The NUSAP1 gene spans 60,257 bases located on chromosome 15, at cytogenetic bands 15q14 (according to HGNC), 15q15.1 (according to Entrez Gene), 15q15.1 (by Ensembl)

Both the mouse Nusap1 and human NUSAP1 genes are selectively expressed in proliferating but not in differentiated cells. Murine Nusap1 was originally identified as a selectively expressed transcript in proliferating but not in differentiated mouse MC3T3E1 osteoblasts by differential display. The mouse cDNA full-length sequence contains a single ORF of 1,281 bp that encodes a protein of 427 aminoacids. Cloning of the human NUSAP1 cDNA and comparison with EST (expressed sequence tags) databases (https://www.ncbi.nlm.nih.gov/genbank/dbest) showed high conservation of NUSAP1 in vertebrates (Raemaekers et al., 2003). In particular the human NUSAP1 cDNA contains an ORF of 1323 bp encoding a protein of 441 aminoacids (source Uniprot). The intron/exon structure for human NUSAP1 is available at https://www.ncbi.nlm.nih.gov/gene?cmd=Retrieve&dopt=Graphics&list_uids=51203. The NUSAP1 gene is overexpressed in several carcinoma types and in tumours of the central nervous system (see below). This is likely linked, at least in part, to transcriptional regulation by the cell cycle transcription factor E2F1 (Gulzar et al., 2013) and the MYC proto-oncogene (Hussain et al., 2008).

7 potential products may be generated from alternatively spliced isoforms (from UniProt/SwissProt), but isoform 1 (https://www.uniprot.org/uniprot/Q9BXS6#Q9BXS6-1) is regarded as the canonical protein. The human NUSAP1 protein (441 aminoacids) has a calculated molecular mass of 48.6 kDa (isoelectric point, 9.9). The apparent molecular mass is 55 kDa, and the difference between expected and observed may be accounted for, at least in part, by phosphorylation by cell cycle kinases.
Biological overview: NUSAP1 (Nuclear and Spindle Associated Protein) is a cell-cycle dependent protein and is the only product of the NUSAP1 gene, which is selectively expressed in proliferating cells (Raemaekers et al., 2003). Both the mRNA and protein abundance peak in the G2 and M phases of the cell cycle and sharply decrease at mitotic exit.
NUSAP1 is a microtubule-associated protein (MAP) and has microtubule-bundling and stabilizing activity in vitro. This underlies its main function as a regulator of the mitotic apparatus in dividing cells (Raemaekers et al., 2003; Ribbeck et al., 2006). At mitotic onset, NUSAP1 localises to the spindle microtubules in the region that comes in contact with chromosomes, and stabilises their interaction, thus contributing to regulate chromosome segregation to daughter cells.
Deregulated abundance of NUSAP1 is associated with misassembly or misfunction of the mitotic spindle and contributes to generate genetic instability in daughter cells through faulty mitosis. NUSAP1 also has roles in invasion (Gordon et al., 2017) and in functional interactions with pro-oncogenic pathways. High levels of NUSAP1 are often associated with poor prognosis.

The human protein NUSAP1 contains several domains conserved in the mouse protein, first described by Raemaekers et al. (2003).
- a helix-extension-helix domain (aa 10-44), containing a potential SAP motif - the latter, named after three proteins that share it, i.e. SAF-A/B, Acinus and PIAS, is found in various nuclear proteins involved in transcription, DNA repair, and RNA processing. The SAP motif, which is essential for DNA binding in the SAF-A/B protein, is also thought to act as the DNA-binding motif in many nuclear proteins (https://www.ebi.ac.uk/interpro/entry/InterPro/IPR003034/), and is reported to confer DNA-interacting capacity to the NH2 terminal (N-ter) region of NUSAP1 (Verbakel et al., 2011),
- a bipartite nuclear localisation signal, NLS (aa 194-211) that can interact with KPNA1 and KPNB1 (importin-alpha and -beta),
- the microtubule-binding domain with a minimal essential core located between residues 243-367,
- a KEN box (aa 383-390), required for protein degradation (Li et al., 2007),
- a highly charged domain with a predicted helical structure, called the charged helical domain (ChHD; aa 410-433), in the COOH terminal (C-ter) region,
- the C-ter region containing the MCAK-binding domain (MCBD), (aa 433-441), responsible for direct binding to KIF2C (or MCAK, mitotic centromere-associated kinesin) and implicated in control of the depolymerisation activity of MCAK at the kinetochore region and, thus, for regulating the stabilisation of kinetochore-microtubules attachments during prometaphase and metaphase (Li et al., 2016).
Phosphorylation sites for various cell cycle kinases, including AURKB (Aurora-B) (Hussain et al., 2008; Ozlu et al., 2010), AURKA (Aurora-A) (Sardon et al., 2010), CDK1 (Chou et al., 2011) and ATM (Xie et al., 2011) have been experimentally identified.

