Collaborative Research: Connecting the sequence logic of RNA splicing to nuclear localization

在真核细胞中，成熟的信使RNA(信使RNA)是由新转录的前信使RNA通过称为RNA剪接的关键步骤产生的。在剪接过程中，部分前信使核糖核酸(称为内含子)被移除，其余部分(外显子)连接到…上但并不是每个前-信使核糖核酸都去除了每个内含子。这个合作项目的目的是了解Pre-mRNA中决定特定mRNAs中哪些外显子被移除的序列。PI将部署高通量测试，其中数百万个序列被并行测试，并使用可解释的机器学习技术分析结果。该项目还将揭示有助于剪接决策的新机制。具体地说，它将揭示细胞核旁边的某些小体，即所谓的“核斑点”，在RNA剪接中所起的作用。此外，拟议的研究将为研究生和本科教育跨学科课程的开发和传播提供大量资源。该项目将为来自不同背景和不同教育阶段的学生和学者提供招聘、参与和职业发展。它将通过培训代表不足的少数群体以及女研究生和博士后研究人员，增加代表不足的群体对生物和计算研究和教育的参与。该项目还将包括对来自不同背景的高中生和本科生的推广。RNA剪接以依赖于序列的方式发生，遵循“剪接代码”中编码的逻辑。除了剪接反应所需的保守序列(5‘和3’剪接位点、分支点)外，剪接调控元件中还编码了额外的逻辑。这些序列元件被各种剪接因子蛋白识别，包括SR蛋白和异质性核糖核蛋白(HnRNPs)。SR蛋白和hnRNP蛋白以序列和上下文相关的方式调节剪接决定。具体地说，如果SR蛋白的结合基序位于外显子，则SR蛋白倾向于促进剪接；如果SR蛋白的结合基序位于内含子，则倾向于抑制剪接；而hnRNP蛋白则具有相反的作用。然而，上下文依赖的监管效应背后的机制仍有待充分揭示。有趣的是，剪接因子相对于核斑点显示出不同的亚细胞位置；SR蛋白富含在核斑点中，而hnRNP蛋白均匀分布在核质中。这些观察结果使我们假设，RNA相对于核斑点的定位在序列特征和剪接结果之间起到了桥梁作用。本项目将检验这一假设，并通过结合两种互补的方法来探索剪接的机制和核斑点的功能：(1)使用基于可解释的机器学习的大规模平行报告分析(MPRA)来确定序列和剪接结果之间的关系；(2)通过成像RNA底物、剪接体成分和剪接因子来表征序列和相对于核斑点的定位之间的关系。这项研究将揭示有助于剪接决策的新机制，特别是通过序列编码的斑点内RNA定位。该项目由分子和细胞生物科学部的细胞动力学和功能和遗传机制计划共同资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

In eukaryotic cells, mature messenger RNAs (mRNA) are generated from newly transcribed pre-mRNA through an essential processing step called RNA splicing. During splicing, parts of the pre-mRNA (known as introns) are removed, and the remaining parts (exons) are joined together…but not every intron is removed from every pre-mRNA. This collaborative project aims to understand the sequences in pre-mRNA that determine which exons are removed in specific mRNAs. The PIs will deploy high-throughput assays where millions of sequences are tested in parallel, and analyzing the results using interpretable machine learning techniques. This project will also reveal novel mechanisms contributing to splicing decisions. Specifically, it will reveal the role that certain small bodies next to the nucleus, known as “nuclear speckles,” play in RNA splicing. In addition, the proposed research will provide substantial resources for the development and dissemination of an interdisciplinary curriculum for graduate and undergraduate education. The project will offer recruitment, involvement, and career development of students and scholars from diverse backgrounds and at different educational stages. It will increase the participation of underrepresented groups in biological and computational research and education via the training of underrepresented minorities and female graduate and postdoctoral researchers. The project will also include outreach to high school students and undergraduates from diverse backgrounds.RNA splicing occurs in a sequence-dependent manner following logic encoded in the “splicing code”. Besides the conserved sequences (5’ and 3’ splice sites, branchpoint) that are required for the splicing reaction, additional logic is encoded in splicing regulatory elements. These sequence elements are recognized by various splicing factor proteins, including SR proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs). SR proteins and hnRNP proteins regulate splicing decisions in a sequence- and context-dependent manner. Specifically, SR proteins tend to promote splicing if their binding motifs are in exons and tend to repress splicing if their binding motifs are in introns; whereas hnRNP proteins have the opposite effect. However, the mechanisms underlying context-dependent regulatory effects remain to be fully revealed. Interestingly, splicing factors exhibit distinct subcellular locations relative to nuclear speckles; SR proteins are enriched in nuclear speckles, whereas hnRNP proteins are uniformly distributed in the nucleoplasm. These observations lead us to hypothesize that positioning of RNAs with respect to nuclear speckles serves as a bridge between sequence features and splicing outcomes. This project will test this hypothesis, and explore the mechanisms of splicing and the function of nuclear speckles by combining two complementary approaches: (1) Identifying the relationship between sequences and splicing outcomes using interpretable machine learning-based analysis of massively parallel reporter assays (MPRA); (2) Characterizing the relationship between sequences and positioning relative to nuclear speckles through imaging RNA substrates, spliceosomal components, and splicing factors. This research will reveal novel mechanisms contributing to splicing decision, specifically through sequence-encoded intra-speckle RNA positioning.This project is co-funded by the Cellular Dynamics and Function and Genetic Mechanisms programs of the Molecular and Cellular Biosciences Division.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.