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:
- 金额:$ 2.01万
- 依托单位:
- 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:
- 关键词:5&apos Splice SiteAblationAdultAffectAgeAlternative SplicingAntibodiesAtaxiaAutomobile DrivingBiologicalBrainBrain DiseasesBrain regionBreedingCandidate Disease GeneCell CycleCerebellar AtaxiaCerebellumCessation of lifeChildCodeDataDevelopmentDevelopmental Delay DisordersDevelopmental GeneDiseaseElementsEmbryoEmbryonic DevelopmentEngineeringEnvironmentEukaryotaEventExcisionExhibitsExperimental DesignsFailureFunctional disorderGene ExpressionGenesGenetic TranscriptionGenetic VariationGenomeGenotypeHarvestHealthHippocampus (Brain)HumanHuman DevelopmentImageImpairmentIn Situ HybridizationIndividualIntronsKnowledgeLeadLeukocytesMacromolecular ComplexesMammalsMediatingMessenger RNAMethodsMinorMolecularMusMutant Strains MiceMutationNucleotidesOntologyOrganismPartner in relationshipPathway interactionsPatientsPatternPeutz-Jeghers SyndromePoint MutationProcessProtein Export PathwayProtein IsoformsProteinsQuantitative Reverse Transcriptase PCRRNARNA SplicingReportingReverse Transcriptase Polymerase Chain ReactionRoleSTK11 geneSiteSmall Nuclear RNASpliced GenesSpliceosomesStainsSyndromeSystemTechniquesTechnologyTestingTimeTissuesTranscriptTransgenic MiceUridineVariantWorkbaseconsanguineous familydisease phenotypeearly onsetembryo tissuegenome editinggrowth hormone deficiencyhuman diseaseindexinginsightinterestmRNA Precursormouse modelmutantnext generation sequencingnovelosteodysplastic primordial dwarfismpostnataltooltranscriptometranscriptome sequencingtranscriptomics
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。
项目成果
期刊论文数量(0)
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