Genetic analysis of human RNA binding motif protein 48 (RBM48) reveals an essential role in U12-type intron splicing
Genetic analysis of human RNA binding motif protein 48 (RBM48) reveals an essential role in U12-type intron splicing
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DOI：
10.1093/genetics/iyac129
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发表时间：
2022-08-30
期刊：
影响因子：
3.3
通讯作者：
Lal，Shailesh
中科院分区：
文献类型：
--
作者：
Siebert，Amy E.;Corll，Jacob;Lal，Shailesh
U12型或次要内含子存在于大多数多细胞真核生物中，在具有次要剪接体的物种中占所有内含子的约0.5%。虽然U12型内含子的进化保守性的生物学意义是有争议的，突变破坏U12剪接在植物和动物中引起发育缺陷。在人类造血干细胞中，U12剪接缺陷破坏了骨髓谱系的正确分化，并与骨髓增生异常综合征相关，使个体易患急性髓性白血病。玉米RNA结合基序蛋白48（RBM 48）直系同源物中的突变体具有异常的U12型内含子剪接。人RBM 48最近被生物化学纯化为次要剪接体的一部分，并显示出识别U6 atac snRNA的5′端。在这份报告中，我们使用CRISPR/Cas9介导的人K-562细胞中RBM 48的消融来显示RBM 48的遗传功能。RNA-seq分析比较野生型和突变型K-562基因型发现，在RBM 48突变体中，48%的含有次要内含子的基因具有显著的U12型内含子保留。将这些结果与玉米bm 48突变体进行比较，确定了在两个物种中被破坏的含有次要内含子的基因的子集。大多数这些含有正向小内含子的基因的突变已被报道会导致植物和动物的发育缺陷。我们的研究结果提供了遗传学证据，证明人类RBM 48突变体的主要缺陷是异常U12型内含子剪接，而人类和玉米RNA-seq数据的比较确定了可能介导U12型剪接缺陷突变表型的候选基因。
U12-type or minor introns are found in most multicellular eukaryotes and constitute ∼0.5% of all introns in species with a minor spliceosome. Although the biological significance for the evolutionary conservation of U12-type introns is debated, mutations disrupting U12 splicing cause developmental defects in both plants and animals. In human hematopoietic stem cells, U12 splicing defects disrupt proper differentiation of myeloid lineages and are associated with myelodysplastic syndrome, predisposing individuals to acute myeloid leukemia. Mutants in the maize ortholog of RNA binding motif protein 48 (RBM48) have aberrant U12-type intron splicing. Human RBM48 was recently purified biochemically as part of the minor spliceosome and shown to recognize the 5′ end of the U6atac snRNA. In this report, we use CRISPR/Cas9-mediated ablation ofRBM48in human K-562 cells to show the genetic function of RBM48. RNA-seq analysis comparing wild-type and mutant K-562 genotypes found that 48% of minor intron-containing genes have significant U12-type intron retention inRBM48mutants. Comparing these results to maizerbm48mutants defined a subset of minor intron-containing genes disrupted in both species. Mutations in the majority of these orthologous minor intron-containing genes have been reported to cause developmental defects in both plants and animals. Our results provide genetic evidence that the primary defect of humanRBM48mutants is aberrant U12-type intron splicing, while a comparison of human and maize RNA-seq data identifies candidate genes likely to mediate mutant phenotypes of U12-type splicing defects.