Department. Of Biochemistry and Gene Center, Ludwig-Maximilians-Universität München, München, Germany.
RNA Biol. 2022;19(1):68-77. doi: 10.1080/15476286.2021.2014694. Epub 2021 Dec 31.
DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured cells.
DNA 双链断裂是基因组中最具毒性的损伤之一，因此准确修复它们非常重要。最近的研究发现，局部转录可以在断裂处起始，并形成一种非编码转录本，称为损伤诱导的长非编码 RNA（dilncRNA），它有助于协调修复所需的 DNA 交易。我们提供了基于 RNA 聚合酶 II 转录 dilncRNA 的新生 RNA 测序证据，并且对于内含子基因中的 DNA 断裂，这种转录更为有效，这与内含子下游更高的损伤诱导 siRNA 水平一致。因此，剪接体刺激 RNA 聚合酶 II 向断裂处的募集，而不仅仅是促进 sense 和 antisense RNA 的退火以形成 siRNA 前体。相比之下，RNA 聚合酶 III 新生 RNA 文库中没有与切割位点相对应的读段，并且选择性抑制 RNA 聚合酶 III 不会降低损伤诱导的 siRNA 的产量。最后，T8 序列下游的损伤诱导 siRNA 密度没有变化，该序列终止了 RNA 聚合酶 III 的转录。因此，我们在培养的 细胞中没有发现 RNA 聚合酶 III 参与 dilncRNA 转录的证据。
