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

在真核细胞中，成熟的信使RNA（mRNA）通过称为RNA剪接的基本加工步骤从新转录的前mRNA产生。 在剪接过程中，前体mRNA的一部分（称为内含子）被移除，其余部分（外显子）连接在一起......但不是每个内含子都从每个前体mRNA中移除。 这个合作项目旨在了解前mRNA中的序列，这些序列决定了特定mRNA中哪些外显子被去除。 PI将部署高通量检测，并行测试数百万个序列，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定的新机制。 具体来说，它将揭示某些靠近细胞核的小物体（称为“核斑点”）在RNA剪接中的作用。 此外，拟议的研究将为研究生和本科生教育的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。 它将通过培训代表性不足的少数民族和女性研究生和博士后研究人员，增加代表性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大学生的外联活动。RNA剪接以依赖于序列的方式发生，遵循“剪接密码”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蛋白和异质核核糖核蛋白（hnRNP）。 SR蛋白和hnRNP蛋白以序列和上下文依赖的方式调节剪接决定。 具体而言，SR蛋白倾向于促进剪接，如果它们的结合基序是在外显子和倾向于抑制剪接，如果它们的结合基序是在内含子，而hnRNP蛋白具有相反的效果。 然而，背景依赖性调控效应的机制仍有待充分揭示。 有趣的是，剪接因子表现出不同的亚细胞位置相对于核斑点; SR蛋白富集在核斑点，而hnRNP蛋白均匀分布在核质。 这些观察使我们假设RNA相对于核斑点的定位作为序列特征和剪接结果之间的桥梁。 本项目将验证这一假设，并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）使用基于可解释机器学习的大规模并行报告分析（MPRA）来识别序列和剪接结果之间的关系;（2）通过RNA底物、剪接体组分和剪接因子的成像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定的新机制，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以及遗传机制项目共同资助。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.