Intronic Cis-Element DR8 in hTERT Is Bound by Splicing Factor SF3B4 and Regulates hTERT Splicing in Non-Small Cell Lung Cancer.

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 to 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.

在包括非小细胞肺癌（NSCLC）细胞在内的大多数人类肿瘤中，人端粒酶逆转录酶（hTERT）基因的剪接以产生全长（FL）转录本对于端粒酶活性以及端粒依赖的细胞永生化是必需的。将hTERT剪接为FL异构体的分子机制在很大程度上仍然未知。我们之前报道过，在NSCLC细胞中，当被NOVA1和PTBP1结合时，一种被称为“直接重复8”（DR8）的内含子8顺式元件可促进FL hTERT剪接、端粒酶活性以及端粒长度的维持。然而，一些NSCLC细胞和患者肿瘤样本缺乏NOVA1表达。这在关于在NOVA1阴性情况下促进端粒酶的剪接因子和顺式元件的知识方面留下了空白。我们报道DR8在NOVA1阴性和阳性的肺癌环境中调节FL hTERT剪接。我们鉴定出剪接因子3b亚基4（SF3B4）作为一种RNA反式作用因子，其在肺腺癌（LUAD）肿瘤中的表达相较于相邻正常组织有所增加，并且预示着LUAD患者的不良预后。与正常肺上皮细胞不同，正常肺上皮细胞在SF3B4蛋白部分减少的情况下仍能继续生长，而在肺癌细胞中，SF3B4的敲低会降低hTERT剪接、端粒酶活性、端粒长度以及细胞生长。还证明了在NOVA1阴性和阳性的NSCLC细胞中，SF3B4都能与hTERT前体mRNA的DR8区域结合。这些发现提供了证据，表明DR8是反式作用因子调节NSCLC细胞中FL hTERT剪接的关键结合枢纽。这些研究有助于确定在癌症发生过程中对端粒酶活性产生至关重要的基因调控机制。