# RBM22-depletion delays progression through all steps of cell cycle and increases ploidy in myeloid cells

Eloïse Le Hir-Reynaud, Benoît Soubise, Abrahan Molina Mendoza, Cassandra

Konan, Séverine Commet, Nadia Gueganic, Corinne Tous, Laurent Corcos,

Nathalie Douet-Guilbert, Marie-Bérengère Troadec

► To cite this version:

Eloïse Le Hir-Reynaud, Benoît Soubise, Abrahan Molina Mendoza, Cassandra Konan, Séverine Commet, et al.. RBM22-depletion delays progression through all steps of cell cycle and increases ploidy in myeloid cells. Biochimica et Biophysica Acta - Molecular Cell Research, 2025, 1872 (6), pp.119965. 10.1016/j.bbamcr.2025.119965. hal-05058097

HAL Id: hal-05058097

https://hal.science/hal-05058097v1

Submitted on 6 May 2025

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Research Paper

# RBM22-depletion delays progression through all steps of cell cycle and increases ploidy in myeloid cells

Eloïse Le Hir-Reynaud<sup>a,1</sup>, Benoît Soubise<sup>a,1</sup>, Abrahan Molina Mendoza<sup>a</sup>, Cassandra Konan<sup>a</sup>, Séverine Commet<sup>a,b</sup>, Nadia Gueganic<sup>a,b</sup>, Corinne Tous<sup>a,b</sup>, Laurent Corcos<sup>a</sup>, Nathalie Douet-Guilbert<sup>a,b</sup>, Marie-Bérengère Troadec<sup>a,b,*</sup>

<sup>a</sup> Univ Brest, Inserm, EFS, UMR 1078, GGB, F-29200 Brest, France

<sup>b</sup> CHU Brest, service de génétique, laboratoire de génétique chromosomique, Brest, France

## ABSTRACT

RNA-Binding Motif 22 (RBM22) is a splicing factor and a transcription regulator that plays important roles in cancer. Our goal was to document further the implication of RBM22 in cell cycle progression. Using normal human haematopoietic stem and progenitor cells and myeloid cell lines (MDS-L, HL-60), we demonstrated that RBM22 depletion reduces proliferation by delaying the progression of the G1-phase, S-phase and G2/M phase. RBM22 depletion alters mitosis, generating endomitosis and alters megakaryocyte differentiation. Altogether, we propose, for the first time, RBM22 as an essential actor of the cell cycle regulation in human haematopoietic stem and progenitor cells and myeloid cells. We demonstrated that RBM22 alteration is partially responsible for the phenotype of cytopenia of myeloid cell lineages observed in myelodysplastic syndromes (MDS) with a partial deletion of chromosome 5 (MDS with del(5q)) where one allele of RBM22 is lost. We hypothesise that the impact of RBM22 on cell cycle progression could explain some phenotypic features of other cancers.

## 1. Introduction

RNA-Binding Motif (RBM) 22 (RBM22, also known as ZC3H16 [1], FSAP47 [2] and FLJ10290 [3]) is a 10,318 base-pair gene located on the reverse strand of the long arm of chromosome 5 at the 5q33.1 locus in the human genome. RBM22 belongs to the RBM family of genes that is characterised by at least one RNA-Recognition Motif (RRM) in their sequence. This family is composed of approximately fifty genes involved in RNA metabolism. RBM22 contains 11 exons and encodes a protein of 420 amino acids with a molecular weight of 46.9 kDa (review in [4]).

RBM22 is highly conserved during evolution. For instance, it can be found in yeast (as Cwc2 and Ecm2), drosophila (CG14641) and mammals, but its pleiotropic roles still need to be fully characterised.

RBM22 is an essential splicing factor which stabilises the catalytic core of the spliceosome. Different phenotypes have been reported when the RBM22 gene or RBM22 protein function is deleted or decreased. In yeast, the mutation of *Cwc2*, the RBM22 homolog, leads to lethality or growth failure [5]. Development abnormalities were reported in Drosophila [6] and zebrafish [7]. In mouse myeloid lineages, Rbm22 has been shown to be essential for cell survival [8]. RBM22 is described as a tumour suppressor gene or oncogene in solid tumours. In haematopoietic neoplasms, Ebert *et al*. showed that RBM22 depletion affects

myeloid differentiation of human haematopoietic stem and progenitor cells (HSPC) expressing the CD34 surface marker [9]. In mice, haploinsufficiency of *Rbm22* alters haematopoiesis by altering the differentiation into B cells [10]. Haematopoiesis is a complex process where the cell cycle, apoptosis and differentiation steps alternate.

