PDF(Pubmed)
Abstract：
UNASSIGNED: N4-acetylcytidine (ac4C) is a post-transcriptional RNA modification catalyzed by N-acetyltransferase 10 (NAT10), a critical factor known to influence mRNA stability. However, the role of ac4C in visual development remains unexplored.
UNASSIGNED: Analysis of public datasets and immunohistochemical staining were conducted to assess the expression pattern of nat10 in zebrafish. We used CRISPR/Cas9 and RNAi technologies to knockout (KO) and knockdown (KD) nat10, the zebrafish ortholog of human NAT10, and evaluated its effects on early development. To assess the impact of nat10 knockdown on visual function, we performed comprehensive histological evaluations and behavioral analyses. Transcriptome profiling and real-time (RT)-PCR were utilized to detect alterations in gene expression resulting from the nat10 knockdown. Dot-blot and RNA immunoprecipitation (RIP)-PCR analyses were conducted to verify changes in ac4C levels in both total RNA and opsin mRNA specifically. Additionally, we used the actinomycin D assay to examine the stability of opsin mRNA following the nat10 KD.
UNASSIGNED: Our study found that the zebrafish NAT10 protein shares similar structural properties with its human counterpart. We observed that the nat10 gene was prominently expressed in the visual system during early zebrafish development. A deficiency of nat10 in zebrafish embryos resulted in increased mortality and developmental abnormalities. Behavioral and histological assessments indicated significant vision impairment in nat10 KD zebrafish. Transcriptomic analysis and RT-PCR identified substantial downregulation of retinal transcripts related to phototransduction, light response, photoreceptors, and visual perception in the nat10 KD group. Dot-blot and RIP-PCR analyses confirmed a pronounced reduction in ac4C levels in both total RNA and specifically in opsin messenger RNA (mRNA). Additionally, by evaluating mRNA decay in zebrafish treated with actinomycin D, we observed a significant decrease in the stability of opsin mRNA in the nat10 KD group.
UNASSIGNED: The ac4C-mediated mRNA modification plays an essential role in maintaining visual development and retinal function. The loss of NAT10-mediated ac4C modification results in significant disruptions to these processes, underlining the importance of this RNA modification in ocular development.
UNASSIGNED: Analysis of public datasets and immunohistochemical staining were conducted to assess the expression pattern of nat10 in zebrafish. We used CRISPR/Cas9 and RNAi technologies to knockout (KO) and knockdown (KD) nat10, the zebrafish ortholog of human NAT10, and evaluated its effects on early development. To assess the impact of nat10 knockdown on visual function, we performed comprehensive histological evaluations and behavioral analyses. Transcriptome profiling and real-time (RT)-PCR were utilized to detect alterations in gene expression resulting from the nat10 knockdown. Dot-blot and RNA immunoprecipitation (RIP)-PCR analyses were conducted to verify changes in ac4C levels in both total RNA and opsin mRNA specifically. Additionally, we used the actinomycin D assay to examine the stability of opsin mRNA following the nat10 KD.
UNASSIGNED: Our study found that the zebrafish NAT10 protein shares similar structural properties with its human counterpart. We observed that the nat10 gene was prominently expressed in the visual system during early zebrafish development. A deficiency of nat10 in zebrafish embryos resulted in increased mortality and developmental abnormalities. Behavioral and histological assessments indicated significant vision impairment in nat10 KD zebrafish. Transcriptomic analysis and RT-PCR identified substantial downregulation of retinal transcripts related to phototransduction, light response, photoreceptors, and visual perception in the nat10 KD group. Dot-blot and RIP-PCR analyses confirmed a pronounced reduction in ac4C levels in both total RNA and specifically in opsin messenger RNA (mRNA). Additionally, by evaluating mRNA decay in zebrafish treated with actinomycin D, we observed a significant decrease in the stability of opsin mRNA in the nat10 KD group.