The NUSAP1 gene is transcribed from late S to G2 phases of cell cycle, yielding highest protein abundance in M phase. At mitotic exit protein levels decrease via the proteasome/ ubiquitination system, involving the ubiquitin ligase APC/C/Cdh1 (Li et al., 2007), the major regulator of protein ubiquitin conjugation in late mitosis (Raemaekers et al., 2003). After cytokinesis and in the next G1 phase, NUSAP1 is degraded after ubiquitination by the M-specific ubiquitin ligase APC/C (Anaphase-promoting complex /cyclosome) in complex with its activator Cdh1 (Li et al., 2007). The protein is expressed in different proliferating tissues (e.g. bone marrow, lymph nodes, testis) and is overexpressed in different cancer types (see below).

NUSAP1 localisation is cell cycle-dependent by immunofluorescence (IF) assays. When NUSAP1 first appears in interphase G2 cells, it localises within the nucleus and concentrates at nucleoli. Cell fractionation experiments confirmed that nuclear/nucleolar localisation (Raemaekers et al., 2003). In mitosis, after nuclear envelope breakdown, soluble NUSAP1 is released in the cytoplasm and localises on microtubules in prometaphase, gradually concentrating at microtubule plus (+) ends near chromosomes until anaphase. In late anaphase, the soluble NUSAP1 fraction becomes barely detectable, and virtually the entire NUSAP1 pool associates to thick microtubules bundles around chromosomes, forming characteristic NUSAP1 spikes in telophase (Raemaekers et al., 2003).

As a microtubule-associated protein, NUSAP1 plays key roles in mitosis. For clarity, we will separately summarise NUSAP1 roles in a) cell cycle progression; b) microtubules-associated activity; c) spindle formation and dynamics; d) DNA-binding activity.
a) Cell cycle progression
NUSAP1 is essential to proper cell cycle progression. Indeed, NUSAP1 depletion following RNA interference delays entry into mitosis and arrests cycling cells at the G2/M transition. Under this condition, dramatic mitotic defects are depicted by IF, e.g. abnormal spindles in prometaphase and altered chromosome condensation. Mitotic cells that still proceeded towards chromosome segregation with disorganised spindles underwent abnormal anaphase and defective cytokinesis (Raemaekers et al., 2003). On the other hand, NUSAP1 overexpression also leads to mitotic arrest (Li et al., 2007) due to the formation of thick microtubule bundles that impair the dynamic activity of the spindle. These results indicate the critical importance of regulated levels of NUSAP1 protein to ensure proper cell division.
b) Regulation of microtubule activity
- NUSAP1 binds and stabilises microtubules
Microtubule sedimentation assays separate polymerised microtubules from free dimers of alpha/beta tubulin, and they are used to study microtubule-associated proteins (MAPs). In these assays, NUSAP1 binds directly to polymerised microtubules (Raemaekers et al., 2003). NUSAP1 can effectively cross-link microtubules into asters and thick fibers, an essential step in assembly of the mitotic apparatus. Moreover, NUSAP1 binding protects microtubules against depolymerisation and helps guiding the microtubules towards the chromosomal DNA, an essential step prior to chromosome segregation (Ribbeck et al., 2006).
- Importins and the GTPase RAN regulate the activity of NUSAP1 on microtubules
NUSAP1 interacts with nuclear import receptors: importin alpha, which recognises the NLS; Importin beta and IPO7 (Importin 7) (the actual import vectors for NUSAP1), in both biochemical experiments (Ribbeck et al., 2006) and in intact cells (Di Francesco et al., 2018). In interphase, this interaction mediates NUSAP1 import in nuclei.
In vitro assays indicate that NUSAP1 functions are differentially affected by Importin beta and importin 7. Indeed, the presence of Importin beta alone impaired NUSAP1 function in the production of aster-like microtubule structures, while not affecting the production of long tubulin fibers; on the contrary, Importin 7 alone prevented NUSAP1 activity in generating long fibers and only aster structures were produced. RANGTP addition reversed the inhibitory effect exerted by either importin on NUSAP1 functions on microtubules, suggesting that the release of free NUSAP1 from Importin beta and -7 interactions is RANGTP-dependent (Ribbeck et al., 2006).
- Phosphorylation of NUSAP1 regulates its functions
NUSAP1 is phosphorylated by the major cell cycle-dependent kinase, CDK1, at threonine 300 and 338 in the microtubule-binding domain. In in vitro microtubule sedimentation assays, phosphorylated NUSAP1 (ph-NUSAP1) fails to bind microtubules, suggesting that CDK1-mediated phosphorylation negatively regulates NUSAP1 interaction with microtubules. During the cell cycle, ph-NUSAP1 is detectable in nucleoli in prophase; later, after nuclear envelope breakdown, it diffuses in the cytosol and around condensed chromosomes; finally, the signal disappears during progression from anaphase to telophase, (Chou et al., 2011).
NUSAP1 can also be phosphorylated by Aurora kinases. Aurora-A was shown to be able to phosphorylate NUSAP1 at Serine 240; although the functional significance of this phosphorylation remains unclear, it may have roles during microtubule assembly (Sardon et al., 2010). NUSAP1 was also identified in a proteomic screening for Aurora-B substrates; this phosphorylation putatively involves several serines or threonines in the N-ter region and is proposed to regulate NUSAP1 activity at the spindle midzone at cytokinesis (Ozlu et al., 2010).
c) Spindle formation and dynamics
- NUSAP1 regulates spindle formation
Xenopus egg extract provide an informative model system to study spindle assembly and function. They derive from unfertilised eggs arrested in metaphase II of meiosis and contain all maternal factors which only need be organised and activated at fertilisation. Immunodepletion experiments show that NUSAP1 is essential for spindle formation. Indeed, in NUSAP1-depleted extracts, abnormal spindles formed, which showed a low microtubule density and a distorted bipolar organisation (Ribbeck et al., 2006). Instead, excess NUSAP1 blocked the onset of spindle formation. When NUSAP1 was added after the spindle had already started to form, the microtubules became strongly bundled into prominent fibers, resulting in a distorted configuration. Thus, the amount of NUSAP1 regulates the balance of microtubules in the spindle (Ribbeck et al., 2006).
-NUSAP1 regulates spindle dynamics
NUSAP1 interacts with MCAK, a kinesin with microtubule-depolymerizing activity, required to correct any misattachments between microtubules and kinetochores occurring during spindle formation. Li and colleagues demonstrated that NUSAP1 regulates the depolymerizing activity of MCAK, assigning to NUSAP1 a novel role in microtubule dynamics (Li et al., 2016). It has also been discovered that NUSAP1 also contributes to orchestrate the alignment and orientation of chromosomes in the metaphase plate by regulating the activity of Kid, a chromokinesin that guides the anti-poleward movements of chromosomes: thanks to its ability to use and hydrolyze ATP, Kid generates a force along the spindle defined the ejection force. NUSAP1 binds Kid and facilitates its association with microtubules, thus governing the pole ejection force generated by Kid (Li et al., 2016).
- NUSAP1 interacts with the nucleoporin and E3 SUMO ligase RANBP2
Mass spectrometry-based analysis of NUSAP1 interacting partners revealed a novel interaction with RANBP2, a nucleoporin endowed with SUMO E3 ligase activity, and its partners, the RANGTP hydrolysis-activating enzyme RANGAP1 and the SUMO conjugating enzyme UBE2I (Ubc9) (Mills et al., 2017). Proximity ligation assays (PLA) localised the interaction on microtubules (Mills et al., 2017).
In summary, NUSAP1 controls the mitotic apparatus in several ways: it regulates microtubule stability both directly, via its own microtubule-bundling activity, and indirectly, by regulating the MCAK microtubule-depolymerizing enzyme; it also regulates Kid-generated pole ejection forces in the spindle. These functions converge to regulate proper chromosome segregation during mitosis.
d) DNA-binding activity
NUSAP1 can also interact with the DNA via the SAP domain in the N-ter region (Verbakel et al., 2011). In vitro, the SAP domain preferentially binds double-stranded DNA. During mitosis, wild-type but not SAP-deleted NUSAP1 (NuSAPΔSAP) localises not only to chromosome proximal microtubules, but also to chromosome arms, indicating that the SAP domain mediates the association of NUSAP1 with chromatin in early mitosis. In interphase nuclei, both WT and NuSAPΔSAP are found in nucleoli, but only wild-type NUSAP1 also assumes its canonical localisation to the periphery of the nucleoplasm, where active chromatin domains reside; possible roles of NUSAP1 in transcription or in the DNA damage response have therefore been suggested, as found for other SAP domain-containing proteins (Aravidin and Koonin 2000).