To allow proliferation and differentiation, each cell follows a cycle in which checkpoint proteins play a crucial role. They halt progression if DNA damage is detected, allowing for repair before cell division [11]. Globally, three checkpoints occur during the cell cycle: (i) the DNA damage checkpoint, governed in part by the p53 protein, which assesses DNA integrity before its replication (late G1) and before cell division (late G2); (ii) the replication stress checkpoint occurs during the S phase and monitors the loss of replicative fork integrity; (iii) the spindle assembly checkpoint ensures the complete spindle assembly before cell division during mitosis [12]. There is also the restriction point, described fifty years ago, allowing the triggering of the entry into cell cycle [13,14]. Each checkpoint and cell cycle phase is particularly controlled by specific protein complexes composed of a cyclin and a cyclin-dependent kinase (CDK). The expression of cyclins varies during the cell cycle, while the expression of CDK is constant; it is their phosphorylation state which allows these complexes to be activated or not. For instance, CDK1, inactive when phosphorylated on threonine 14 or

<footnote>* Corresponding author at: Univ Brest, Inserm, EFS, UMR 1078, GGB, F-29200 Brest, France.
E-mail address: marie-berengere.troadec@univ-brest.fr (M.-B. Troadec).</footnote>

<footnote n="1">¹ co-first authors</footnote>
tyrosine 15, is activated when phosphorylated on threonine 161, where it is implicated in DNA damage checkpoints and allows entry into mitosis [15]. CDK4 is activated when phosphorylated on threonine 172 and participates in the passage of the restriction point and in the progression through the G1 phase [16].

RBM22 depletion has been shown to cause cell cycle defects in HeLa cells, resulting in longer mitosis and no cell division, without affecting cell viability [3]. These authors identified 37 genes essential for cell division, among which RBM22 is found (identified as FLJ10290). The goal of the present study is to go further and to generalise the characterisation of the implication of RBM22 in cell cycle progression by studying other cellular contexts than HeLa cells. Very few data were available on this function of RBM22. RBM22 haploinsufficiency (the loss of one allele) has been observed in myelodysplastic neoplasm (also called myelodysplastic syndrome) with a partial deletion of the long arm of chromosome 5 (MDS with del(5q)), as the RBM22 gene is located within the commonly deleted region of MDS with del(5q) [9,17,18]. MDS patients are characterised by important cytopenia in the myeloid lineages (low levels of red blood cells (anaemia), white blood cells (leukopenia) and/or platelets (thrombocytopenia)) due to defects in proliferation and apoptosis of haematopoietic stem and progenitor cells (HSPC). Therefore, we focused on the impact of RBM22 depletion on cell proliferation in MDS with del(5q) due to this recurrent phenotype on proliferation.

We chose to focus on human haematopoietic stem and progenitor cells and myeloid haematological cells (normal human CD34+ HSPC, human MDS-L cell line, human HL-60 cell line) also due to previous descriptions of phenotypic effects of RBM22 depletion in haematological cells [8,9]. We showed that the depletion of RBM22 reduces cell proliferation of myeloid cells. Surprisingly, RBM22 depletion alters every step of the cell cycle by delaying the progression through the G1-phase, S-phase and G2/M phase. RBM22 depletion also induces a mitosis defect, including endomitosis (multiplication of the number of nuclei in one cell) and an alteration of megakaryocyte differentiation of the MDS-L cell line. Altogether, we propose for the first time RBM22 as an essential actor of the cell cycle regulation in human haematopoietic stem and progenitor cells and myeloid cells.

## 2. Material and methods

### 2.1. Cell lines

The MDS-L cell line, derived from blasts obtained from a patient with a 5q- MDS syndrome, was kindly provided by Pr. Kaoru Tohyama [19]. It was cultured in RPMI1640, 10 % FBS, 2 mM L-glutamine, and 1 % penicillin/streptomycin with 50 ng/mL recombinant human IL-3 (Miltenyi Biotech, 130-095-069). The HL-60 cell line was cultured in IMDM, 20 % FBS, 2 mM L-glutamine and 1 % penicillin/streptomycin. The cell lines were maintained at a concentration of 0.5*10⁶ cells/mL in a humidified incubator at 37 °C and 5 % CO₂.

### 2.2. Lentiviral-particle production and cell line transduction

Lentiviral particles were produced by co-transfection of HEK293T cells with a psPAX2, a pVSV-G and a pLK0-TET-ON-puro expressing either a small hairpin (sh) shRNA directed against RBM22 to deplete RBM22 expression (shRBM22, 5'-CCGGGGAATCAATGATCCTGTAGCTCTCGAGAGCTACAGGATCATTGATTCCTTTTTG) or a scrambled (shScrambled, 5'-CCGGCCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGGTTTTTG, corresponding to the control condition) and containing 5'- and 3'-LTRs. Transfection was made with 16 µL of Lipofectamine™ 3000 reagent (ThermoFisher Scientific, L3000015) and 2.1, 1.1 and 3.2 µg of each plasmid, respectively. Lentiviral particles were collected 48 h after transfection, centrifuged for 3 min at 1200g and filtered on a 0.45 µm nitrocellulose membrane. Transduction of MDS-L cells was performed immediately after the

purification of lentiviral particles, and Polybrene (Merck-Millipore, TR-1003-G) was added at a final concentration of 4 µg/mL. Puromycin (Invivogen, 58582) was added 48 h after transduction at a final concentration of 2.5 µg/mL for selection. Doxycycline was added to the medium at a final concentration of 3 µg/mL to induce the expression of the shRNA when needed. The efficacy of the shRNA induction was assessed by qPCR and Western blot. For HL-60 cells, the control condition corresponds to cells transduced without induction of the expression of the shRNA.

