Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing in Transgenic Mice
Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing in Transgenic Mice
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突变体 U2AF1 表达改变转基因小鼠的造血和前体 mRNA 剪接
DOI：
10.1182/blood.v124.21.827.827
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发表时间：
2014
期刊：
影响因子：
20.3
通讯作者：
M. Walter
中科院分区：
文献类型：
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作者：
C. Shirai;J. Ley;B. S. White;J. Tibbitts;J. Shao;M. Ndonwi;Sanghyun P. Kim;Brian A. Wadugu;Theresa Okeyo;T. Graubert;M. Walter
我们的研究小组和其他研究人员发现，11%的骨髓增生异常综合征（MDS）患者的U2 AF 1杂合错义突变复发。U2 AF 1基因编码一个参与内含子3 '剪接位点识别的剪接因子，这表明前体mRNA剪接的扰动在MDS发病机制中起作用。为了研究最常见的U2 AF 1突变U2 AF 1（S34 F）对体内造血和前体mRNA剪接的影响，我们建立了位点特异性、单拷贝、强力霉素诱导的U2 AF 1（WT）和U2 AF 1（S34 F）转基因小鼠。为了研究突变体U2 AF 1（S34 F）的细胞自主效应，我们在诱导转基因表达之前将转基因供体骨髓移植到野生型受体小鼠中。转基因诱导4周后，与仅U2 AF 1（WT）和rtTA受体对照组相比，U2 AF 1（S34 F）受体小鼠外周血中的总WBC减少（分别为4.3 vs 7.11和7.13 K/µl，p≤0.01），但骨髓细胞结构或脾脏大小无显著变化（n=9-11）。与对照小鼠（n=9-11）相比，U2 AF 1（S34 F）受体小鼠的成熟细胞谱系分布受到干扰，包括外周血（p≤0.05）和骨髓（p≤0.01）中单核细胞和B细胞减少。骨髓单核细胞减少早在5天发生，与对照组相比，与膜联蛋白V+（p≤0.05）和磷酸化H2 AX（p≤0.05）增加相关，表明这些细胞的丢失可能是由于细胞凋亡。此外，与仅U2 AF 1（WT）和rtTA对照组相比，通过CFU-C甲基纤维素测定和流式细胞术检测c-Kit+/谱系-细胞以及共同髓系祖细胞（CMP），U2 AF 1（S34 F）受体小鼠骨髓和脾脏中祖细胞数量增加（p≤0.05，n=5-10）。通过流式细胞术测量的骨髓KLS（c-Kit+/Lineage-/Sca-1+）细胞，U2 AF 1（S34 F）受体小鼠的骨髓造血干细胞（HSC）频率也增加（p≤0.05）。早在5天就观察到U2 AF 1（S34 F）受体小鼠中骨髓KLS细胞的增加，并且与U2 AF 1（WT）对照相比，与KLS细胞中更高水平的细胞内Ki 67（细胞增殖的标志物）相关（体内p <0.05）。接下来，我们在U2 AF 1（S34 F）-和U2 AF 1（WT）-移植的小鼠（各n=3）中转基因诱导5天后对分选的骨髓CMP进行无偏RNA测序。我们鉴定了460个剪接点，与U2 AF 1（WT）对照（FDR U2 AF 1突变）相比，U2 AF 1（S34 F）样品中差异表达。为了优先考虑改变的连接以进行进一步分析，我们用来自具有和不具有U2 AF 1突变的AML患者样品以及过表达U2 AF 1（S34 F）或U2 AF 1（WT）的原代人CD 34+细胞的RNA测序数据对小鼠CMP连接结果进行了分析。在所有3个数据集中，我们在已知参与癌症和干细胞生物学的2个基因中发现了同源失调的连接：H2 AFY和MED 24。我们使用具有突变型U2 AF 1（S34 F）与U2 AF 1（WT）的MDS患者骨髓样品通过RT-PCR验证了H2 AFY和MED 24同种型表达的一致变化（p）。总之，这些结果表明突变型U2 AF 1表达有助于在具有U2 AF 1突变的患者中观察到的改变的造血和前mRNA剪接。本研究还确定了U2 AF 1突变所特有的基因亚型表达的变化，这些变化可能对MDS发病机制具有功能意义。没有相关的利益冲突需要申报。
Our group and others discovered recurrent heterozygous missense mutations in U2AF1 in 11% of patients with myelodysplastic syndromes (MDS). The U2AF1 gene encodes a splicing factor involved in intronic 3’-splice site recognition, which suggests that perturbations in pre-mRNA splicing play a role in MDS pathogenesis. To study the effects of the most common U2AF1 mutation, U2AF1(S34F), on hematopoiesis and pre-mRNA splicing in vivo , we created site-specific, single-copy, doxycycline-inducible U2AF1(WT) and U2AF1(S34F) transgenic mice. To examine the cell-autonomous effects of mutant U2AF1(S34F), we transplanted transgenic donor bone marrow into wild type recipient mice prior to induction of transgene expression. Following 4 weeks of transgene