Pre-mRNA splicing regulation is critical for controlling macrophage activation

项目总结 尽管前信使核糖核酸剪接对基因表达结果有重大影响，但我们知之甚少。 关于剪接体本身是如何在细胞重新编程过程中被修改和调节的。先天免疫细胞 就像巨噬细胞在感觉到病原体等“危险信号”时会重新编程基因表达一样， 细胞器损伤或化学信号，以对抗检测到的威胁。虽然发生的变化 在巨噬细胞激活过程中转录的特征很好，几乎什么都不知道如何预 MRNA的剪接受免疫刺激的调节。这个项目的长期目标是揭示 巨噬细胞激活修饰剪接体，并将这些变化与先天免疫基因联系起来 表达结果。剪接体是一个复杂的、动态的大分子机器。它有能力 识别内含子并催化其去除依赖于大量识别特定基因的RNA结合蛋白 外显子和内含子中的序列来“阅读”剪接密码。这项提议的中心假设是 在巨噬细胞激活过程中，剪接因子的翻译后修饰指导着 专门的剪接体，其特征是具有一组不同的蛋白质-蛋白质相互作用，促进 先天免疫基因表达程序。为了支持这一模型，磷酸蛋白质组学实验表明 30个剪接因子，其中许多具有已知的调节作用，在特定的特定位置被磷酸化或去磷酸化 巨噬细胞依赖脂多糖激活后的丝氨酸残基。实验 询问这样一个因子hnRNP M表明，内毒素处理触发去磷酸化伴随着 它在细胞核内的重新分布。ShRNA介导的巨噬细胞击倒改变hnRNP M的丢失 许多前mRNAs的选择性剪接，并导致重要先天免疫的过度诱导 转录本，包括强有力的炎症介质IL-6和关键的病毒限制因子Mx1。这 Proposal对这些观察结果进行了扩展，在全球范围内研究了以下剪接体的变化 巨噬细胞激活。它将结合高通量方法，包括亲和纯化-质量 光谱学、磷蛋白质组学、RNA-seq和RNA片段-seq与靶向遗传和生化 涉及特定剪接因子驱动先天免疫基因表达变化的实验。这 研究计划将填补我们在巨噬细胞后如何调控剪接的关键知识空白 激活和加深我们对剪接体如何阅读和解释剪接密码的理解 在先天免疫激活期间，也在其他细胞重新编程期间，包括分化、应激、 饥饿和致癌。

PROJECT SUMMARY Despite the substantial impact pre-mRNA splicing has on gene expression outcomes, little is known about how the spliceosome itself is modified and regulated during cellular reprogramming. Innate immune cells like macrophages reprogram gene expression when they sense a “danger signal,” such as a pathogen, organelle damage, or chemical signal, to combat the detected threat. While changes that occur transcriptionally during macrophage activation are well characterized, almost nothing is known about how pre- mRNA splicing is regulated following immune stimuli. The long-term goal of this project is to uncover how macrophage activation modifies the spliceosome and to connect these changes with innate immune gene expression outcomes. The spliceosome is a complex and dynamic macromolecular machine. Its ability to recognize introns and catalyze their removal relies on numerous RNA binding proteins that recognize specific sequences in exons and introns to “read” the splicing code. The central hypothesis of this proposal is that during macrophage activation, post-translational modification of splicing factors directs assembly of a specialized spliceosome characterized by a distinct cohort of protein-protein interactions that promotes the innate immune gene expression program. In support of this model, phosphoproteomic experiments reveal that 30+ splicing factors, many with known regulatory roles, are phosphorylated or dephosphorylated at specific serine residues following lipopolysaccharide (LPS)-dependent activation of macrophages. Experiments interrogating one such factor, hnRNP M, show that LPS treatment triggers dephosphorylation concomitant with its redistribution in the nucleus. Loss of hnRNP M by shRNA-mediated knockdown in macrophages alters alternative splicing of a number of pre-mRNAs and leads to hyper-induction of important innate immune transcripts, including the potent inflammatory mediator IL-6 and the key viral restriction factor Mx1. This proposal expands upon these observations, looking globally at changes to the spliceosome following macrophage activation. It will combine high-throughput approaches, including affinity purification-mass spectrometry, phosphoproteomics, RNA-seq, and RNA CLIP-seq with targeted genetic and biochemical experiments to implicate specific splicing factors in driving innate immune gene expression changes. This research program will fill key gaps in our knowledge of how splicing is regulated following macrophage activation and further our understanding of how the spliceosome reads and interprets the splicing code not only during innate immune activation but also during other cellular reprogramming, including differentiation, stress, starvation, and carcinogenesis.