UNASSIGNED: The ac4C-mediated mRNA modification plays an essential role in maintaining visual development and retinal function. The loss of NAT10-mediated ac4C modification results in significant disruptions to these processes, underlining the importance of this RNA modification in ocular development.
摘要：
■N4-乙酰胞苷（ac4C）是由N-乙酰转移酶10（NAT10）催化的转录后RNA修饰，已知影响mRNA稳定性的关键因素。然而,ac4C在视觉发育中的作用仍未被探索。
■进行公共数据集和免疫组织化学染色的分析以评估nat10在斑马鱼中的表达模式。我们使用CRISPR/Cas9和RNAi技术敲除（KO）和敲除（KD）nat10，人类NAT10的斑马鱼直系同源，并评估其对早期发育的影响。为了评估nat10击倒对视功能的影响，我们进行了全面的组织学评估和行为分析.利用转录组分析和实时(RT)-PCR来检测由nat10敲低引起的基因表达的改变。进行斑点印迹和RNA免疫沉淀(RIP)-PCR分析,以特异性地验证总RNA和视蛋白mRNA中ac4C水平的变化。此外,我们使用放线菌素D测定法来检查nat10KD后视蛋白mRNA的稳定性。
■我们的研究发现，斑马鱼NAT10蛋白与人类对应物具有相似的结构特性。我们观察到nat10基因在斑马鱼早期发育过程中在视觉系统中显著表达。斑马鱼胚胎中nat10的缺乏导致死亡率和发育异常增加。行为和组织学评估表明nat10KD斑马鱼有明显的视力障碍。转录组学分析和RT-PCR鉴定了与光转导相关的视网膜转录本的大量下调，光响应,光感受器，nat10KD组的视觉感知。点印迹和RIP-PCR分析证实了总RNA和特别是视蛋白信使RNA（mRNA）中ac4C水平的显着降低。此外,通过评估用放线菌素D处理的斑马鱼的mRNA衰减，我们观察到nat10KD组视蛋白mRNA的稳定性显着降低。
■ac4C介导的mRNA修饰在维持视觉发育和视网膜功能中起着至关重要的作用。NAT10介导的ac4C修饰的丧失导致这些过程的显著中断，强调这种RNA修饰在眼部发育中的重要性。
■进行公共数据集和免疫组织化学染色的分析以评估nat10在斑马鱼中的表达模式。我们使用CRISPR/Cas9和RNAi技术敲除（KO）和敲除（KD）nat10，人类NAT10的斑马鱼直系同源，并评估其对早期发育的影响。为了评估nat10击倒对视功能的影响，我们进行了全面的组织学评估和行为分析.利用转录组分析和实时(RT)-PCR来检测由nat10敲低引起的基因表达的改变。进行斑点印迹和RNA免疫沉淀(RIP)-PCR分析,以特异性地验证总RNA和视蛋白mRNA中ac4C水平的变化。此外,我们使用放线菌素D测定法来检查nat10KD后视蛋白mRNA的稳定性。
■我们的研究发现，斑马鱼NAT10蛋白与人类对应物具有相似的结构特性。我们观察到nat10基因在斑马鱼早期发育过程中在视觉系统中显著表达。斑马鱼胚胎中nat10的缺乏导致死亡率和发育异常增加。行为和组织学评估表明nat10KD斑马鱼有明显的视力障碍。转录组学分析和RT-PCR鉴定了与光转导相关的视网膜转录本的大量下调，光响应,光感受器，nat10KD组的视觉感知。点印迹和RIP-PCR分析证实了总RNA和特别是视蛋白信使RNA（mRNA）中ac4C水平的显着降低。此外,通过评估用放线菌素D处理的斑马鱼的mRNA衰减，我们观察到nat10KD组视蛋白mRNA的稳定性显着降低。
■ac4C介导的mRNA修饰在维持视觉发育和视网膜功能中起着至关重要的作用。NAT10介导的ac4C修饰的丧失导致这些过程的显著中断，强调这种RNA修饰在眼部发育中的重要性。
