权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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.

摘要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，更高的生物。在哺乳动物中，选择性剪接由两种大分子复合物控制：（U2依赖性）和次要（U12依赖性）剪接体。虽然U2介导的剪接体已经被虽然经过广泛的研究，但对U12介导的剪接体仍知之甚少。最近在小鼠中的研究表明一个次要剪接体成分丢失导致早期胚胎死亡。虽然完整在人类中尚未观察到任何次要剪接体单位的缺失，存在9种疾病表型与轻微的剪接体功能障碍有关我们的实验室是第一个报告C84 T核苷酸开关在 RNU 12导致人类早发性小脑共济失调（EOCA）和发育迟缓。RNU 12编码富含尿苷的U12小核RNA（snRNA），通过内含子启动次要剪接体功能前mRNA的识别。来自纯合子RNU 12 C84 T/C84 T患者的白细胞表现出异常表达共济失调相关的小内含子基因（MIGs）和内含子保留增加，暗示了小内含子剪接缺陷和EOCA之间的关系我们的初步数据表明，RNU 12 C84 T变体损害U12介导的小脑特异性前mRNA的剪接，而RNU 12的完全缺失导致由于关键的早期发育基因的异常剪接导致的早期胚胎致死。这一假设将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的基因编辑的小鼠模型中，其包含mRnu 12中的C84 T变体（mRnu 1284 T），或79 bp的缺失失活U12 snRNA（mRnu 12-）。AIM 1将评估C84 T RNU 12突变在选择性通过对小脑进行批量RNA测序，与皮质和 mRnu 1284 T/84 T小鼠和对照在不同发育时间点的海马。使用转录组学工具，我们将评估基因表达的变化，异构体的使用，小内含子保留和剪接位点转移。候选转录本将使用qRT-PCR和原位方法在基因型、组织和发育中进行验证杂交方法AIM 2将通过致死率确定RNU 12总损失对胚胎存活的影响来自mRnu 12 +/- x mRnu 12 +/-交配对的胚胎的研究。胚胎将被检查和基因分型，发育阶段以评估mRnu 12-/-胚胎何时死亡。在mRnu 12-/-丢失之前的阶段，我们将使用SMART-Seq 2分析mRnu 12突变体和对照之间的mRNA表达和剪接差异技术和转录组分析。候选转录本的表达模式将使用胚胎组织的RT-PCR和免疫组化染色。总之，这些目标将阐明关键的 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。