Fig 2: The C-terminal tail specifies nuclear immobilization and limits aggregation. (A) Immunofluorescence images showing female murine fibroblasts transfected with full-length mouse GFP-845, or derived constructs m845Δ15 (lacking the C-terminal 15 amino acids), m845DDD or m845AAA (green). Cells were co-stained for H3K27me3 (red). DNA is shown in blue, bar is 10 microns. (B) Frequency of nuclei containing large (non-Xi) CIZ1 aggregates, shown as percentage of cells transfected. Results are shown with (+) and without (−) prefixation detergent wash (Det.). n = transfected nuclei counted. (C) Schematic showing the C-terminal portion of CIZ1, GFP-mC275 and derived GFP-mC275 DDD, and their use in 48 h transient expression experiments to assess their ability to assemble into detergent-resistant structures [4]. Below, example images of transfected nuclei. Histograms show retention frequency in male (N = 2) and female (N = 2) CIZ1 null primary embryonic fibroblasts that were transfected (green). n denotes technical replicates for each cell population, with number of nuclei scored in parentheses. (D) Illustration displaying the effect of AURKB site cluster phosphomimic (P) on CIZ1’s association with chromatin and associated detergent-resistant nuclear structures.

Fig 3: AURKB site modification in mitosis. (A) Western blot showing denatured proteins in whole cell lysates collected from untransfected D3T3 cells, or populations expressing full-length mouse GFP-m845WT or GFP-m845DDD, after immunostaining for CIZ1-N or CIZ1-C (tail epitope), or β-actin, and GFP as indicated. (B) Western blot showing denatured endogenous proteins in chemically treated D3T3 cells to achieve cell cycle enrichment in mitosis (M, nocodazole), S phase (S, thymidine), or a phosphatase-suppressed state (okadaic acid). Immunoblotting for C-terminal tail and N-terminal CIZ1 indicates a reduction in tail epitope, compared to untreated cells, during arrest in metaphase, or after phosphatase inhibition. Histone H3 is shown as a loading control. Cy, cycling. (C) Illustration showing data interpretation in which the CIZ1 tail AURKB site cluster is phosphorylated in mitosis, driving dispersal of CIZ1 from Xi assemblies. (D) Female D3T3 cells in stages of mitosis as indicated, immunostained for CIZ1-N or CIZ1-C and co-stained for SAFA. Right, histograms show frequency of retention in interphase (I), prophase (P), metaphase (M), or anaphase (A), where N indicates replicate analyses and n nuclei scored. Lower, by metaphase CIZ1-N and CIZ1-C are significantly different (P < .00017), student’s t-test. (E) Experimental overview of in vitro kinase reactions using purified recombinant human CIZ1 C-terminal fragment C179 and purified AURKB. Middle, products analysed by western blot with C-terminal CIZ1 epitope-defined antibodies, showing changes in reactivity in response to exposure to increasing concentrations of AURKB kinase. Graph shows band intensities relative to untreated C179 control. Products were also analysed by mass spectrometry (Supplementary Fig. S2C).

Fig 4: Interaction between CIZ1 dimer and RNA is regulated by AURKB sites in the C-terminal tail. (A) Schematic of h/m CIZ1 showing conserved domains, in yellow (acidic domain), orange (MH3 dimerization domain), and blue (unstructured tail h37/m38 C-terminal amino acids). Below, human C-terminal (C179) fragments, and derived mutants used as bait fragments in interaction studies, including C179Δtail and phosphomimic C179DDD. Below, C-terminal fragment encompassing the Zn finger motifs used for modelling (green, C305). Left, SDS–PAGE gels showing purified protein preparations stained with Coomassie Blue, or probed with anti-CIZ1 mAb 87, which recognizes all three proteins, or anti-CIZ1 tail pAb, which recognizes an epitope deleted in C179Δtail and mutated in C179DDD. (B) SEC-MALLS, showing normalized UV absorbance at 280 nm and molar mass (dotted line) for human CIZ1-C179 (blue), and human CIZ1-C179Δtail (orange). (C) SEC-MALLS chromatogram showing normalized UV absorbance at 280 nm and molar mass (dotted line) for equivalent murine fragment C181 (black), and derived deletion mutant lacking the matrin 3 homology domain (C181ΔMH3, yellow). (D) Summary of measured molecular masses, indicating that the C-terminal fragment forms a stable dimer that is dependent on the MH3 domain but not the tail region. (E) AlphaFold dimer structure predictions of MH3 domain, showing human CIZ1 aa 779–838 uniprot Q9ULV3-1 (blue) and murine CIZ1 aa 725–785 uniprot Q8VEH2 (cyan). The domain forms a tight dimer with monomer–monomer interactions involving main chain hydrogen bonding between β-strands of the two MH3-type Zn finger motifs. (F) Example electrophoretic mobility shift assays (EMSA) showing the effect of C179, C179Δtail, and C179DDD on the mobility of digoxygenin (DIG)-labelled Xist repeat E RNA probe (left, 0.66 nM) or GAPDH RNA (right, 0.65 nM). Below, immunoblots of EMSA membranes using CIZ1 anti-MH3 domain antibody. Above, murine Xist structure [49] and the derived Xist repeat E RNA probe used in EMSAs. Right, quantification of binding based on the fraction of shifted probe, derived from three replicate experiments (see also Supplementary Fig. S4). Graphs show means ± SEM. (G) AlphaFold-Multimer [47, 48] structure prediction of human C-terminal aa 592–898 (hC306), showing the highest-ranking prediction, in which the acidic domains (yellow) are exposed and the unstructured tails (blue) extend from the core. (H) Model, depicting CIZ1 homodimers interacting with chromosome-associated RNAs via its C-terminal tails, with N-terminal PLD domains available for association with other proteins or other RNAs (left). Right, shows AURKB-mediated phosphorylation driving release from chromosome-associated RNA. In vitro in interphase this results in PLD-driven CIZ1 aggregation.