CTC1調控細胞質内端粒核糖核酸顆粒

Abstract

有一部分的癌細胞利用ALT機制來延長其端粒長度。雖然ALT機制内的分子機轉尚待充分闡明，但目前可知其主要是通過同源重組來延長端粒長度。端粒RNA(TERRA)為一長鏈非編碼RNA，其在ALT細胞中高度表達。根據iDRIP實驗結果指出，在ALT cell内，CTC1 為其中之一會與TERRA相互作用的蛋白質。 CTC1可以與STN1和TEN1結合形成CST複合物，該複合物會與DNA聚合酶α-引物酶相互作用，促進端粒的有效複製，有助於端粒的維持。當CTC1基因發生突變時會導致Coats Plus, 先天性角化不全（DC）和相關的骨髓衰竭綜合症。 在這項研究中，我利用人類骨肉瘤上皮細胞(U2-OS) 來探討TEERA 與CTC1 之間的相互作用。我發現當CTC1表現量降低的時候，並不會影響APB的形成但增加了細胞質中的TERRA顆粒。推測是CTC1表現量不足所導致的結果，因為當STN1表現量降低 ，並不會增加細胞質中TERRA 顆粒。我也發現，一些TERRA顆粒會與GRSF1和mtSSB 的位置重疊，這可能表明某些TERRA顆粒會被傳送至線粒體。除此之外，我也發現抑制CTC1表現後，導致TERRA表達水平略微增加，並分泌更多的含cfTERRA的胞外體至細胞外。此外CST複合體的缺陷也會刺激免疫反應的增加，使TNF-alpha, IL-6, INF-beta 的表現量增加。 總結來說，我發現CTC1在會調節細胞質中的TERRA顆粒生成，但是這個功能似乎與CST複合體功能沒有直接關係，值得一提的是CST複合體的缺陷卻可以引起cfTERRA的增加，引發免疫反應。此外，TERRA顆粒所代表的生物意義則需進一步地探討。

Some cancer cells and immortal cells can employ alternative lengthening of telomeres (ALT) mechanism to elongate their telomeres. Although the molecular details of ALT mechanism remain to be fully elucidated, it is acceptable that ALT pathway utilizes homologous recombination to extend telomeres. TERRA is a long non-coding RNA and is highly expressed in ALT cells. According to the result of TERRA-iDIRP mass spectrometry, CTC1 is a TERRA interacting protein in ALT cells. CTC1 can associate with STN1 and TEN1 to form CST complex, which is associated with DNA polymeraseα-primase, promoting efficient telomere replication and contributing to telomere maintenance. Mutations in CTC1 gene cause Coats Plus, dyskeratosis congenita (DC), and related bone marrow failure syndromes. In this study, I used U2-OS cells to investigate the interplay between CTC1 and TERRA. I found that CTC1 depletion doesn’t affect APB formation but leads to an increase of TERRA granules in the cytoplasm. The increased cytoplasmic TERRA granules were only observed in CTC1 deficient cells but not in STN1 deficient cells. Some of the TERRA granules are colocalized with GRSF, mtSSB and Tom20, suggesting that TERRA granules can localize to mitochondria. And also, CST complex deficiency slightly increases TERRA expression level, secretes more cfTERRA-containing exosomes, and induces immune responses. Altogether, I discovered CTC1 plays an important role in regulating TERRA granules in the cytoplasm, however, this function is independent of CST complex activity. It is likely that cfTERRA secretion caused by CST complex dysfunction does not have a strong link with cytosolic TERRA granules accumulation. The biological function of cytosolic TERRA granules is needed to be further investigated.