Study of the RNA splicing in THUMPD2 knockout HeLa S3 cells

Splicing is one of the most important stages in the maturation of the majority of RNA in eukaryotic cells. In this work, we studied the effect of m2G72 modification in U6 snRNA on splicing rate and accuracy in HeLa S3 cells. High-throughput polyA+ RNA-seq was performed for THUMPD2 methyltransferase gene knockout cells. This methyltransferase catalyzes the indicated N2-methylation of U6 snRNA. To study the splicing kinetics knockout cells were incubated for 10 and 20 minutes with 5-ethynyl uridine (EU), followed by nascent RNA purification and high throughput sequencing. Each experiment was done in 3 biological replicates.

RNA剪接（splicing）是真核细胞中绝大多数RNA成熟过程的关键步骤之一。本研究针对海拉S3（HeLa S3）细胞内U6小核RNA（U6 snRNA）的m2G72修饰对剪接效率与准确性的影响展开探究。研究对THUMPD2甲基转移酶基因敲除细胞开展了高通量polyA+ RNA测序（polyA+ RNA-seq），该酶可催化U6 snRNA的指定位点N2-甲基化修饰。为解析剪接动力学特征，研究人员将基因敲除细胞与5-乙炔基尿苷（5-ethynyl uridine，EU）共孵育10分钟及20分钟，随后完成新生RNA纯化与高通量测序。所有实验均设置3次生物学重复。