (SUMO) is a post-translational modification that modulates protein interactions and localisation. RANBP2 is a large nucleoporin endowed with SUMO E3 ligase and SUMO-stabilising activity, and is implicated in some cancer types. RANBP2 is part of a larger complex, consisting of SUMO-modified RANGAP1, the GTP-hydrolysis activating factor for the GTPase RAN. During mitosis, the RANBP2–SUMO-RANGAP1 complex localises to the mitotic spindle and to kinetochores after microtubule attachment. Here, we address the mechanisms that regulate this localisation and how they affect kinetochore functions. Using proximity ligation assays, we find that nuclear transport receptors importin-β and CRM1 play essential roles in localising the RANBP2–SUMO-RANGAP1 complex away from, or at kinetochores, respectively. Using newly generated inducible cell lines, we show that overexpression of nuclear transport receptors affects the timing of RANBP2 localisation in opposite ways. Concomitantly, kinetochore functions are also affected, including the accumulation of SUMO- conjugated topoisomerase-IIα and stability of kinetochore fibres. These results delineate a novel mechanism through which nuclear transport receptors govern the functional state of kinetochores by regulating the timely deposition of RANBP2

The Mitotic Apparatus and Kinetochores in Microcephaly and Neurodevelopmental Diseases

Regulators of mitotic division, when dysfunctional or expressed in a deregulated manner (over- or underexpressed) in somatic cells, cause chromosome instability, which is a predisposing condition to cancer that is associated with unrestricted proliferation. Genes encoding mitotic regulators are growingly implicated in neurodevelopmental diseases. Here, we briefly summarize existing knowledge on how microcephaly-related mitotic genes operate in the control of chromosome segregation during mitosis in somatic cells, with a special focus on the role of kinetochore factors. Then, we review evidence implicating mitotic apparatus- and kinetochore-resident factors in the origin of congenital microcephaly. We discuss data emerging from these works, which suggest a critical role of correct mitotic division in controlling neuronal cell proliferation and shaping the architecture of the central nervous system

Aurora B SUMOylation Is Restricted to Centromeres in Early Mitosis and Requires RANBP2

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis

Aurora B SUMOylation Is Restricted to Centromeres in Early Mitosis and Requires RANBP2

Conjugation with the small ubiquitin-like modifier (SUMO) modulates protein interactions and localisation. The kinase Aurora B, a key regulator of mitosis, was previously identified as a SUMOylation target in vitro and in assays with overexpressed components. However, where and when this modification genuinely occurs in human cells was not ascertained. Here, we have developed intramolecular Proximity Ligation Assays (PLA) to visualise SUMO-conjugated Aurora B in human cells in situ. We visualised Aurora B-SUMO products at centromeres in prometaphase and metaphase, which declined from anaphase onwards and became virtually undetectable at cytokinesis. In the mitotic window in which Aurora B/SUMO products are abundant, Aurora B co-localised and interacted with NUP358/RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilising activity. Indeed, in addition to the requirement for the previously identified PIAS3 SUMO ligase, we found that NUP358/RANBP2 is also implicated in Aurora B-SUMO PLA product formation and centromere localisation. In summary, SUMOylation marks a distinctive window of Aurora B functions at centromeres in prometaphase and metaphase while being dispensable for functions exerted in cytokinesis, and RANBP2 contributes to this control, adding a novel layer to modulation of Aurora B functions during mitosis

Novel quinoline derivatives and uses thereof

The invention relates to preparation of new quinoline-4-carboxamide derivatives, pharmaceutical compositions containing thereof and the use of the same as inhibitors of Ndc80 kinetochore subcomplex-MT binding and of kinetochore-MT binding, more preferably for use in the treatment of cell proliferative disorders including cancers and drug-resistant cancer. The example compound I was prepared using f 2-phenylquinoline-4-carboxylic acid as starting material(data given)

Importin-β/karyopherin-β1 modulates mitotic microtubule function and taxane sensitivity in cancer cells via its nucleoporin-binding region

The nuclear transport receptor importin-β/karyopherin-β1 is overexpressed in cancers that display genomic instability. It is regarded as a promising cancer target and inhibitors are being developed. In addition to its role in nucleo-cytoplasmic transport, importin-β regulates mitosis, but the programmes and pathways in which it operates are defined only in part. To unravel importin-β's mitotic functions we have developed cell lines expressing either wild-type or a mutant importin-β form in characterised residues required for nucleoporin binding. Both forms similarly disrupted spindle pole organisation, while only wild-type importin-β affected microtubule plus-end function and microtubule stability. A proteome-wide search for differential interactors identified a set of spindle regulators sensitive to mutations in the nucleoporin-binding region. Among those, HURP (hepatoma up-regulated protein) is an importin-β interactor and a microtubule-stabilising factor. We found that induction of wild type, but not mutant importin-β, under the same conditions that destabilise mitotic microtubules, delocalised HURP, indicating that the spatial distribution of HURP along the spindle requires importin-β's nucleoporin-binding residues. Concomitantly, importin-β overexpression sensitises cells to taxanes and synergistically increases mitotic cell death. Thus, the nucleoporin-binding domain is dispensable for importin-β function in spindle pole organisation, but regulates microtubule stability, at least in part via HURP, and renders cells vulnerable to certain microtubule-targeting drugs

Circular RNA ZNF609/CKAP5 mRNA interaction regulates microtubule dynamics and tumorigenicity

Circular RNAs (circRNAs) are widely expressed in eukaryotes and are regulated in many biological processes. Although several studies indicate their activity as microRNA (miRNA) and protein sponges, little is known about their ability to directly control mRNA homeostasis. We show that the widely expressed circZNF609 directly interacts with several mRNAs and increases their stability and/or translation by favoring the recruitment of the RNA-binding protein ELAVL1. Particularly, the interaction with CKAP5 mRNA, which interestingly overlaps the back-splicing junction, enhances CKAP5 translation, regulating microtubule function in cancer cells and sustaining cell-cycle progression. Finally, we show that circZNF609 downregulation increases the sensitivity of several cancer cell lines to different microtubule-targeting chemotherapeutic drugs and that locked nucleic acid (LNA) protectors against the pairing region on circZNF609 phenocopy such effects. These data set an example of how the small effects tuned by circZNF609/CKAP5 mRNA interaction might have a potent output in tumor growth and drug response

Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores

Abstract Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of centromere transcripts (cenRNAs). The latter are ensured by the downregulation of RNA polymerase II (RNAPII) activity, and cenRNA turnover by the nuclear exosome. Using S. cerevisiae, we now add protein kinase Rio1 to this scheme. Yeast cenRNAs are produced either as short (median lengths of 231 nt) or long (4458 nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII accessibility and promotes cenRNA degradation by the 5’−3’exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs. These exist as short transcripts (225 nt) at levels that are minimally two orders of magnitude higher than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulate, CEN nucleosomes and kinetochores misform, causing chromosome instability. The latter phenotypes are also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved