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.