To perform an experiment with a second shRNA directed against RBM22 and a rescue experiment, MDS-L cells were transiently transduced twice, 3 days apart, after the production of lentiviral particles as described previously (shRBM22 n°2: 5'-CCATATATCCGAATGACCAAATCAAGAGTTTGGTCATTCGGATATATGGTTTTTTTG).

### 2.3. Cord blood CD34+ cell isolation, culture, transduction and differentiation

Human umbilical cord blood samples were obtained after informed consent and under a protocol approved by the institutional ethical board. Total mononuclear cells were enriched by Ficoll density gradient centrifugation, and CD34 + HSPCs were immunomagnetically isolated using anti-CD34 microbeads and MACS Columns MS (Miltenyi Biotech, 130-100-453, 130-042-201). The cells were then cultured in Stem-MACS™ expansion medium complemented with StemMACS™ expansion cocktail (Miltenyi Biotech, 130-100-473, 130-100-843) for 2-3 days, at a concentration of 1*10⁶ cells/mL the first day and 0.5*10⁶ cells/mL afterwards. Cells were transduced at day 2 and day 3 with 25 MOI of lentiviral particles (produced by the AniRA lentivector production facility, Lyon, France) each time, and LentiBoost-P® (Sirion Biotech) was added at a final concentration of 1:100 of the total volume. Half of the medium was replaced before the second transduction.

### 2.4. Megakaryocytic differentiation monitoring

Megakaryocytic differentiation of the MDS-L cell line was tracked by flow cytometry (CytoFLEX S, Beckman Coulter). The state of differentiation was appreciated with anti-human CD41-PE (Biolegend, 303706) antibody. Cell doublets were excluded on an FSC-A/FSC-H plot.

### 2.5. Cell cycle assays

Prior to the cell cycle assays and after 10 days of shRNA induction, cells were partially synchronised either by a double blockade in the G2/M phase with 4 µg/mL nocodazole (Merck, SML1665) over two consecutive nights or at the end of the G1 phase by a 24-h blockade with 250 nM palbociclib (Euromedex, AB-M6167). The cell cycle was assessed either with the FxCycle™ PI/RNAse Staining Solution (ThermoFisher Scientific, 13433329) or the BD Pharmingen™ APC BrDU kit (BD Biosciences, 552598) following the supplier protocols.

### 2.6. Cell proliferation and viability assay, apoptosis assay

One million five hundred thousand MDS-L cells and one million HL-60 cells were plated at a concentration of 0.5*10⁶ cells/mL in medium containing doxycycline but no puromycin. Cell proliferation and viability were assessed by cell counting on a Malassez counting cell, diluting the cells in Trypan blue (1:1), on days 3, 5, 7 and 10. Medium was completely renewed at each time point by centrifugation. Cell concentration was readjusted to 0.5*10⁶ cells/mL on day 3 for MDS-L by increasing the total volume. Half of the total cells were kept after counting on day 7 for MDS-L and on days 5 and 7 for HL-60 at 0.5*10⁶ cells/mL. For MDS-L, an apoptosis assay was performed at the end of the proliferation assay (10 days of shRNA induction), using the Annexin-V FITC kit (Miltenyi Biotech, 130-092-052) and following the provider
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** RBM22 depletion alters cell proliferation. (A) mRNA levels of RBM22 in shRBM22 MDS-L cells compared to D0. (B) Protein levels of RBM22 in shRBM22 MDS-L cells compared to D0 (left panel) and Western-Blot of the evolution of RBM22 protein level through time of shRBM22 induction in MDS-L (right panel). (C) Proliferation curves of control and shRBM22 MDS-L cells by cell counting (n = 6). (D) Viability of control and shRBM22 MDS-L cells assessed by cell counting with Trypan blue (n = 6). (E) Apoptosis assay of control and shRBM22 MDS-L cells by flow cytometry. Percentages of living (AnnV-/PI-) and apoptotic (AnnV+) cells are represented on the left panel, and representative cytometry plots are on the right panel (n = 3). (F) Proliferation curves of control and shRBM22 HL-60 cells by cell counting (n = 3). (G) Division time of shRBM22 CD34 + -HSPCs compared to control HSPCs. The shRBM22 CD34 + -HSPCs have a reduction by 25 % of RBM22 mRNA expression compared to control HSPCs. All tests are Student t-tests, except for (1C), (1E) and (1F), which are Mann-Whitney tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
recommendations.

## 2.7. RNA isolation, retrotranscription, PCR and quantitative PCR (qPCR)

Total RNA was extracted and purified from fresh cells or thawed dry pellets using the NucleoSpin™ RNA Plus kit (Macherey-Nagel™, 740984) following the provider's protocol. Purified RNA was quantified on a NanoVue spectrophotometer. Retrotranscription was made with 250 ng or 500 ng of total RNA, using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, 4368813). qPCR was performed with the PowerSYBR® Green PCR Master Mix (Applied Biosystems, 4367659). Primers were the following: GAPDH 5'-GAGTCAACG-GATTTGGTCGT-3' / 5'-TTGATTTGGAGGGATCTCG-3', RBM22 5'-TTCATCCAGTTTGCACACG-3 / 5'-TTCTTTTCCTCTGGCTGCCT-3'. They were previously validated, and their concentration ratio was optimised by an efficacy assay. The expression of RBM22 was normalised on GAPDH, and the Fold Change (FC) of their expression between conditions was assayed by the ΔΔCt method.

