108-109年度「國立臺灣大學與中央研究院創新性合作計畫」
端粒RNA 與CST 蛋白體的交互作用來調控端粒的完整性
TERRA-CST interaction in regulating telomere integrity
計畫成員
國立臺灣大學分細所 朱雪萍 助理教授
中央研究院分生所 陳律佑 助研究員
計畫執行期間：2019.1.1 – 2020.12.31
優良研究成果
—研究非典型染色體末端延長模式榮登<自然通訊> 期刊
染色體的末端稱之為端粒 (Telomere)，可以維持染色體的穩定性，端粒的長度是維持細胞的正常分裂與生長重要的因素，與老化及癌症有著密切的關係。端粒的延長可利用端粒酶的活性來執行，但是也可不利用端粒酶的活性， 稱之為非典型端粒延長模式（Alternative Lengthening of Telomeres）。
這種方式在演化上是高度保留的，在許多真核生物都可發現。但是關於非典型端粒延長模式是如何啟動的，科學界還不是很了解。此種方式約可在癌細胞中可以發現，目前臨床上發現患有這類使用非典型端粒延長模式的癌症病人，以目前的治療方式成效不佳，對於使用非典型端粒延長的癌細胞一籌莫展。
近年來發現染色體的末端可以轉錄出RNA (命名為TERRA)，這種不轉譯成蛋白質的RNA稱之為非編碼RNA。其研究團隊利用RNA 標靶結合Cas9 的系統 （RNA-targeting Cas9）來清除 TERRA RNA，降低TERRA RNA使得癌細胞的端粒縮短，證明了TERRA RNA 可以調控此種非典型端粒延長模式。
其研究團隊利用新開發出來的技術，純化RNA與蛋白質複合體並結合定量質譜，以系統化的方式（iDRiP-MS, identification of direct RNA interacting proteins—mass spectrometry）來找尋 TERRA RNA所結合的蛋白質，找到許多參與DNA 修復機制的蛋白質都可以與TERRA RNA作用，其中一個蛋白質稱之為XPF， XPF在使用非典型端粒延長模式的癌細胞中大量與端粒結合， TERRA RNA 會與端粒形成 DNA:RNA 雜交，其特殊結構會誘導XPF到端粒上，並產生DNA 斷裂，進而促進DNA 損傷的訊息，以啟動同源重組 (homologous recombination) 的修復機制與DNA生成機制來延長端粒，抑制XPF蛋白質的表現，會降低染色體末端的DNA生合成，並縮短此類癌細胞的端粒長度與抑制癌細胞的生長。
這項研究成果證明TERRA RNA 與XPF 在非典型端粒延長模式啟動機制的重要性，提供了對於治療此類癌症的新契機。
Break before Synthesis
The maintenance of telomere length is closely related to the processes of cancers and aging. How do telomeres extend their length? It has been well-known that an enzyme called telomerase can lengthen telomeres. However, some cancers do not rely on telomerase activity to lengthen their telomeres. They utilized “Alternative lengthening of Telomeres (ALT)”, a mechanism that includes a break-induced replication to extend telomeres and is highly conserved in many eukaryotes. Patients with ALT cancers have a higher risk of death compared with those having non-ALT cancers.
How do cells initiate the breaks at telomeres? Dr. Hsueh-Ping (Catherine) Chu’s team in the Molecular and Cellular Biology department at NTU discovered that TERRA R-loops and XPF are the drivers. TERRA is a long non-coding RNA, which contains telomeric repeat sequences and forms DNA:RNA hybrids at telomeres. The DNA:RNA hybrid and a displaced single-stranded DNA form an R-loop structure. TERRA R-loops are specifically enriched at telomeres in cancer cells utilizing the ALT mechanism. The research team uncovered that TERRA R-loops trigger telomere clustering and activate DNA damage response by recruiting XPF. Such DNA damage response at telomeres is required for inducing homologous recombination and telomere synthesis in ALT cancer cells.
They developed an RCas9 system to specifically deplete TERRA RNA without editing telomeric DNA in ALT cells and found that TERRA depletion shortens telomere length in ALT cancer cells. They systematically identified TERRA interacting proteins in ALT cells and revealed that TERRA interacts with a large subset of proteins involved in the DNA repair pathway. Interestingly, TERRA interacts with several nucleotide excision repair factors including XPF, an enzyme that cuts DNA. They showed that TERRA R-loops recruit XPF to telomeres, leading to DNA double-strand breaks to activate break-induced telomere synthesis at ALT telomeres.
Targeting XPF by small interference RNAs inhibits cell growth in ALT cancer cells and reduces telomere lengthening. These findings provide new insights into ALT cancer therapy.
This research was published in Oct 2022. Nature communications
Chia-Yu Guh1, #, Hong-Jhih Shen1, #, Liv WeiChien Chen1,#, Pei-Chen Chiu1, #, I-Hsin Liao1, Chen-Chia Lo1, Yunfei Chen1, Yu-Hung Hsieh1, Ting-Chia Chang1, Chien-Ping Yen1, Yi-Yun Chen2, Tom Wei-Wu Chen3, Liuh-Yow Chen4, Ching-Shyi Wu5, Jean-Marc Egly6,7 and Hsueh-Ping Catherine Chu 1,*. XPF activates break-induced telomere synthesis. Nature Communications (SCI), 2022 Oct 2;13(1):5781. *Corresponding author.
https://www.nature.com/articles/s41467-022-33428-0
圖說文字
染色體的末端轉錄出TERRA RNA，與端粒DNA 形成迴圈 (R-loop)，其特殊結構會誘導XPF內切酶到端粒上，並產生DNA 斷裂，促進DNA 損傷的訊息，以啟動DNA生成機制來延長端粒—此為非典型端粒延長模式 。
