m6A RNA modification of TERRA lncRNA drives R-Loop formation and is required for telomere maintenance in ALT tumors [I]

Telomerase-negative tumors can maintain telomere length by alternative lengthening of telomeres (ALT) but the mechanism of telomere maintenance in ALT cells is not well understood. A significant portion of the relapse Neuroblastoma (NB) tumors are positive for ALT which suggests better dissecting the ALT mechanism could provide novel therapeutic opportunities. TERRA RNA which is derived from the telomere ends is localized to telomeres in R-loop dependent manner and is essential for telomere maintenance. In the present study, we provide evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA RNA by methyl transferase METTL3 is essential for telomere maintenance in ALT cells and loss of TERRA m6A/METTL3 leads to accumulation of DNA damage over telomere. Our data suggest that m6A modification in TERRA RNA is required for R-loop formation and telomere targeting of TERRA. We observed that R-loop enriched TERRA is abundantly m6A modified and m6A mediated recruitment of hnRNPA2B1 to TERRA RNA is essential for R-loop formation. Together our study suggests that m6A-mediated R-loop formation could be a widespread mechanism utilized by other chromatin-interacting lncRNAs. We also show treating ALT positive NB cells with small molecule METTL3 inhibitor leads to compromised telomere targeting of TERRA and accumulation of DNA damage over telomere, suggesting METTL3 inhibition could be a therapeutic opportunity for ALT positive NB. m6A RNA immunoprecipitation (m6A-RIP seq) using Illumina short-reads and direct RNA Nanopore sequencing in U2OS and NB cells.

端粒酶阴性肿瘤可通过端粒替代延长（ALT，alternative lengthening of telomeres）维持端粒长度，但ALT细胞中端粒维持的具体机制尚未完全阐明。复发性神经母细胞瘤（NB，Neuroblastoma）中有相当比例的肿瘤呈ALT阳性，这提示深入解析ALT机制可带来全新的治疗机遇。源自端粒末端的端粒重复序列RNA（TERRA RNA）以依赖R环（R-loop）的方式定位于端粒，且对端粒维持至关重要。本研究证实，由甲基转移酶METTL3介导的TERRA RNA内部腺嘌呤N6位甲基化修饰（m6A）是ALT细胞端粒维持的必需条件；TERRA m6A/METTL3的缺失会导致端粒区域DNA损伤积累。我们的研究数据表明，TERRA RNA的m6A修饰是R环形成以及TERRA靶向端粒的必要前提。研究发现，富集于R环的TERRA存在大量m6A修饰，且m6A介导的异质性细胞核核糖蛋白A2B1（hnRNPA2B1）向TERRA RNA的招募对R环形成至关重要。综上，本研究提示m6A介导的R环形成可能是其他染色质相互作用型长链非编码RNA（lncRNAs）所广泛利用的机制。本研究还证实，使用METTL3小分子抑制剂处理ALT阳性NB细胞，会破坏TERRA的端粒靶向能力并导致端粒区域DNA损伤积累，这表明METTL3抑制有望成为ALT阳性NB的潜在治疗手段。本研究针对U2OS细胞与NB细胞，采用基于Illumina短读长测序的m6A RNA免疫共沉淀测序（m6A-RIP-seq）以及纳米孔直接RNA测序技术开展了相关实验。