Pre-mRNA splicing regulation is critical for controlling macrophage activation

项目摘要 尽管前体mRNA剪接对基因表达结果有实质性影响， 关于剪接体本身在细胞重编程过程中是如何被修改和调节的。先天免疫细胞 就像巨噬细胞在感受到“危险信号”时，如病原体， 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 在巨噬细胞活化过程中的转录是很好的特点，几乎没有什么是已知的， mRNA剪接受免疫刺激调节。这个项目的长期目标是揭示 巨噬细胞活化修饰剪接体，并将这些变化与先天免疫基因 表达结果。剪接体是一个复杂的动态大分子机器。的能力 识别内含子并催化它们的去除依赖于许多RNA结合蛋白，这些蛋白识别特异性 外显子和内含子中的序列来“读取”剪接密码。这一提议的核心假设是， 在巨噬细胞活化过程中，剪接因子的翻译后修饰指导巨噬细胞的组装， 一种特殊的剪接体，其特征是一组独特的蛋白质-蛋白质相互作用， 先天免疫基因表达程序。为了支持这一模型，磷酸化蛋白质组学实验表明， 30+剪接因子，许多具有已知的调节作用，在特定的位置磷酸化或去磷酸化。 丝氨酸残基后脂多糖（LPS）依赖性活化的巨噬细胞。实验 对这样一种因子hnRNP M的研究表明，LPS处理引发了伴随着hnRNP M的去磷酸化， 它在细胞核中的再分布。巨噬细胞中通过shRNA介导的敲低引起的hnRNP M的丢失改变了 许多前体mRNA的选择性剪接，并导致重要的先天免疫的过度诱导， 转录，包括有效的炎症介质IL-6和关键的病毒限制因子Mx 1。这 一项提案扩展了这些观察结果，从全球范围内观察剪接体的变化， 巨噬细胞活化它将结合联合收割机高通量的方法，包括亲和纯化-质量 质谱、磷酸化蛋白质组学、RNA-seq和RNA CLIP-seq， 实验表明，特定的剪接因子在驱动先天免疫基因表达的变化。这 一项研究计划将填补我们对巨噬细胞吞噬后剪接如何调节的知识中的关键空白。 激活，并进一步了解剪接体如何读取和解释剪接密码，不仅 在先天免疫激活期间，也在其他细胞重编程期间，包括分化，应激， 饥饿和致癌作用。

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.