Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 12;11(1):19.
doi: 10.1186/s13045-018-0558-8.

SF3B1 deficiency impairs human erythropoiesis via activation of p53 pathway: implications for understanding of ineffective erythropoiesis in MDS

Yumin Huang  1   2 , John Hale  3 , Yaomei Wang  2   4 , Wei Li  2   4   5 , Shijie Zhang  4 , Jieying Zhang  2   6 , Huizhi Zhao  4 , Xinhua Guo  2 , Jing Liu  6 , Hongxia Yan  3 , Karina Yazdanbakhsh  7 , Gang Huang  8 , Christopher D Hillyer  3 , Narla Mohandas  3 , Lixiang Chen  9 , Ling Sun  10 , Xiuli An  11   12   13
Affiliations

SF3B1 deficiency impairs human erythropoiesis via activation of p53 pathway: implications for understanding of ineffective erythropoiesis in MDS

Yumin Huang et al. J Hematol Oncol. .

Abstract

Background: SF3B1 is a core component of splicing machinery. Mutations in SF3B1 are frequently found in myelodysplastic syndromes (MDS), particularly in patients with refractory anemia with ringed sideroblasts (RARS), characterized by isolated anemia. SF3B1 mutations have been implicated in the pathophysiology of RARS; however, the physiological function of SF3B1 in erythropoiesis remains unknown.

Methods: shRNA-mediated approach was used to knockdown SF3B1 in human CD34+ cells. The effects of SF3B1 knockdown on human erythroid cell differentiation, cell cycle, and apoptosis were assessed by flow cytometry. RNA-seq, qRT-PCR, and western blot analyses were used to define the mechanisms of phenotypes following knockdown of SF3B1.

Results: We document that SF3B1 knockdown in human CD34+ cells leads to increased apoptosis and cell cycle arrest of early-stage erythroid cells and generation of abnormally nucleated late-stage erythroblasts. RNA-seq analysis of SF3B1-knockdown erythroid progenitor CFU-E cells revealed altered splicing of an E3 ligase Makorin Ring Finger Protein 1 (MKRN1) and subsequent activation of p53 pathway. Importantly, ectopic expression of MKRN1 rescued SF3B1-knockdown-induced alterations. Decreased expression of genes involved in mitosis/cytokinesis pathway including polo-like kinase 1 (PLK1) was noted in SF3B1-knockdown polychromatic and orthochromatic erythroblasts comparing to control cells. Pharmacologic inhibition of PLK1 also led to generation of abnormally nucleated erythroblasts.

Conclusions: These findings enabled us to identify novel roles for SF3B1 in human erythropoiesis and provided new insights into its role in regulating normal erythropoiesis. Furthermore, these findings have implications for improved understanding of ineffective erythropoiesis in MDS patients with SF3B1 mutations.

Keywords: Apoptosis; Human erythropoiesis; P53; SF3B1.

