Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan.
Department of Applied Biological Sciences, Faculty of Agriculture, Setsunan University, Hirakata, Japan.
Genes Cells. 2021 Jan;26(1):18-30. doi: 10.1111/gtc.12817. Epub 2020 Dec 2.
Primary RNA transcripts are processed in a plethora of ways to become mature functional forms. In one example, human spliceosomal U snRNAs are matured at their 3'-end by an exonuclease termed TOE1. This process is important because mutations in TOE1 gene can cause a human genetic disease, pontocerebellar hypoplasia (PCH). Nevertheless, TOE1 may not be the only maturation exonuclease for U snRNAs in the cell. Here, we biochemically identify two exonucleolytic factors, Interferon-stimulated gene 20-kDa protein (ISG20) and the nuclear exosome as such candidates, using a newly developed in vitro system that recapitulates 3'-end maturation of U1 snRNA. However, extensive 3'-end sequencing of endogenous U1 snRNA of the knockdown (KD) cells revealed that these factors are not the maturation factors per se. Instead, the nascent transcripts of the spliceosomal U snRNAs as well as of unstable U1 variants were found to increase in quantity upon KD of the factors. These results indicated that ISG20 and the nuclear exosome promote the degradation of nascent spliceosomal U snRNAs and U1 variants, and therefore implied their role in the quality control of newly synthesized U snRNAs.
初级 RNA 转录物通过多种方式加工成为成熟的功能形式。例如，人类剪接体 U snRNA 通过一种称为 TOE1 的外切核酸酶在其 3'-末端成熟。这个过程很重要，因为 TOE1 基因的突变会导致人类遗传疾病——桥小脑发育不良（PCH）。然而，TOE1 可能不是细胞中 U snRNA 成熟的唯一外切核酸酶。在这里，我们使用新开发的体外系统，对干扰素刺激基因 20kDa 蛋白 (ISG20) 和核外切酶进行生化鉴定，该系统可以模拟 U1 snRNA 的 3'-末端成熟。然而，对敲低 (KD) 细胞内源性 U1 snRNA 的广泛 3'-末端测序表明，这些因子本身并不是成熟因子。相反，在这些因子的 KD 后，剪接体 U snRNA 和不稳定的 U1 变体的新生转录本的数量增加。这些结果表明，ISG20 和核外切酶促进新生剪接体 U snRNA 和 U1 变体的降解，因此暗示它们在新合成的 U snRNA 的质量控制中发挥作用。
