Role of the U-12 dependent Minor Spliceosome in Early Embryo Development and Brain Disease

U-12 依赖性小剪接体在早期胚胎发育和脑疾病中的作用

基本信息

批准号：
10493118
负责人：
Taylor Floyd
金额：
$ 2.01万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-30 至 2022-11-01
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10493118
关键词：
5&apos Splice Site Ablation Adult Affect Age Alternative Splicing Antibodies Ataxia Automobile Driving Biological Brain Brain Diseases Brain region Breeding Candidate Disease Gene Cell Cycle Cerebellar Ataxia Cerebellum Cessation of life Child Code Data Development Developmental Delay Disorders Developmental Gene Disease Elements Embryo Embryonic Development Engineering Environment Eukaryota Event Excision Exhibits Experimental Designs Failure Functional disorder Gene Expression Genes Genetic Transcription Genetic Variation Genome Genotype Harvest Health Hippocampus (Brain)Human Human Development Image Impairment In Situ Hybridization Individual Introns Knowledge Lead Leukocytes Macromolecular Complexes Mammals Mediating Messenger RNA Methods Minor Molecular Mus Mutant Strains Mice Mutation Nucleotides Ontology Organism Partner in relationship Pathway interactions Patients Pattern Peutz-Jeghers Syndrome Point Mutation Process Protein Export Pathway Protein Isoforms Proteins Quantitative Reverse Transcriptase PCR RNA RNA Splicing Reporting Reverse Transcriptase Polymerase Chain Reaction Role STK11 gene Site Small Nuclear RNA Spliced Genes Spliceosomes Stains Syndrome System Techniques Technology Testing Time Tissues Transcript Transgenic Mice Uridine Variant Work base consanguineous family disease phenotype early onset embryo tissue genome editing growth hormone deficiency human disease indexing insight interest mRNA Precursor mouse model mutant next generation sequencing novel osteodysplastic primordial dwarfism postnatal tool transcriptome transcriptome sequencing transcriptomics

项目摘要

ABSTRACT Alternative splicing is an imperative process that contributes to cellular specialization and systems complexity of higher organisms. In mammals, alternative splicing is controlled by two macromolecular complexes: the major (U2-dependent) and minor (U12-dependent) spliceosomes. While the U2-mediated spliceosome has been extensively investigated, the U12-mediated spliceosome remains little understood. Recent studies in mice indicate that loss of one minor spliceosome component causes early embryonic lethality. Although complete absence of any minor spliceosome units has yet to be observed in humans, there are 9 disease phenotypes associated with minor spliceosome dysfunction. Our lab was the first to report that a C84T nucleotide switch in RNU12 causes human Early-Onset Cerebellar Ataxia (EOCA) and developmental delay. RNU12 encodes the uridine-rich U12 small nuclear RNA (snRNA), which initiates minor spliceosome function through intron recognition in pre-mRNAs. Leukocytes from homozygous RNU12C84T/C84T patients exhibited aberrant expression of ataxia-related minor intron-containing genes (MIGs) and elevated intron retention, implicating an association between deficient minor intron splicing and EOCA. Our preliminary data suggests that the RNU12 C84T variant impairs U12-mediated splicing of cerebellar-specific pre-mRNAs, while complete RNU12 absence causes early embryonic lethality due to aberrant splicing of key early developmental genes. This hypothesis will be tested using next generation sequencing techniques, transcriptomics, embryo and brain development studies in novel gene-edited mouse models, which contain either the C84T variant in mRnu12 (mRnu1284T), or a 79bp deletion inactivating U12 snRNA (mRnu12—). AIM 1 will assess the role of the C84T RNU12 mutation in selective mis-splicing of cerebellar transcripts through bulk RNA-sequencing of the cerebellum, contrasted with cortex and hippocampus of mRnu1284T/84T mice and controls at different developmental timepoints. Using transcriptomic tools, we will evaluate changes in gene expression, isoform usage, minor intron retention and splice site shift. Candidate transcripts will be validated across genotypes, tissues and development using qRT-PCR and in situ hybridization methods. AIM 2 will determine the impact of total RNU12 loss on embryo survival through lethality studies of embryos from mRnu12+/— x mRnu12+/— mating pairs. Embryos will be examined and genotyped across developmental stages to evaluate when mRnu12—/— embryos die. At a stage prior to mRnu12—/— loss, we will analyze differences in MIG expression and splicing between mRnu12— mutants and controls using SMART-Seq2 technology and transcriptome analyses. Expression patterns of candidate MIG transcripts will be validated using RT-PCR and immunohistochemical staining of embryo tissues. Together, these Aims will illuminate the critical role of U12-mediated spliceosome function in mammalian development and its relevance to disease.

抽象的选择性剪接是一个必要的过程，有助于细胞的专业化和系统的复杂性在哺乳动物中，选择性剪接由两个大分子复合物控制：主要的。（U2 依赖性）和次要（U12 依赖性）剪接体，而 U2 介导的剪接体已被经过广泛研究，U12 介导的剪接体仍然知之甚少。表明一种次要剪接体成分的丢失会导致早期胚胎死亡。尚未在人类中观察到任何微小剪接体单位的缺失，有 9 种疾病表型我们的实验室第一个报告了 C84T 核苷酸转换。 RNU12 会导致人类早发性小脑性共济失调 (EOCA) 和发育迟缓。富含尿苷的 U12 小核 RNA (snRNA)，通过内含子启动次要剪接体功能来自纯合子 RNU12C84T/C84T 患者的前 mRNA 的识别表现出异常表达。共济失调相关的小内含子基因 (MIG) 和内含子保留升高，表明存在关联我们的初步数据表明，RNU12 C84T 变体存在缺陷的小内含子剪接和 EOCA 之间的关系。损害 U12 介导的小脑特异性前 mRNA 剪接，而 RNU12 完全缺失会导致由于关键早期发育基因的异常剪接，导致早期胚胎死亡。使用下一代测序技术、转录组学、胚胎和大脑发育研究进行测试在新型基因编辑小鼠模型中，该模型包含 mRnu12 中的 C84T 变体 (mRnu1284T) 或 79bp 缺失失活 U12 snRNA (mRNA AIM 1) 将评估 C84T RNU12 突变在选择性中的作用。通过对小脑进行批量 RNA 测序，发现小脑转录本的错误剪接，与皮质和使用转录组学观察 mRnu1284T/84T 小鼠和对照小鼠在不同发育时间点的海马。工具，我们将评估基因表达、异构体使用、次要内含子保留和剪接位点移位的变化。候选转录本将使用 qRT-PCR 和原位在基因型、组织和发育方面进行验证 AIM 2 将通过致死率确定总 RNU12 损失对胚胎存活的影响。对 mRnu12+/— x mRnu12+/— 交配对胚胎的研究将对胚胎进行检查和基因分型。评估 mRnu12—/— 胚胎何时死亡的发育阶段在 mRnu12—/— 损失之前的阶段，我们将进行评估。使用 SMART-Seq2 分析 mRnu12 突变体和对照之间 MIG 表达和剪接的差异候选 MIG 转录本的表达模式将使用技术和转录组分析进行验证。 RT-PCR 和胚胎组织的免疫组织化学染色一起，这些目标将阐明这一关键点。 U12 介导的剪接体功能在哺乳动物发育中的作用及其与疾病的相关性。