induction, U2AF1(S34F)-recipient mice have reduced total WBCs in the peripheral blood compared to U2AF1(WT)- and rtTA only-recipient controls (4.3 vs 7.11 and 7.13 K/µl, respectively, p≤0.01), but no significant changes in bone marrow cellularity or spleen size (n=9-11). U2AF1(S34F)-recipient mice have a perturbed mature cell lineage distribution, including reduced monocytes and B cells in both peripheral blood (p≤0.05) and bone marrow (p≤0.01) when compared to control mice (n=9-11). Reduction of bone marrow monocytes occurs as early as 5 days and is associated with increased Annexin V+ (p≤0.05) and phospho-H2AX (p≤0.05) compared to controls, suggesting loss of these cells may be due to apoptosis. In addition, U2AF1(S34F)-recipient mice have increased numbers of progenitors in both bone marrow and spleen by CFU-C methylcellulose assay and flow cytometry for c-Kit+/Lineage- cells, as well as common myeloid progenitors (CMPs), when compared to U2AF1(WT) and rtTA only controls (p≤0.05, n=5-10). U2AF1(S34F)-recipient mice also have an increase in the frequency of bone marrow hematopoietic stem cells (HSCs) measured by flow cytometry for bone marrow KLS (c-Kit+/Lineage-/Sca-1+) cells (p≤0.05). The increase in bone marrow KLS cells in U2AF1(S34F)-recipient mice is seen as early as 5 days and is associated with higher levels of intracellular Ki67 (a marker of cell proliferation) in KLS cells compared to U2AF1(WT) controls (p in vivo . Next, we performed unbiased RNA sequencing on sorted bone marrow CMPs following 5 days of transgene induction in U2AF1(S34F)- and U2AF1(WT)-transplanted mice (n=3 each). We identified 460 splicing junctions that were differentially expressed in U2AF1(S34F) samples compared to U2AF1(WT) controls (FDR U2AF1 mutations. To prioritize altered junctions for further analysis, we intersected mouse CMP junction results with RNA sequencing data from AML patient samples with and without U2AF1 mutations and primary human CD34+ cells over-expressing U2AF1(S34F) or U2AF1(WT). Across species and present in all 3 datasets, we identified homologous dysregulated junctions in 2 genes known to be involved in cancer and stem cell biology: H2AFY and MED24 . We validated concordant changes in both H2AFY and MED24 isoform expression by RT-PCR using MDS patient bone marrow samples that have mutant U2AF1(S34F) versus U2AF1(WT) (p Together, these results suggest that mutant U2AF1 expression contributes to the altered hematopoiesis and pre-mRNA splicing observed in patients with U2AF1 mutations. This study also identifies changes in gene isoform expression unique to U2AF1 mutations that may have functional significance for MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.