RNA modifications, alternative splicing and circular RNA landscape in the mouse brain: inosine and beyond [RNA-seq]

RNA modifications, including adenosine-to-inosine (A-to-I) editing, are fundamental for normal brain physiology, as they participate in multiple biological processes and pathological conditions. These modifications can alter RNA secondary structures and affinity for RNA-binding proteins, thereby influencing RNA processing mechanisms such as alternative splicing. In this study, we investigated A-to-I RNA editing and its impact on alternative splicing regulation in the mouse brain using ADAR enzyme-deficient mice. We analyzed the brain transcriptome of ADAR2 knockout (Adar2/Gria2R/R), as well as double ADAR1 catalytically inactive / ADAR2 knockout (Adar1E861A/E861A Ifih/ Adar2/Gria2R/R) mice compared to wild-type controls, identifying alternative splicing events associated with the absence of ADAR1 and ADAR2 specific A-to-I editing. Total RNA from whole brains from adult 34-month-old male mice was used for RNA-seq as described in the Methods section of this publication. Three biological replicates for each genotype were sequenced. ADAR2 knockout (Adar2/Gria2R/R), double ADAR1 catalytically inactive/ADAR2 knockout (Adar1E861A/E861A Ifih/Adar2/Gria2R/R) and wild-type controls. All mice were C57BL/6J on a background.RNA修饰，包括腺苷到肌苷（A-to-I）编辑，对于维持正常大脑生理功能至关重要，其参与了诸多生物学过程与病理状态。这类修饰可改变RNA二级结构以及与RNA结合蛋白的结合亲和力，进而影响可变剪接等RNA加工过程。本研究利用ADAR（双链RNA腺苷脱氨酶）缺陷型小鼠，探究了小鼠大脑中的A-to-I RNA编辑现象及其对可变剪接调控的影响。我们以野生型小鼠为对照，分析了ADAR2敲除（Adar2/Gria2R/R）小鼠与ADAR1催化失活/ADAR2双敲除（Adar1E861A/E861A Ifih/Adar2/Gria2R/R）小鼠的大脑转录组，鉴定出与ADAR1缺失及ADAR2特异性A-to-I编辑相关的可变剪接事件。本研究采用本论文方法部分所述的实验方案，对成年34月龄雄性小鼠的全脑总RNA进行RNA测序（RNA-seq）。每种基因型设置3个生物学重复样本进行测序，本次实验涵盖的小鼠模型包括ADAR2敲除（Adar2/Gria2R/R）、ADAR1催化失活/ADAR2双敲除（Adar1E861A/E861A Ifih/Adar2/Gria2R/R）以及野生型对照，所有受试小鼠均为C57BL/6J背景品系。