Knockdown of spliceosome U2AF1 significantly inhibits the development of human erythroid cells

Abstract U2AF1 (U2AF35) is the small subunit of the U2 auxiliary factor (U2AF) that constitutes the U2 snRNP (small nuclear ribonucleoproteins) of the spliceosome. Here, we examined the function of U2AF1 in human erythropoiesis. First, we examined the expression of U2AF1 during in vitro human erythropoiesis and showed that U2AF1 was highly expressed in the erythroid progenitor burst‐forming‐unit erythroid (BFU‐E) cell stage. A colony assay revealed that U2AF1 knockdown cells failed to form BFU‐E and colony‐forming‐unit erythroid (CFU‐E) colonies. Our results further showed that knockdown of U2AF1 significantly inhibited cell growth and induced apoptosis in erythropoiesis. Additionally, knockdown of U2AF1 also delayed terminal erythroid differentiation. To explore the molecular basis of the impaired function of erythroid development, RNA‐seq was performed and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results showed that several biological pathways, including the p53 signalling pathway, MAPK signalling pathway and haematopoietic cell lineage, were involved, with the p53 signalling pathway showing the greatest involvement. Western blot analysis revealed an increase in the protein levels of downstream targets of p53 following U2AF1 knockdown. The data further showed that depletion of U2AF1 altered alternatively spliced apoptosis‐associated gene transcripts in CFU‐E cells. Our findings elucidate the role of U2AF1 in human erythropoiesis and reveal the underlying mechanisms.

mitoses to produce basophilic, polychromatic and orthochromatic erythroblasts. Orthochromatic erythroblasts enucleate to become reticulocytes, which subsequently mature into red blood cells. [1][2][3][4] The surface markers CD34, IL-3R, CD36 and GPA have been defined for BFU-E and CFU-E cells in human early erythropoiesis. 5 During terminal erythroid differentiation, the expression levels of GPA and band 3 increase and that of a4 integrin decreases. 6 Thus, we used these surface markers to monitor the erythropoiesis process in vitro.
A series of genes and cell factors are involved in the regulation of erythroid differentiation. In contrast to extensive studies on the role of transcription factors and cytokines, [7][8][9] little is known about the contribution of splicing to the regulation of normal erythropoiesis. The spliceosome is essential for removing noncoding introns from precursor messenger RNAs (pre-mRNAs), which consist of the small nuclear ribonucleoproteins (snRNPs) U1, U2, U5 and U4-U6. 10,11 Three essential sequences, the 5' splice site (5' ss), 3' splice site (3' ss) and branch point, are required for splicing. The process of spliceosome assembly is fully accomplished when a series of RNA splicing events are achieved. Splice site recognition is promoted by enhancers or prevented by silencers to regulate splicing throughout spliceosome assembly. Additionally, some specific enhancers and silencers are essential for the regulation of alternative splicing in the cell differentiation stage. [12][13][14] The cytotoxic granule-associated RNA binding protein T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 bind to U-rich motifs downstream of 5' ss in the regulation of the alternative splicing of exons. 15 The RNA recognition motifs 1 (RRM1) and the C-terminal glutamine-rich (Q) domain are required for facilitating U1 snRNP recruitment to 5' ss. The Q-rich domain of TIA1 interacts with the N-terminal region of U1-C to recruit U1 snRNP to 5' ss, and this process is enhanced by RRM1. 16 A paralogue of U2AF65, RBM39, is involved in the pre-mRNA splicing pathways in humans and Drosophila. 17,18 U2AF1 (also named U2AF35) is the U2 small nuclear RNA cofactor, which includes zinc finger domain, UHM and RS domain structures.
Three conserved mRNA isoforms, termed U2AF1a (NM_006758), U2AF1b (NM_001025203) and U2AF1c (NM_001025204), were generated by alternative splicing of the U2AF1 gene. U2AF is a heterodimer composed of the small subunit U2AF1 and large subunit U2AF2 (U2AF65), among which U2AF1 is involved in the recognition of the AG at 3' ss and U2AF2 contacts the polypyrimidine tract. 19,20 U2AF1 binds to the AG dinucleotide interacting with both U2AF2 and SR protein to establish the earliest splicing complex E. Then, the U2AF complex replaces SF1 bind to the BPS with one of its subcomponents, SF3B1, to generate the splicing A complex. 21 Mutations in spliceosome components have been detected in myelodysplastic syndromes (MDS). Reports on analysis of MDS patients have revealed a high incidence of U2AF1-S34F missense mutation. 21,22 The study shows that overexpression of this U2AF1-S34F mutant impairs erythroid differentiation and skews granulomonocytic differentiation towards granulocytes in human haematopoietic progenitors. 23 Depletion of U2AF1 affects the alternative splicing of the Dscam gene transcript in Drosophila. 18 Additionally, knockdown of U2AF1 alters alternatively spliced isoforms of Cdc25B and Cdc25C transcripts and retard mitotic progression in cancer cell. 24,25 Recently study revealed some spliceosome components, such as SF3B1 and SRSF2 deficiency impairs human erythropoiesis. 26,27 But the functions of depletion U2AF1 in erythropoiesis remain unknown.
To determine whether U2AF1 plays a key role in erythropoiesis, we knock down U2AF1 in erythroid cells derived from human CD34 + haematopoietic stem cells. We showed that depletion of U2AF1 impaired cell growth, led to apoptosis and delayed erythroid differentiation. Bioinformatics and biochemical analyses demonstrated that knockdown of U2AF1 induced apoptosis involving the p53 pathway and altered the alternative splicing of apoptosis-associated gene transcripts. Our findings reveal a critical function of the U2AF1 and suggest that U2AF1 regulates alternative splicing during human erythropoiesis.

