Department of Molecular and Cellular Biology and Genome Center, University of California, Davis, Davis, CA.
J Cell Biol. 2021 Jun 7;220(6). doi: 10.1083/jcb.202004079.
The S9.6 antibody is broadly used to detect RNA:DNA hybrids but has significant affinity for double-stranded RNA. The impact of this off-target RNA binding activity has not been thoroughly investigated, especially in the context of immunofluorescence microscopy. We report that S9.6 immunofluorescence signal observed in fixed human cells arises predominantly from ribosomal RNA, not RNA:DNA hybrids. S9.6 staining was unchanged by pretreatment with the RNA:DNA hybrid-specific nuclease RNase H1, despite verification in situ that S9.6 recognized RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to RNase T1, which specifically degrades RNA. Additional imaging and biochemical data indicate that the prominent cytoplasmic and nucleolar S9.6 signal primarily derives from ribosomal RNA. Importantly, genome-wide maps obtained by DNA sequencing after S9.6-mediated DNA:RNA immunoprecipitation (DRIP) are RNase H1 sensitive and RNase T1 insensitive. Altogether, these data demonstrate that imaging using S9.6 is subject to pervasive artifacts without pretreatments and controls that mitigate its promiscuous recognition of cellular RNAs.
S9.6 抗体广泛用于检测 RNA:DNA 杂交体，但对双链 RNA 具有显著的亲和力。这种非靶向 RNA 结合活性的影响尚未得到彻底研究，特别是在免疫荧光显微镜检查的背景下。我们报告说，在固定的人类细胞中观察到的 S9.6 免疫荧光信号主要来自核糖体 RNA，而不是 RNA:DNA 杂交体。尽管 S9.6 识别 RNA:DNA 杂交体并且 RNase H1 是活性的，但 S9.6 染色并未因预处理 RNA:DNA 杂交体特异性核酸内切酶 RNase H1 而改变。然而，S9.6 染色对 RNase T1 非常敏感，RNase T1 特异性降解 RNA。其他成像和生化数据表明，细胞质和核仁中突出的 S9.6 信号主要来自核糖体 RNA。重要的是，在用 S9.6 介导的 DNA:RNA 免疫沉淀 (DRIP) 后获得的全基因组图谱对 DNA 测序是 RNase H1 敏感的，而对 RNase T1 不敏感。总的来说，这些数据表明，在没有预处理和控制措施的情况下，使用 S9.6 进行成像会受到普遍的假象影响，这些假象会减轻其对细胞 RNA 的混杂识别。
