Supplementary MaterialsSupplemental data jci-127-91363-s001
April 22, 2021
Supplementary MaterialsSupplemental data jci-127-91363-s001. approximately 11% of patients with MDS (6, 17), making one of the most commonly mutated genes in this disease. Mutations in also occur in the closely related condition AML at a frequency of approximately 4% (18), as well as in lung adenocarcinoma and other cancers (18, 19). mutations are associated with a worse overall survival in MDS patients and a higher risk of transformation to AML (11, 20, 21). mutations almost exclusively occur in 2 highly conserved amino acid positions, S34 and Q157, within the 2 2 zinc finger domains of the protein (6). There is clear evidence in yeast showing that the zinc finger domains in U2AF1 recognize RNA (6). The high percentage of sequence identity in the zinc finger domains between yeast and human U2AF1 (6) suggests that the zinc finger domains in human U2AF1 are also RNA binding (22). The presence of missense mutational hotspots and the absence of nonsense/frameshift mutations suggest that mutations are gain-of-function or change-of-function/neomorphic mutations (6). Abnormal RNA splicing, with cassette exon splicing being the most frequent type of event, has been reported in bone tissue marrow examples from S34F mutant weighed against WT examples (18, 23, 25). Lately, Shirai et al. generated a doxycycline-inducible transgenic mouse model of S34F mutation displaying some phenotypes that are closely associated with MDS (26). This transgenic murine model sheds light around the role of this mutation in altering hematopoesis and pre-mRNA splicing in the mouse (26). An investigation of the lineage-specific effect of S34F mutation on human hematopoiesis could provide new insights into the molecular pathogenesis of S34F mutation exhibits lineage specificity in altering pre-mRNA splicing of downstream target genes, resulting in different phenotypes in the different myeloid lineages that are involved in MDS. Results Expression of U2AF1S34F in hematopoietic progenitors. To study the impact of the S34F mutation on erythroid and granulomonocytic differentiation, we first overexpressed the S34F mutant (WT (= 8). (F) Image of erythroid cell pellets on day 14 of culture for visual WIN 55,212-2 mesylate determination of hemoglobinization (= 8). (G) Number of BFU-E colonies obtained from hematopoietic CD34+ progenitors transduced with EV, = 7). Scale bars: 100 m. (I) Cell counts for values in panels BCD, G, and J were computed by repeated-measures 1-method ANOVA with Tukeys post-hoc check. values in -panel I were computed by 2-method ANOVA with Bonferronis post check. * 0.05, ** 0.01, and *** 0.001. Open up in another window Body 2 Appearance of U2AF1S34F skews granulomonocytic differentiation toward granulocytes.(A) Expression degrees of U2AF1S34F and U2AF1WT proteins in transduced granulomonocytic cells in time 11. Anti-U2AF1 and anti-FLAG antibodies had been utilized to measure total U2AF1 proteins and exogenous U2AF1S34F or RECA U2AF1WT proteins made by the vector, respectively. (B) Median fluorescence strength (MFI) of forwards scatter (a sign of cell size) of granulomonocytic cells on times 11 and 14. (C) Percentage of Compact disc11b+ cells in granulomonocytic civilizations on times 11 and 14. (D) Cell matters for = 7). (J) Consultant pictures of May-Grnwald-GiemsaCstained granulomonocytic cells on time 20 (= 7). The reddish colored arrows indicate eosinophils. Size pubs: 25 m. (K) Percentage of eosinophils per 100 cells on time 20. (L) Amount of CFU granulocytes-macrophages (CFU-GM), CFU granulocytes (CFU-G), and CFU macrophages (CFU-M) extracted from hematopoietic Compact disc34+ progenitors transduced with EV, beliefs in sections B, C, ECH, L and K were calculated by repeated-measures 1-method ANOVA with Tukeys post-hoc check. values in -panel D were computed by 2-method ANOVA WIN 55,212-2 mesylate with Bonferronis post check. * 0.05, ** 0.01, and *** 0.001. U2AF1S34F impairs erythroid differentiation. To research the result of S34F mutation, as WIN 55,212-2 mesylate assessed by pyrosequencing (Body 3A). Replicate MATS (rMATS), a computational device created for the recognition of differential substitute splicing from replicate RNA-seq data (31), was useful for RNA-seq data evaluation. A complete of 506 splicing occasions (347 genes) and 439 splicing occasions (300 genes) had been determined in WT (TCT) and S34F mutant (TTT) mRNA in erythroid and granulomonocytic colonies, dependant on pyrosequencing. (B and C) Aberrant splicing occasions connected with S34 mutations and erythroid colonies and granulomonocytic colonies inside our research; (I) beliefs in -panel B and C had been computed by Fishers specific check with Bonferronis modification. * 0.05 and ** 0.01. A3SS, substitute 3 splice site; A5SS, substitute 5 splice site; MXE, exclusive exon mutually; RI,.