## 2.8. Western-blot assays

Cell pellets were lysed on ice for 30 min in 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 1 % Triton and 1 % protease inhibitors. One percent phosphatase inhibitors were added when working on phosphorylated proteins of the cell cycle. Cell extracts were then centrifuged at 12,000g, 4 °C, for 10 min, and supernatants were kept at -20 °C. Samples of 30-40 µg protein were boiled at 95 °C for 5 min in Laemmli Buffer and separated on 10 % polyacrylamide SDS-PAGE gels. Proteins were then transferred on a nitrocellulose membrane at 100 V for 1 h 30, and membranes were blocked with 5 % skimmed milk-Tris. Buffered Saline-Tween20 (TBST: 25 mM Tris, 130 mM NaCl, 2.7 mM KCl, 0.1 % Tween20) or 5 % BSA-TBST for phosphorylated proteins, and washed in TBST. Blots were probed with primary antibodies against RBM22 (Abcam, 157105, 1:5000), β-actin (Abcam, 20272, 1:2000), phospho-CDK4 (T172, ThermoFisher, 750 15772546), CDK4 (Abcam, ab68266), CDK1 (Abcam, ab133327), phospho-CDK1 (Y15, Abcam, ab275958) or Cyclin B1 (Abcam, ab32053) diluted either in 5 % milk-TBST or 5 % BSA-TBST and washed in TBST. They were then incubated with horseradish peroxidase-conjugated secondary antibodies, washed in TBST, and proteins were detected using a homemade ECL solution [20].

## 2.9. Immunofluorescence

Cell membranes were labelled with the BioTracker™ 490 Green Cytoplasmic Membrane Dye (Merck, SCT106, 1:200) diluted in culture medium for 30 min at 37 °C and washed in medium. They were subsequently cytospun on a slide at 800 rpm for 5 min and fixed in 4 % paraformaldehyde for 20 min at 4 °C in a humid chamber. Cells were then washed in PBS and incubated in 1 µg/mL Hoechst solution (Invitrogen, H3570) for 15 min, at room temperature, in a humid chamber and washed in PBS. Coverslips were finally mounted with Vectashield® mounting medium.

## 2.10. Fluorescence in situ hybridisation

Two hundred thousand cells were spread on a slide and subsequently fixed in homemade Carnoy's solution (75 % ethanol, 25 % glacial acetic acid) overnight at room temperature. Cells were then permeabilised by incubating the slides in a bath of 2× SSC (Sigma, S6639) / 0.4 % NP40 (Merck, 492016) for 30 min at 37 °C. They were then dehydrated by successive baths in 70 %, 90 %, and 100 % ethanol for 3 min each. After drying the slides, they were pre-heated to 37 °C and subsequently labelled on a ThermoBrite (Abbott Molecular) with 5 µL of pre-heated probes, diluted at 1:10 in the provided buffer, directed against chromosome 7 centromere (Chromosome 7 Alpha Satellite Probe, CytoCell,

LPE007G) and chromosome 10 centromere (Chromosome 10 Alpha Satellite Probe, CytoCell, LPE010R). Slides were temporarily sealed with a coverslip and flexible mounting adhesive (Marabu), and hybridisation was performed on the ThermoBrite at 75 °C for 2 min. Slides were then conserved at 37 °C, in the dark, for 24-72 h. Coverslips were then discarded, and slides were washed in a bath of 0.4× SSC/0.3 % NP40 at 72 °C for 45 s and subsequently in a bath of 2× SSC/0.1 % NP40 at RT for 20 s. Coverslips were mounted with DAPI and sealed. Slides were conserved at 4 °C in the dark until observation.

## 2.11. Statistical analysis

All experiments counted at least 3 independent replicates ($n \ge 3$) except for the rescue experiment ($n = 1$). Dataset normality was assessed by Shapiro-Wilk tests. The difference between two variables was assayed by unpaired parametric Student t-tests or non-parametric Mann-Whitney tests. Statistical significance was considered for $p < 0.05$. Statistical analyses were performed on GraphPad Prism 10.0 (GraphPad Software, San Diego, California, USA). Tests used are indicated in the legend of each figure. ns: non-significant; *: $p < 0.05$; **: $p < 0.005$; ***: $p < 0.0005$; ****: $p < 0.0001$.