| Cell culture and lentivirus infection
Human peripheral blood samples were obtained from Xiangya Hospital of Central South University. Isolated CD34 + cells and sorted cells were cultured as previously described. 6 HEK293T cells were plated in DMEM (Gibco) with 10% FBS (Gibco) at 37°C in 5% CO 2 . The control shRNA and two U2AF1 shRNAs were purchased from Sigma. All vectors were co-transfected with the packaging vectors pCMV and pucMDG into HEK 293T cells for lentivirus production. The shRNA-mediated knockdown was performed as previously described. 28

| Quantitative Real-Time RT-PCR
Total RNA was isolated from cells using the RNeasy mini kit

| Colony assay
To determine the CFU-E and BFU-E potential, 200 cultured or sorted cells were resuspended in 1 mL of MethoCult classic medium (H4434) or MethoCult medium with EPO only (H4330) and plated in triplicate as previously described. 5,29 CFU-E colonies were counted on day 7, and BFU-E colonies were counted on day 14.

| Western blotting
The cultured cells were harvested and washed with PBS, followed by the addition of RIPA buffer (Thermo Fisher) and protease inhibitor cocktail (Roche) for 30 minutes on ice and collection of protein extracts by centrifugation. The protein concentration was measured using a Pierce ® BCA protein assay kit (Thermo Fisher). Next, 40 µg of total protein was analysed by electrophoresis by 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The blots were blocked in 5% non-fat milk and incubated with antibodies overnight at 4°C. The secondary antibody was purchased from Amersham Biosciences.

| RNA-seq and bioinformatics analysis
RNA-sequencing was performed as previously described. RNA was extracted from sorted Luciferase control and U2AF1 knockdown CFU-E cells. cDNA libraries were prepared using the Illumina TruSeq kit and sequenced using the Illumina HiSeq 4000 system. Quality control was performed before sequencing, and high-quality reads from sequencing were aligned with Tophat2 following the detailed protocol. 30 The cuffdiff and cummerbund R packages were used for the analysis and extraction of differentially expressed genes. 31 Gene Ontology analysis and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were applied to annotate the biological functions. 32 Cufflinks was used to assemble transcripts with the human hg19 genomes, and spliceR was used to annotate and extract differentially spliced genes.

