Endogenous epitope tagging of eEF1A2 in mice reveals early embryonic expression of eEF1A2 and subcellular compartmentalisation of neuronal eEF1A1 and eEF1A2

IF 2.6 3区 医学 Q3 NEUROSCIENCES
Faith C.J. Davies , Grant F. Marshall , Eleanor Pegram , Danni Gadd , Catherine M. Abbott
{"title":"Endogenous epitope tagging of eEF1A2 in mice reveals early embryonic expression of eEF1A2 and subcellular compartmentalisation of neuronal eEF1A1 and eEF1A2","authors":"Faith C.J. Davies ,&nbsp;Grant F. Marshall ,&nbsp;Eleanor Pegram ,&nbsp;Danni Gadd ,&nbsp;Catherine M. Abbott","doi":"10.1016/j.mcn.2023.103879","DOIUrl":null,"url":null,"abstract":"<div><p>All vertebrate species express two independently-encoded forms of translation elongation factor eEF1A. In humans and mice eEF1A1 and eEF1A2 are 92 % identical at the amino acid level, but the well conserved developmental switch between the two variants in specific tissues suggests the existence of important functional differences. Heterozygous mutations in eEF1A2 result in neurodevelopmental disorders in humans; the mechanism of pathogenicity is unclear, but one hypothesis is that there is a dominant negative effect on eEF1A1 during development. The high degree of similarity between the eEF1A proteins has complicated expression analysis in the past; here we describe a gene edited mouse line in which we have introduced a V5 tag in the gene encoding eEF1A2. Expression analysis using anti-V5 and anti-eEF1A1 antibodies demonstrates that, in contrast to the prevailing view that eEF1A2 is only expressed postnatally, it is expressed from as early as E11.5 in the developing neural tube. Two colour immunofluorescence also reveals coordinated switching between eEF1A1 and eEF1A2 in different regions of postnatal brain. Completely reciprocal expression of the two variants is seen in post-weaning mouse brain with eEF1A1 expressed in oligodendrocytes and astrocytes and eEF1A2 in neuronal soma. Although eEF1A1 is absent from neuronal cell bodies after development, it is widely expressed in axons. This expression does not appear to coincide with myelin sheaths originating from oligodendrocytes but rather results from localised translation within the axon, suggesting that both variants are transcribed in neurons but show completely distinct subcellular localisation at the protein level. These findings will form an underlying framework for understanding how missense mutations in eEF1A2 result in neurodevelopmental disorders.</p></div>","PeriodicalId":18739,"journal":{"name":"Molecular and Cellular Neuroscience","volume":"126 ","pages":"Article 103879"},"PeriodicalIF":2.6000,"publicationDate":"2023-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Molecular and Cellular Neuroscience","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1044743123000738","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"NEUROSCIENCES","Score":null,"Total":0}
引用次数: 1

Abstract

All vertebrate species express two independently-encoded forms of translation elongation factor eEF1A. In humans and mice eEF1A1 and eEF1A2 are 92 % identical at the amino acid level, but the well conserved developmental switch between the two variants in specific tissues suggests the existence of important functional differences. Heterozygous mutations in eEF1A2 result in neurodevelopmental disorders in humans; the mechanism of pathogenicity is unclear, but one hypothesis is that there is a dominant negative effect on eEF1A1 during development. The high degree of similarity between the eEF1A proteins has complicated expression analysis in the past; here we describe a gene edited mouse line in which we have introduced a V5 tag in the gene encoding eEF1A2. Expression analysis using anti-V5 and anti-eEF1A1 antibodies demonstrates that, in contrast to the prevailing view that eEF1A2 is only expressed postnatally, it is expressed from as early as E11.5 in the developing neural tube. Two colour immunofluorescence also reveals coordinated switching between eEF1A1 and eEF1A2 in different regions of postnatal brain. Completely reciprocal expression of the two variants is seen in post-weaning mouse brain with eEF1A1 expressed in oligodendrocytes and astrocytes and eEF1A2 in neuronal soma. Although eEF1A1 is absent from neuronal cell bodies after development, it is widely expressed in axons. This expression does not appear to coincide with myelin sheaths originating from oligodendrocytes but rather results from localised translation within the axon, suggesting that both variants are transcribed in neurons but show completely distinct subcellular localisation at the protein level. These findings will form an underlying framework for understanding how missense mutations in eEF1A2 result in neurodevelopmental disorders.

小鼠eEF1A2的内源性表位标记揭示了eEF1A2的早期胚胎表达和神经元eEF1A1和eEF1A2的亚细胞区隔化
所有脊椎动物都表达两种独立编码的翻译延伸因子eEF1A。在人类和小鼠中,eEF1A1和eEF1A2在氨基酸水平上有92%的相同性,但在特定组织中这两种变体之间的发育转换非常保守,这表明存在重要的功能差异。eEF1A2的杂合突变导致人类神经发育障碍;致病机制尚不清楚,但有一种假说认为,在发育过程中对eEF1A1有显性负面影响。eEF1A蛋白之间的高度相似性在过去具有复杂的表达分析;这里我们描述了一种基因编辑的小鼠系,其中我们在编码eEF1A2的基因中引入了V5标签。使用抗V5和抗eEF1A1抗体的表达分析表明,与eEF1A2仅在出生后表达的主流观点相反,它早在E11.5就在发育中的神经管中表达。双色免疫荧光还揭示了出生后大脑不同区域eEF1A1和eEF1A2之间的协调转换。这两种变体在断奶后的小鼠大脑中完全相互表达,其中eEF1A1在少突胶质细胞和星形胶质细胞中表达,eEF1A2在神经元胞体中表达。尽管eEF1A1在发育后不存在于神经元细胞体中,但它在轴突中广泛表达。这种表达似乎与源自少突胶质细胞的髓鞘不一致,而是由轴突内的局部翻译引起的,这表明两种变体都在神经元中转录,但在蛋白质水平上表现出完全不同的亚细胞定位。这些发现将为理解eEF1A2的错义突变如何导致神经发育障碍形成一个潜在的框架。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
求助全文
约1分钟内获得全文 求助全文
来源期刊
CiteScore
5.60
自引率
0.00%
发文量
65
审稿时长
37 days
期刊介绍: Molecular and Cellular Neuroscience publishes original research of high significance covering all aspects of neurosciences indicated by the broadest interpretation of the journal''s title. In particular, the journal focuses on synaptic maintenance, de- and re-organization, neuron-glia communication, and de-/regenerative neurobiology. In addition, studies using animal models of disease with translational prospects and experimental approaches with backward validation of disease signatures from human patients are welcome.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信