School of Kinesiology, University of Michigan, Ann Arbor, Michigan.
Biostatistics Department and School of Public Health, University of Michigan, Ann Arbor, Michigan.
Mol Cancer Res. 2022 Oct 4;20(10):1574-1588. doi: 10.1158/1541-7786.MCR-21-0058.
Splicing of the hTERT gene to produce the full-length (FL) transcript is necessary for telomerase enzyme activity and telomere-dependent cellular immortality in the majority of human tumors, including non-small cell lung cancer (NSCLC) cells. The molecular machinery to splice hTERT to the FL isoform remains mostly unknown. Previously, we reported that an intron 8 cis-element termed "direct repeat 8" (DR8) promotes FL hTERT splicing, telomerase, and telomere length maintenance when bound by NOVA1 and PTBP1 in NSCLC cells. However, some NSCLC cells and patient tumor samples lack NOVA1 expression. This leaves a gap in knowledge about the splicing factors and cis-elements that promote telomerase in the NOVA1-negative context. We report that DR8 regulates FL hTERT splicing in the NOVA1-negative and -positive lung cancer contexts. We identified splicing factor 3b subunit 4 (SF3B4) as an RNA trans-factor whose expression is increased in lung adenocarcinoma (LUAD) tumors compared with adjacent normal tissue and predicts poor LUAD patient survival. In contrast to normal lung epithelial cells, which continued to grow with partial reductions of SF3B4 protein, SF3B4 knockdown reduced hTERT splicing, telomerase activity, telomere length, and cell growth in lung cancer cells. SF3B4 was also demonstrated to bind the DR8 region of hTERT pre-mRNA in both NOVA1-negative and -positive NSCLC cells. These findings provide evidence that DR8 is a critical binding hub for trans-factors to regulate FL hTERT splicing in NSCLC cells. These studies help define mechanisms of gene regulation important to the generation of telomerase activity during carcinogenesis.
Manipulation of a core spliceosome protein reduces telomerase/hTERT splicing in lung cancer cells and results in slowed cancer cell growth and cell death, revealing a potential therapeutic strategy.
非小细胞肺癌（NSCLC）细胞中端粒酶酶活性和端粒依赖性细胞永生所必需的 hTERT 基因拼接需要产生全长（FL）转录本。拼接 hTERT 到 FL 同种型的分子机制在大多数人类肿瘤中仍然大多未知。先前，我们报道了一个称为“DR8”的内含子 8 顺式元件通过在 NSCLC 细胞中与 NOVA1 和 PTBP1 结合促进 FL hTERT 拼接、端粒酶和端粒长度维持。然而，一些 NSCLC 细胞和患者肿瘤样本缺乏 NOVA1 表达。这使得在 NOVAl 阴性背景下促进端粒酶的拼接因子和顺式元件的知识存在空白。我们报告了 DR8 在 NOVAl 阴性和阳性 NSCLC 细胞环境中调节 FL hTERT 拼接。我们确定了剪接因子 3b 亚基 4（SF3B4）作为一种 RNA 反式因子，其在肺腺癌（LUAD）肿瘤中的表达与相邻正常组织相比增加，并预测 LUAD 患者的预后不良。与正常肺上皮细胞不同，后者在 SF3B4 蛋白部分减少的情况下继续生长，SF3B4 敲低降低了 hTERT 拼接、端粒酶活性、端粒长度和肺癌细胞的生长。SF3B4 还被证明在 NOVAl 阴性和阳性 NSCLC 细胞中结合 hTERT 前体 mRNA 的 DR8 区域。这些发现提供了证据表明 DR8 是反式因子结合的关键枢纽，可调节 NSCLC 细胞中 FL hTERT 的拼接。这些研究有助于定义在致癌发生过程中端粒酶活性产生的重要基因调控机制。
核心剪接体蛋白的操作减少了肺癌细胞中端粒酶/ hTERT 的拼接，导致癌细胞生长缓慢和细胞死亡，揭示了一种潜在的治疗策略。