# 3. Results

## 3.1. RBM22 depletion reduces cell proliferation without affecting viability

We focused on the impact of RBM22 haploinsufficiency on cell proliferation in myelodysplastic neoplasm (MDS) with del(5q) because one allele of this gene is lost in this neoplasm and because the cell phenotype (cytopenia in the myeloid lineages: anaemia, leukopenia, and/or thrombocytopenia) implicates a slowdown of cell proliferation. We performed proliferation assays on a model of myeloid cell, the MDS-L cell line expressing either the shRBM22 or shScrambled in a doxycycline-inducible manner. MDS-L cells contain only one copy of the RBM22 gene. With this model, we decided to worsen the diminution of RBM22 protein level to study its effect. We show that it drastically reduces cell proliferation (Fig. 1A-C). Interestingly, this reduction of proliferation was not due to an increased cell death, as shown by cell counting in Trypan blue and apoptosis assays by flow cytometry (Fig. 1D-E). We also showed a significant decrease in proliferation in a different model of human myeloid cells, the HL-60 cells, when RBM22 was depleted by shRBM22 (Fig. 1F, Supplementary Fig. 1A-B). These results are in concordance with a study describing a mitotic arrest and cytokinesis defect in HeLa cells when RBM22 expression is reduced, without affecting viability [3].

This result suggests that RBM22 plays a crucial role in cell cycle progression. Concordantly, in normal human CD34+ haematopoietic stem and progenitor cells (HSPC), we also showed that RBM22 depletion (reduction by 25 % of RBM22 mRNA expression) increases cell division time (Fig. 1G) in a range that is comparable to that observed in MDS-L and HL-60 cells. Altogether, these results show that the knockdown of RBM22 in different human cell models mimics the growth phenotype observed in MDS with del(5q) patients' cells.

## 3.2. RBM22 depletion alters cell cycle progression in the G1-phase

We then aimed at understanding how the depletion of RBM22 impacts cell proliferation. As the reduction of proliferation is not due to an increased mortality, we hypothesised that the cells would progress more slowly through their cycle or would be blocked at some stages of the cell cycle. We first focused on the progression through the G1-phase. To do so, we synchronised cells in this phase by a block with palbociclib, which inhibits phosphorylation of CDK4 and CDK6. The blockade was then released, and the progression of cells through their cycle was monitored with propidium iodide in flow cytometry. On the one hand, we observed that significantly fewer cells were synchronised in the G1-phase at T0
<figure><img src="image_3.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Fig. 2. RBM22 depletion slows down the G1-phase of the cell cycle.** (A) Proportion of MDS-L cells in the G1-phase at 0 h, 6 h, 16 h and 24 h after a partial synchronisation with palbociclib (n = 6). (B) Proportion of MDS-L cells in the G1-phase at 0 h, 1 h30, 3 h30 and 5 h30 after a partial synchronisation with nocodazole followed by flow cytometry analysis (Control n = 9, shRBM22 n = 3). (C) Western blot showing the phosphorylation state and expression of pCDK4<sup>Thr172</sup>, CDK4 and β-actin in the MDS-L cell line at 0 h and 1 h30 after a partial synchronisation with nocodazole (n = 3). All tests are Student t-tests and each point is compared to the same time of the control condition.</figcaption></figure>

<figure><img src="image_5.png" /></figure>

when **RBM22** was depleted, suggesting an upstream slowdown (Fig. 2A). As expected, the pool of cells in the G1-phase decreased through time because cells progressed in their cycle. On the other hand, the decrease was slower when **RBM22** was depleted compared to the control condition, suggesting that cells remained blocked in the G1-phase and that this phase progression was also slowed down. Concordantly, we studied cell cycle progression through G1-phase after synchronising the cells in the G2/M phase by a double block with nocodazole, which causes the depolymerisation of microtubules. We observed that the rate of increase of the pool of cells in the G1-phase from T0 to T3h30 is lower when the expression of **RBM22** was decreased (Fig. 2B). This result confirmed the slowdown in G1-phase for **RBM22**-depleted cells. To understand mechanistically what was happening, we studied CDK4 and phospho-CDK4<sup>Thr172</sup> expression in MDS-L cells synchronised in the G2/M phase by Western blot. CDK4 is associated with Cyclin D during the G1-phase, and this complex is activated by phosphorylating CDK4<sup>Thr172</sup> to allow cell cycle progression. First, we observed that CDK4 protein level was higher in **RBM22**-depleted cells compared to control cells (Fig. 2C). This result demonstrates an alteration in CDK4 expression in the context of **RBM22** depletion. However, phospho-CDK4<sup>Thr172</sup> and subsequently the ratio phospho-CDK4<sup>Thr172</sup>/CDK4 was less phosphorylated at T0 when **RBM22** was depleted (Fig. 2C), demonstrating the slowdown of the restriction point activation and explaining the important delay in the G1-phase progression for **RBM22**-depleted cells.

Altogether, these results showed that the depletion of RBM22 induced an alteration of CDK4 expression, a slowdown of the G1-phase, and a delay at the restriction point (cell cycle checkpoint) activation.

### 3.3. RBM22 depletion slows down S-phase progression

To further study the cell cycle, we focused on the progression through the S-phase by a BrdU pulse-chase. We observed that the proportion of BrdU-labelled cells (i.e., in S phase) was significantly higher in **RBM22**-depleted cells as compared to the control, demonstrating an arrest in S-phase when **RBM22** expression was reduced (Fig. 3A, Supplementary Fig. 1C). We also studied this phase after cell synchronisation in G1-phase with palbociclib. We found that, at T0 and T6 h after the block release, the percentage of cells in S-phase was significantly higher in the shRBM22 condition compared to the control, indicating a slowdown of this phase (Fig. 3B).