The NUSAP1 gene has conserved orthologues in chimpanzee, macaque, dog, cat, cow, horse, pig, opossum, mouse, rat, chicken, zebrafish, drosophila and frog (sources NCBI and HGCN, HCOP homology predictions).

Fusion genes: Data from Atlas, Mitelman, Cosmic Fusion, Fusion Cancer, and TCGA fusion databases
CD74 (5q32) / NUSAP1 (15q15.1)
NUSAP1 (15q15.1) / RRNAD1 (1q23.1) (Klijn et al., 2015)
NUSAP1 (15q15.1) / ZNF445 (3p21.31) (Wang et al., 2015)
OIP5 (15q15.1) / NUSAP1 (15q15.1) (Yoshihara et al., 2015)
Expected fusion products by conceptual translation of open reading frames generated by translocations:
ITPKA/NUSAP1 fusion, from translocation t(15;15)(q15;q15) (Gao et al., 2018)
MGA /NUSAP1 fusion, from translocation t(15;15)(q15;q15) (Gao et al., 2018)
Decipher database
The region of chromosome 15 containing, among others, the NUSAP1 locus is involved in sporadic cases of deletions or duplications associated with complex phenotypes; of those, the most selective for NUSAP1 are:
heterozygous deletion 15:41285354-45826511 (removing 4,5 Mbp), associated with facial and intellectual disabilities;
heterozygous deletion 15:41285354-45826511 (removing 6,9 Mbp), associated with microcephaly, intellectual disabilities and other traits.
Overall, these phenotypes are consistent with the notion that mutation or deletion of mitotic genes, when occurring early in development, often cause failed development of the central nervous system (Lang and Gershon, 2018; Degrassi et al., 2019).