PubMed Disclaimer

Conflict of interest statement

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Effects of SF3B1 knockdown on proliferation of erythroid progenitors. a qRT-PCR results showing SF3B1 mRNA expression levels in erythroblasts transduced with lentivirus containing luciferase-shRNA or SF3B1-shRNA, and cultured for 6 days. β-actin was used as internal calculator. Bar plot represents mean ± SD of triplicate samples. b Representative western blotting showing SF3B1 protein levels in erythroblasts transduced with lentivirus containing luciferase-shRNA or SF3B1-shRNA, and cultured for 6 days. c Growth curves of cells transduced with lentivirus containing luciferase-shRNA or SF3B1-shRNA. d Representative flow cytometry profiles of apoptosis as assessed by dual staining of Annexin V and 7AAD at day 6 of culture. e Quantitative analysis of apoptosis from three independent experiments. f Colony-forming ability of cells cultured for 6 days, which contain mixed populations of cells that include BFU-E cells, CFU-E cells, and proerythroblasts. g Colony-forming ability of sorted BFU-E and CFU-E cells using IL-3R, CD34, and CD36 as surface markers [30]. ***P < 0.001
Fig. 2
Fig. 2
Effects of SF3B1 knockdown on terminal erythroid differentiation. a Flow cytometry analysis showing the percentage of GPA-positive cells on day 7. b Flow cytometry analysis showing the expression of α4 integrin and band 3 of erythroid cells cultured for different days as indicated. c Growth curves of cells transduced with lentivirus containing luciferase-shRNA or SF3B1-shRNA. d Representative flow cytometry profiles of apoptosis as assessed by dual staining of Annexin V and 7AAD of cells cultured for 9 days. e Quantitative analysis of apoptosis from three independent experiments. f Representative flow cytometry profiles of cell cycle as assessed by EdU and 7AAD staining of cells cultured for 7 days. g Quantitative analysis of cell cycle from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
Fig. 3
Fig. 3
Generation of erythroblasts with abnormal nucleus upon SF3B1 knockdown. a Representative cytospin images of erythroblasts cultured for 16 days. b Quantitative analysis of abnormally nucleated erythroblasts cultured for 16 days from three independent experiments. c Representative cytospin images of sorted erythroblasts at each distinct developmental stage. d Quantitative analysis of abnormally nucleated polychromatic and orthochromatic erythroblasts from three independent experiments. Scale bar 10 μm. *P < 0.05, **P < 0.01
Fig. 4
Fig. 4
Activation of p53 pathway in SF3B1-knockdown CFU-E cells. a The top 5 up- and downregulated pathways revealed by GSEA analysis of the differentially expressed genes between luciferase and SF3B1 knockdown CFU-E cells. b The mRNA levels of TP53, BAX, p21, and BBC3 revealed by RNA-seq analysis. c The mRNA levels of TP53, BAX, p21, and BBC3 revealed by real-time PCR analysis; β-actin was used as internal control. *P < 0.05, **P < 0.01, ***P < 0.01. d Representative western blotting showing the expression of P53, BAX, p21, and BBC3 in erythroblasts cultured for 7 days
Fig. 5
Fig. 5
Effects of SF3B1 knockdown on splicing in CFU-E cells. a The most frequent splicing events by type after SF3B1 knockdown: ESI, exon skipping/inclusion; MESI, multiple exon skipping/inclusion; ISI, intron skipping/inclusion; A5, alternative 5′ splice site; A3, alternative 3′ splice site; ATSS, alternative transcription start site; ATTS, alternative transcription termination site; MEE, mutually exclusive exons. b Expression of differentially spliced transcripts checked by real-time PCR. c Schematic expression of two MKRN1 transcripts. d Bar graphs showing the large and small isoforms of MKRN1 in luciferase control groups and SF3B1 knockdown groups by the analysis of RNA-seq data. Dot plots represent 3 independent experiments
Fig. 6
Fig. 6
Activation of p53 pathway is due to decreased expression of large isoform of MKRN1. a Representative western blotting showing the expression of SF3B1 and MKRN1 large isoform in cells transduced with luciferase-shRNA and SF3B1-shRNA. b Growth curves of cells transduced with luciferase-shRNA, SF3B1-shRNA2/pGFP, and SF3B1-shRNA2/pGFP-MKRN1. c Quantitative analysis of apoptosis of cells transduced with luciferase-shRNA, SF3B1-shRNA2/pGFP, and SF3B1-shRNA2/pGFP-MKRN1. d Representative western blot analysis showing the expression of p53, BAX, p21, and BBC3 in cells transduced with luciferase-shRNA, SF3B1-shRNA2/pGFP, and SF3B1-shRNA2/pGFP-MKRN1
Fig. 7
Fig. 7
Downregulation of mitosis/cytokinesis pathway in polychromatic and orthochromatic erythroblasts. a RNA-seq expression data of RACGAP1, PRC1, and PLK1 in luciferase and SF3B1 knockdown polychromatic and orthochromatic erythroblasts. Dot plots represent 3 independent experiments. b mRNA expression levels of RACGAP1, PRC1, and PLK1 as assessed by real-time PCR using β-actin as internal calibrator. c Representative images of morphology from DMSO and PLK1 inhibitor treated cells. d Quantification of abnormally nucleated erythroblasts from three independent experiments. **P < 0.01
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ginzburg Y, Rivella S. β-thalassemia: a model for elucidating the dynamic regulation of ineffective erythropoiesis and iron metabolism. Blood. 2011;118:4321–4330. doi: 10.1182/blood-2011-03-283614. - DOI - PMC - PubMed
    1. Wickramasinghe SN, Wood WG. Advances in the understanding of the congenital dyserythropoietic anaemias. Br J Haematol. 2005;131:431–446. doi: 10.1111/j.1365-2141.2005.05757.x. - DOI - PubMed
    1. Lipton JM, Ellis SR. Diamond-Blackfan anemia: diagnosis, treatment, and molecular pathogenesis. Hematol Oncol Clin North Am. 2009;23:261–282. doi: 10.1016/j.hoc.2009.01.004. - DOI - PMC - PubMed
    1. Haldar K, Mohandas N. Malaria, erythrocytic infection, and anemia. Hematol Am Soc Hematol Educ Progr. 2009;2009:87–93. - PMC - PubMed
    1. Ebert BL, Galili N, Tamayo P, Bosco J, Mak R, Pretz J, et al. An erythroid differentiation signature predicts response to lenalidomide in myelodysplastic syndrome. Appelbaum FR, editor. PLoS Med. 2008;5:e35. - PMC - PubMed

Publication types

MeSH terms