Additional timepoints (T16h and T24h) further reinforced our observations: the T16h timepoint showed significantly fewer cells in the shRBM22 condition, confirming a defect of these cells during the end of the G1 phase, which affected their capacity to enter the S phase. The T24h timepoint, conversely, showed significantly more cells in the shRBM22 condition compared to the control. This confirmed that the progression of RBM22-depleted cells through the S phase was also hampered (Fig. 3B).

In order to better understand the impacts of this alteration, we questioned whether the RBM22-depleted cells would present DNA damage. If these damages are not repaired properly before the entrance into the S phase, the speed of the replicative machinery can be reduced or even stopped, thus increasing the time spent by the cells in the S phase. We determined DNA damage by immunofluorescence using an anti-γH2AX antibody, a marker of DNA double-strand breaks (Fig. 3C).
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** RBM22 depletion slows down the S-phase of the cell cycle. (A) Proportion of MDS-L cells in the S phase, labelled by a 1 h BrdU pulse-chase (n = 3). (B) Proportion of MDS-L cells in the S-phase at 0 h, 6 h, 16 h and 24 h after a partial synchronisation with palbociclib followed by flow cytometry analysis (n = 6). (C) Proportion of nuclei in function of γH2AX fluorescence intensity in immunofluorescence, in control MDS-L (left panel) and shRBM22 MDS-L (right panel). The average is represented by a grey bar. All tests are Student t-tests, and each point is compared to the corresponding timepoint of the control condition.</figcaption></figure>

Surprisingly, we observed that the γH2AX fluorescence intensity was lower when RBM22 was depleted, indicating that there were fewer DNA double-strand breaks in this condition. These results indicated that RBM22 depletion induces a slowdown of the S-phase, but this could not be explained by an increase in DNA damage, which was present at lower levels than in the control condition.

### 3.4. RBM22 depletion also alters mitosis

We next focused on the last part of the cell cycle, the G2/M phase. As previously described, we synchronised the cells in the G2/M phase with nocodazole. As expected, we found that the pool of cells in the G2/M phase progressively decreased after nocodazole-block release (Fig. 4A). We showed a tendency of RBM22-depleted cells to remain blocked in the G2/M phase longer, suggesting difficulties in successfully reintegrating the cell cycle. More than 5 h after the block release, there were still significantly more RBM22-depleted cells in the G2/M phase as compared to the control.

To go further, we analyzed Cyclin B1, phospho-CDK1<sup>Tyr15</sup> and CDK1 expression in cells synchronised in the G2/M phase by Western blot. The Cyclin B1 is progressively accumulated during the G2 phase and is degraded at the entrance into mitosis. This cyclin forms a complex with CDK1, which is activated by dephosphorylating CDK1<sup>Tyr15</sup> to allow progression through mitosis. We showed that, in the RBM22-depleted condition, phospho-CDK1<sup>Tyr15</sup> was significantly less phosphorylated at T0 and T1h30 compared to the control, indicating that cells remain blocked longer in mitosis (Fig. 4B). In addition, the analysis of cyclin B1 expression showed that this protein was significantly less expressed at T0 when RBM22 was depleted, confirming that cells remain blocked longer in mitosis (Fig. 4C). Altogether, these results indicate that RBM22 depletion increased the time to complete mitosis.

### 3.5. RBM22 depletion produces polyploid cells

Surprisingly, induced expression of shRBM22 also promoted the emergence of a novel cell population of significantly larger cells, as observed with a phase contrast microscope and confirmed by flow cytometry analysis (Fig. 5A-B). Concordantly, we observed after the nocodazole-block release, but also on unsynchronised cells, a significant increase in the amount of polyploid cells by flow cytometry and FISH (Fig. 5C-F). These results showed that, apart from slowing down mitosis, the depletion of RBM22 also induced a defect in the last steps of mitosis. To confirm these results, we performed flow cytometry on unsynchronised cells with another shRBM22 and by rescuing RBM22 expression (Fig. 5G, Supplementary Fig. 1D). We observed an increase of the ratio of polyploid cells in the MDS-L shRBM22 condition. This ratio is decreased when RBM22 expression is rescued, confirming our previous results. We observed that the depletion of RBM22 increased cell ploidy, characterised by cells with more than one nucleus, rather than inducing an incorrect separation of sister chromatids (Fig. 5H). MDS-L cells can slightly differentiate into megakaryocytes, which are known to be polyploid. In fact, when megakaryocyte progenitors reach the end of their differentiation, they begin to mature with a process of polyploidisation called endomitosis, in which the nucleus does not segregate during mitosis. This, sequentially, gives rise to diploid, tetraploid and more polyploid promegakaryoblasts with multilobulated nuclei [21,22]. Interestingly, we showed that the modulation of RBM22 expression increased megakaryocytic differentiation, with the CD41<sup>+</sup> cell marker as a readout, notably when RBM22 was decreased (Fig. 5I, Supplementary Fig. 1E). This could partially explain the increase in the proportion of polyploid cells, but not all, considering the timespan of just a few hours after the block release, which is not sufficient for the cells to differentiate into CD41<sup>+</sup> cells. Those results reinforce the idea of a cytokinesis defect when RBM22 is reduced. This type of defect was
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

