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. 2019 Aug;23(8):5076-5086.
doi: 10.1111/jcmm.14370. Epub 2019 May 29.

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

Jieying Zhang  1 , Huizhi Zhao  2 , Kunlu Wu  1 , Yuanliang Peng  1 , Xu Han  1 , Huan Zhang  2 , Long Liang  1 , Huiyong Chen  1 , Jingping Hu  1 , Xiaoli Qu  2 , Shijie Zhang  2 , Lixiang Chen  2 , Jing Liu  1
Affiliations

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

Jieying Zhang et al. J Cell Mol Med. 2019 Aug.

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.

Keywords: U2AF1; erythropoiesis; spliceosome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of U2AF1 during human erythroid differentiation. A, RNA‐seq data showing the expression of three different isoforms of U2AF1 at each distinct stage of human erythroid differentiation. B, Representative image of Western blotting showing U2AF1 protein expression levels on days 4, 7, 11 and 15 of culture. C, Two U2AF1 shRNA target regions. D, qRT‐PCR results showing U2AF1 mRNA expression in erythroblasts transduced with lentivirus containing Lucif‐shRNA or U2AF1‐shRNA on day 6 of culture. Actin was used as an internal control. E, Representative image of Western blotting showing U2AF1 protein expression levels in erythroblasts transduced with lentivirus containing Lucif‐shRNA or U2AF1‐shRNA on day 6 of culture (left panel). Quantitative analysis of protein expression levels from three independent experiments is shown (right panel). Actin was used as a loading control. Statistical analysis of data from three independent experiments and bar plot representing means ± SD of triplicate samples. ***P < 0.001
Figure 2
Figure 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
Figure 3
Figure 3
Effects of U2AF1 knockdown on terminal erythroid differentiation. A, Representative images of flow cytometry showing GPA expression in erythroblasts infected with Lucif‐shRNA or U2AF1‐shRNA on day 7 of culture. B, Flow cytometry analysis of band 3 and a4 integrin expression on different days in GPA+ erythroid cells infected with Lucif‐shRNA or U2AF1‐shRNA. C, Growth curves of erythroid cells transduced with LucifshRNA or U2AF1‐shRNA determined by cell counting. D, Quantitative analysis of apoptosis by Annexin V and 7AAD staining in erythroblasts infected with Lucif‐shRNA or U2AF1‐shRNA. Bar plot represents means ± SD of triplicate samples. *P < 0.05, **P < 0.01
Figure 4
Figure 4
Knockdown of U2AF1 impairs enucleation and leads to the generation of erythroblasts with the abnormal nucleus. A, Representative profiles of enucleation as assessed by Syto‐16 staining on day 15 and day 17 of culture. B, Quantitative analysis of enucleation on indicated days from three independent experiments. C, Representative cytospin images of erythroblasts cultured for 15 d. D Quantitative analysis of abnormally nucleated erythroblasts at day 13, day 15 of culture from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Figure 5
Figure 5
Knockdown of U2AF1 affected gene expression in colony‐forming‐unit erythroid (CFU‐E) cells. RNA‐seq data were processed by the BGI standard and showed differentially expressed genes between Lucif‐shRNA and U2AF1‐shRNA. The differentially expressed genes were analysed as an FDR < 0.05 and fold change ≥2. Hierarchical clustering of differentially expressed genes that were up‐regulated at least 2.0‐fold (A) and down‐regulated at least 2.0‐fold (B) between Luciferase and U2AF1 knockdown CFU‐E cells in three independent experiments. Every gene is coloured according to its average mRNA expression level. The differentially expressed genes involved in the pathway are indicated in the right panel. C, Heatmap showing the GSEA analysis of the differentially expressed genes between Luciferase and U2AF1 knockdown CFU‐E cells. D, Representative images of Western blotting showing the expression of p53, BAX, Bcl‐2, BBC3 and p21 in erythroblasts on day 6 of culture (left panel). Quantitative analysis of protein expression levels from three independent experiments is shown (right panel). *P < 0.05, **P < 0.01, ***P < 0.001
Figure 6
Figure 6
Knockdown of U2AF1 affected the alternative splicing of apoptosis‐associated genes in CFU‐E cells. A, The kinds of splicing events. B, The most frequent splicing events after U2AF1 knockdown: exon skipping/inclusion (ESI), multiple exon skipping/inclusion (MESI), intron skipping/inclusion (ISI), alternative 5’ splice site (A5), alternative 3’ splice site (A3), alternative transcription start site (ATSS), alternative transcription termination site (ATTS) and mutually exclusive exons (MEE). C, Schematic expression of differentially spliced transcripts.
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