| Cytospin preparation
1 × 10 5 cells in 100 μL 1 × PBS were spun on slides for 5 minutes at 300 rpm using the cytospin apparatus (Thermo Scientific). Then slides were stained with Giemsa solution (Sigma) according to manufacturer's instructions. The slides were analysed, and the images were acquired using a Leica DM2000 inverted microscope.

| Statistical analysis
All data were processed and statistically analysed using SPSS 18.0 software. Significant differences between groups were determined using the t test, and P < 0.05 was considered to indicate a significant difference.

| U2AF1 expressed during human erythroid differentiation
Here, we analysed the expression of three different U2AF1 isoforms on the sorted cells during human erythropoiesis. The isoforms U2AF1a and U2AF1b were more abundant than U2AF1c, and the expression of U2AF1c was very low ( Figure 1A). The expression of U2AF1a was higher in erythroid progenitor cells than other stages, and its expression was stable during terminal erythroid differentiation. The expression of U2AF1 was further confirmed at the protein level by Western blotting ( Figure 1B). The expression of U2AF1 was knocked down using two different shRNA constructs (1 or 2) to target isoform a. The shRNA1 targeted the U2AF1a isoform-specific exon 8 and shRNA2 target exon 3 ( Figure 1C). The knockdown efficiency of U2AF1 shRNAs was confirmed by qRT-PCR ( Figure 1D) and Western blotting ( Figure 1E).

| Knockdown of U2AF1 impaired cell growth and increased apoptosis in early erythropoiesis
To understand the function of U2AF1 in early erythropoiesis, we transfected CD34 + cells with lentiviruses on day 2 and analysed them after day 3 of culture under our erythroid differentiation conditions. Cell growth was inhibited in U2AF1 knockdown cells at early stages compared with the control (Figure 2A). However, the PI staining results showed that U2AF1 knockdown did not affect cell cycle progression ( Figure 2B,2C). The 7AAD and Annexin V staining and quantitative analysis revealed that the depletion of U2AF1 induced apoptosis ( Figure 2D,2E). To examine whether U2AF1 was involved in the colony-forming ability, we performed colony assays using sorted BFU-E and CFU-E cells. We then plated 200 cells on day 6 of culture in two different MethoCult media. Figure 2F shows that the BEU-E and CFU-E colonies decreased obviously in U2AF1 knockdown sorted cells compared with the control. Taken together, these data showed that U2AF1 knockdown impaired the proliferation of erythroid progenitors, induced apoptosis and reduced the colony forming ability.

| Knockdown of U2AF1 impaired human terminal erythroid development
Human terminal erythroid differentiation was monitored by flow cytometry based on the expression levels of surface markers. 33,34 As shown in Figure 3A, more than 45% of the Lucif-shRNA-transduced cells were GPA-positive, while fewer than 20% of the U2AF1-shRNA transduced cells were GPA-positive. Furthermore, U2AF1 knockdown delayed terminal erythroid differentiation ( Figure 3B).
Next, we examined cell growth from day 7 to day 15 during terminal erythroid differentiation. The data showed that U2AF1 knockdown significantly impaired cell growth starting at day 7 of culture ( Figure 3C). Additionally, the number of apoptotic cells increased in U2AF1-shRNA-transduced compared with Lucif-shRNA-transduced cells from day 7 to day 15 ( Figure 3D).

| Knockdown of U2AF1 impairs enucleation and leads to the generation of erythroblasts with abnormal nucleus
The effect of U2AF1 knockdown on enucleation, we assessed the enucleated cells from day 13 to day 19 and Figure 4A shows the representative profiles. Quantitative analysis reveals that the Lucif-shRNA-transduced cells started to enucleate on day 13 and more than 60% of cells enucleated by day 19. In contrast, the enucleation rate of U2AF1-shRNA2 transduced cells was fewer than 20% on day 19 ( Figure 4B). When examining the effect of U2AF1 knockdown on cell morphology, we found that many erythroblasts exhibited abnormal nuclei at the late stage.
Cytospin analysis of cultured erythroblasts revealed the generation of abnormal nuclei occurred specifically at day 13 and day 15 of culture ( Figure 4C). Quantitative analysis showed that approximately 20% of erythroblasts at day 13 of culture and 40%-50% of erythroblasts had abnormal nuclei at day 15 of culture compared to control ( Figure 4D).