**Fig. 4. RBM22 depletion slows down the G2/M phase of the cell cycle.** (A) Proportion of MDS-L cells in the G2/M-phase at 0 h, 1 h30, 3 h30 and 5 h30 after a partial synchronisation with nocodazole (flow cytometry) (Control n = 9, shRBM22 n = 6). Western blot showing the phosphorylation state and expression of pCDK1<sup>Tyr15</sup>, CDK1 (B, n = 3) and Cyclin B1 and β-actin (C, n = 3) in the MDS-L cell line at 0 h and 1 h30 after a partial synchronisation with nocodazole. All tests are Student t-tests, and each point is compared to the corresponding timepoint of the control condition.
<figure><img src="image_12.png" /><figcaption>A</figcaption></figure>

MDS-L control

<figure><img src="image_13.png" /></figure>

MDS-L shRBM22

<figure><img src="image_14.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>E</figcaption></figure>

MDS-L control

<figure><img src="image_19.png" /></figure>

MDS-L shRBM22

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_23.png" /><figcaption>H</figcaption></figure>

MDS-L control

<figure><img src="image_24.png" /></figure>

MDS-L shRBM22

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>I</figcaption></figure>

<figure><img src="image_29.png" /></figure>

(caption on next page)
**Fig. 5. RBM22 depletion increases cell ploidy by hampering mitosis and alters DNA synthesis.** (A) Representative pictures of larger cells in control and shRBM22 MDS-L cell cultures. Scale bar: 20 µm. (B) Proportion of larger MDS-L cells in the cell culture, assayed by flow cytometry (upper panel); representative cytometry plots are on the lower panel (n = 4). (C) Representative plots obtained in flow cytometry are shown with the correspondence between the different peaks and cell cycle phases indicated (Control n = 9, shRBM22 n = 6). (D) Proportion of polyploid MDS-L cells at 0 h, 1 h30, 3 h30 and 5 h30 after a partial synchronisation with nocodazole, assayed by flow cytometry (n = 3). (E) Representative FISH pictures of polyploid MDS-L cells labelled with probes of chromosomes 7 and 10 (respectively green, normally 1 dot/cell, and red, normally 2 dots/cell). Scale bar: 10 µm. (F) Quantity of polyploid MDS-L cells counted by FISH (n = 3). (G) Ratio of polyploid control and shRBM22 MDS-L cells with RBM22 rescue condition, compared to the control condition (n = 1). (H) Representative pictures of control and shRBM22 MDS-L cells whose membranes and nuclei were stained with green membrane dye and DAPI (blue), respectively. Scale bar: 20 µm. (I) Proportion of CD41+ MDS-L cells assayed by flow cytometry (n = 6). All tests are Student t-tests. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

briefly mentioned after RBM22 depletion in HeLa cells [3].

Altogether, our results indicate that the decrease in RBM22 expression leads to an overall slowing down of the cell cycle and that RBM22 plays a major role in cell cycle progression.

## 4. Discussion

In this study, we show a reduced proliferation of RBM22-haploinsufficient cells, without any increase in cell death. The opposite situation could have artificially lowered the cell proliferation rate. We explain this alteration of proliferation by a slowing down of the cell cycle. This is in accordance with previous studies reporting that RBM22 and its orthologues were essential for cell growth and embryonic development in yeast, Drosophila, zebrafish and mouse, and that its depletion induced a mitotic arrest without inducing cell death in HeLa cells [3,5–8,23].

We also showed that the cell cycle was impacted by different mechanisms. The G1-phase was slowed down concomitantly with a delay in the phosphorylation of CDK4Thr<sup>172</sup> in RBM22-haploinsufficient cells, whereas, unexpectedly, the total level of CDK4 protein was massively increased. This CDK4, in its complex with Cyclin D1, allows cells to progress through the G1-phase when it is phosphorylated in an active way. An increased activity of this complex has been reported in cancers, and CDK4 expression is used as a prognosis indicator in triple negative breast cancers, for instance [24]. Patients treated with CDK4 inhibitors commonly suffer from anaemia and thrombocytopenia [25]. We hypothesise that this reinforces the idea that RBM22 depletion is, at least partially, responsible for anaemia and other cytopenia observed in MDS with del(5q) by decreasing phosphorylation of CDK4<sup>Thr172</sup>.

