Institute of Biochemistry, Justus Liebig University of Giessen, D-35392 Giessen, Germany.
Nucleic Acids Res. 2012 Jul;40(12):5666-78. doi: 10.1093/nar/gks221. Epub 2012 Mar 8.
CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4-6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4-6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons.
CD45 编码一种跨膜蛋白酪氨酸磷酸酶，表达于免疫系统的多种细胞中。通过三个可变外显子 4-6 的组合使用，生成了在其细胞外结构域中存在差异的同工型，从而调节磷酸酶活性和免疫反应。这些 CD45 外显子的选择性剪接涉及两种异质核糖核蛋白，hnRNP L 和其细胞类型特异性的同源蛋白 hnRNP L-like（LL）。为了解决外显子 4-6 的复杂组合剪接问题，我们研究了人类 B 细胞中 hnRNP L/LL 蛋白表达与 CD45 剪接模式的关系，应用了 RNA-Seq。此外，还在 HeLa 细胞中进行了突变和 RNA 结合分析。我们得出结论，hnRNP LL 作为主要的 CD45 剪接抑制剂，其主要靶标是外显子 6 中的两个 CA 元件。在外显子 4 中，一个元件同时被 hnRNP L 和 LL 靶向。相比之下，外显子 5 从未被单独抑制，仅与外显子 4 和 6 共同调节。L/LL 稳定相互作用需要 CD45 RNA，特别是外显子 4 和 6。我们提出了一个新的组合选择性剪接模型：hnRNP L 和 LL 在 CD45 前体 RNA 上合作，桥接外显子 4 和 6，并环出外显子 5，从而实现三个可变外显子的完全抑制。