| Knockdown of U2AF1 affected gene expression in erythroblasts
To further explore the underlying molecular mechanisms for the changes in cell growth and apoptosis, we sorted Luciferase-knockdown and U2AF1-knockdown CFU-E cells for RNA-seq analysis. There were 628 differentially expressed genes, including 282 up-regulated ( Figure 5A) and 346 down-regulated genes ( Figure 5B), between Luciferase-knockdown and U2AF1-knockdown CFU-E cells. Using the KEGG pathways to analyze the 628 differentially expressed genes, we found that these genes were involved in several biological pathways, including the p53 signalling pathway, MAPK signalling pathway and haematopoietic cell lineage. The data from microarray analysis showed that many of the differentially expressed genes were involved in the p53 signalling pathway ( Figure 5C). The p53-induced apoptosis is mediated by transcriptional activation of Bcl-2 family genes such as Bcl-2, BAX and BBC3. 35,36 Western blot analysis revealed increased protein levels of p53, BAX, PUMA (BBC3) and p21 and decreased protein levels of Bcl-2 in U2AF1-knockdown CFU-E cells ( Figure 5D).

| Knockdown of U2AF1 affected the alternative splicing of genes in erythroblasts
Previous research has indicated that U2AF1 is a regulator of alternative splicing and is involved in recognition of the AG at 3' ss. To study the effect of U2AF1 on alternative splicing, we analysed the F I G U R E 2 Effects of U2AF1 knockdown on the proliferation of human erythroid progenitors. A, Growth curves of cells transduced with lentivirus containing Lucif-shRNA or U2AF1-shRNA. B, Representative images of flow cytometry analysis of the cell cycle as assessed by propidium iodide and 7AAD staining of cells on day 6 of culture. C, Quantitative analysis of the cell cycle from three independent experiments is shown. D, Representative images of flow cytometry analysis of apoptosis by Annexin V and 7AAD staining on day 6 of culture. E, Quantitative analysis of apoptosis from three independent experiments on day 6 of culture. F, Colony forming ability of sorted burst-forming-unit erythroid and colony-forming-unit erythroid cells. Statistical analysis of data from three independent experiments and bar plot representing means ± SD of triplicate samples. *P < 0.05, **P < 0.01, ***P < 0.001 RNA-seq data and found that 543 transcripts were differentially spliced in Lucif-shRNA and U2AF1-shRNA CFU-E cells. There were eight kinds of splicing events ( Figure 6A), and notable splicing events were exon skipping/inclusion (ESI), alternative transcription start site (ATSS) and alternative transcription termination site (ATTS) in CFU-E cells ( Figure 6B). To monitor alternative splicing, we analyzed the SNHG1, BAX, MTA1, EIF3B, HNRNPC and HNRNPD genes, which play an important role in apoptosis. As shown in Figure 6C, depletion of U2AF1 affected alternative splicing of these genes transcripts compared with Lucif-shRNA-transduced cells.

| D ISCUSS I ON
RNA splicing is an important post-transcriptional process during which introns are removed and exons are retained from pre-mRNA by spliceosomes. The U2AF family members U2AF1 and U2AF2 have been implicated in the regulation of splicing. It has been previously reported that the expression of the U2AF1a isoform is more abundant than U2AF1b in higher vertebrates. Additionally, the U2AF1c isoform is targeted by RNA surveillance for the introduction of a premature termination codon. 37 In addition, studies have demonstrated that U2AF1a is essential for HeLa cell division and that knockdown of U2AF1a, alone or in conjunction with U2AF1b, has a more significant effect than knockdown of U2AF1b alone. 24    increases p53-dependent apoptosis by impairing hnRNPC regulation of p53 activity. 46,47 In this study, the splicing patterns of these gene were found to be ESI, ISI, ATSS and ATTS. Our findings have implications for the understanding of the relationship between splicing factors and disordered erythropoiesis such as MDS.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflict of interest.