Moreover, we showed a slowdown in the S-phase of the cell cycle when RBM22 expression was decreased. Our results demonstrated that DNA damage was repaired more quickly or that there was no more DNA damage in the RBM22-haploinsufficient condition, suggesting that the slowdown of the S-phase could be due to a defective checkpoint or an alteration of the replicative machinery. To our knowledge, there is no report so far about any direct link between RBM22 or the RBM family or protein with the RRM motif and replicative machinery. However, a recent study showed that RBM22 plays a key role in the coordination of transcription by inhibiting the RNAPII 5' pause release, sustaining a high transcription elongation rate and participating in transcription termination [26]. We can hypothesise that either RBM22 could play a similar role in the replicative machinery or it could modulate the expression of proteins involved in replicative machinery. According to Saldivar *et al.*, γH2AX signals involved by ATR increase when cells enter S-phase, with a maximum in mid-to-late S-phase and a rapid decrease at the end of DNA replication. The signal that we observed in our study may be due to DNA replication rather than DNA damage [27].

In our study, we showed that RBM22-haploinsufficiency also impacts the progression through the G2/M phase, where we showed that the Cyclin B1/CDK1 complex is maintained in an active state by the phosphorylation of the Tyr15 of CDK1, keeping the cells in mitosis longer. Further studies would be necessary to clarify this mechanism.

Furthermore, we showed that the mitotic arrest is associated with increased proportions of polyploid cells, suggesting a cytokinesis defect leading to endomitosis (multiplication of the number of nuclei in one

cell). This is partially explained by the fact that RBM22-haploinsufficient cells differentiate more into megakaryocytes, but due to the short time between the nocodazole-block release and the increase of polyploid cells, most of it undeniably results from the mitosis defect. It has already been shown that the loss or the inhibition of CDK1 activity causes polyploidy in transformed human cell lines *in vitro*, so the increased proportions of polyploid cells could be explained by the altered expression of CDK1 [28]. However, it could be interesting to further investigate the mechanisms involving polyploidy, such as defects in chromosomal segregation or in cytoplasmic cleavage.

RBM22 is an essential component of the spliceosome; it has been repeatedly associated with tumours as an oncogene or a tumour suppressor following the histological context, and several reports indicated that it plays a role in haematopoiesis [29–32]. Kittler showed that RBM22 is essential for cell proliferation [3]. Here, we show that RBM22 is also endowed with a major role in the regulation of the cell cycle. Because of its implication in the splicing process, there is a strong possibility that RBM22 impacts target genes such as CDK1 or CDK4, or it could also induce a slowdown of every step of the cell cycle because of its haploinsufficiency. Few target genes of RBM22 are known; however, the programmes it is involved in ([33]; [34]) could clearly account for at least some of the effects on cell division. The precise mechanism of cell cycle slowing down by RBM22 will be further investigated.

The alteration of RBM22 expression has been associated with phenotypic features of some cancers, such as triple-negative breast cancers and glioblastoma. In these diseases, the knockdown of RBM22 allowed the decrease of viability and proliferation of the cancer cells [4,29,30]. As stated above, in haematological neoplasms, RBM22 is the second gene among the 40 genes of the commonly deleted region of myelodysplastic neoplasm (MDS) with partial deletion of chromosome 5 (del(5q)) affecting the most the haematopoietic myeloid differentiation in human CD34+ HSPC [9]. In light of the results, the role of RBM22 in the pathophysiology of del(5q) MDS deserves to be investigated.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbamcr.2025.119965.

### CRediT authorship contribution statement

**Eloïse Le Hir-Reynaud:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis.
**Benoît Soubise:** Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis.
**Abrahan Molina Mendoza:** Writing – review & editing, Investigation.
**Cassandra Konan:** Writing – review & editing, Investigation.
**Séverine Commet:** Writing – review & editing, Investigation.
**Nadia Gueganic:** Writing – review & editing, Investigation.
**Corinne Tous:** Writing – review & editing, Investigation.
**Laurent Corcos:** Writing – review & editing, Methodology.
**Nathalie Douet-Guilbert:** Writing – review & editing, Formal analysis, Conceptualization.
**Marie-Bérengère Troadec:** Writing – review & editing, Writing – original draft, Supervision, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.
Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We greatly thank the patients, patients' families and mothers for sample donations and the teams of Brest hospital maternity for their time. We thank our colleagues from the team ASTRE for technical assistance and scientific advice. We thank all the colleagues from the chromosomal genetics laboratory from CHRU Brest, France, in particular Dr. Audrey Basinko and Dr. Frédéric Morel. We thank Leïla Baudet for her technical contribution. We thank Dr. Kaoru Tohyama from Kawasaki Medical School, Japan, for kindly providing us the MDS-L cell line. We thank Philippe Brunet de la Grange for his advice on HSPC transduction. We acknowledge the contribution of the AniRA lentivector production facility from the CELPHEDIA Infrastructure and SFR Biosciences (UAR3444/CNRS, US8/Inserm, ENS de Lyon, UCBL, France), especially Gisèle Froment, Didier Nègre and Caroline Costa.

This project was supported by grants from the Ligue Contre le Cancer Grand Ouest (MBT, NDG), the association Halte au Cancer (MBT), the Groupe Français des Myélodysplasies (MBT, NDG), the association Gaetan Saleün (MBT) and the association Connaître et Combattre les Myélodysplasies (MBT), l'Institut National du Cancer (InCa PLBIO INCa_18469; MBT), Association Fondation de l'Avenir and Fonds INNOVEO Brest.

Data availability

Data will be made available on request.

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