人類細胞中 TERRA 轉錄體之全面分析

Abstract

TERRA（Telomeric repeats containing RNAs），是起源於端粒序列的長非編碼RNA，已知是一種表觀遺傳調節因子，並在端粒功能中扮演重要角色。在人類基因組中，已知次端粒具有GpG islands含有重複的61-29-37 nt序列模式，具有啟動子功能並與TERRA轉錄相關聯。儘管已經識別了這些TERRA啟動子，TERRA轉錄體於每條染色體上的精確轉錄起始位點、全長RNA序列和表達水平仍然不清楚。在這項研究中，我利用探針純化了U2OS細胞中的TERRA，並採用Illumina和Nanopore平台测序，所得之序列讀值不只有端粒重複序列，也包含特定染色體的次端粒序列，能夠定義和量化特定染色體末端的TERRA轉錄區域。通過將讀數對應到提供完整人類端粒序列組合的CHM13基因組，發現大多數TERRA轉錄體來自帶有GpG islands啟動子的次端粒區域，另外，本研究也發現ITSs區域會表現的TERRA。本研究還構建了染色體末端的TERRA表達區域，並開發了一個RNA-seq pipeline，使用來自人類骨肉瘤和與血液組織的RNA-seq數據集來測量染色體特異性TERRA水平。通過這個分析，闡明了TERRA水平與ALT活性和衰老之間的相關性。意外的是，本研究中還發現了TERRA與核糖體RNA的融合轉錄體，表明端粒序列插入到人類基因組的核醣體基因作當中。這些發現提供了對TERRA轉錄體的全面描述，並為未來研究TERRA功能和轉錄調節奠定了基礎。

Telomeric repeats containing RNA, TERRA, a long non-coding RNA that is transcribed from telomeres, which is known as an epigenetic regulator and plays a crucial role in telomere functions. In humans, subtelomeric GpG islands containing the 61-29-37 repetitive sequence motif show promoter activity and regulate TERRA transcription. Despite the discovery of these TERRA promoters, the precise transcriptional start sites, full sequences, and the expression of TERRA from different telomeres remain unclear. In this study, TERRA was purified using C-rich antisense probes (TERRA-capture) in U2OS cells, and both short- and long-read sequencing methods, Illumina and Nanopore platforms, respectively, were employed. Long-read direct RNA sequencing enables the identification and quantification of TERRA transcription regions on specific chromosome ends. By aligning the reads to the CHM13 reference genome, which provides comprehensive subtelomere information, I found that the majority of TERRA transcripts are derived from chromosome ends with 61-29-37 repeat promoters in the subtelomeric regions. In addition, TERRA originating from the interstitial telomeric repeat sequences was also identified. Moreover, TERRA expression profiles were constructed for each chromosome end, and a new bioinformatic pipeline was developed to measure chromosome-specific TERRA levels using RNA-seq datasets from human osteosarcomas and blood cells collected from people of different ages. Through this analysis, telomere-specific TERRA expression levels were quantified. A positive correlation between TERRA level and alternative lengthening of telomeres (ALT) activity was observed. Interestingly, TERRA expression in human blood cells is associated with human aging. Unexpectedly, chimeric transcripts of TERRA and ribosomal RNA were discovered in cancer cell lines, suggesting the insertion of telomeric sequences into rDNA in the human genome in cancers. These findings provide a comprehensive characterization of the TERRA transcriptome and establish a foundation for future studies on the functions and transcriptional regulation of TERRA.