Avogaro Laura, Oss Pegorar Claudio, Bettin Nicole, Cusanelli Emilio
Laboratory of Cell Biology and Molecular Genetics, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento.
Laboratory of Cell Biology and Molecular Genetics, Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento; Department of Life Sciences, University of Trieste.
J Vis Exp. 2019 Jan 17(143). doi: 10.3791/58790.
Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.
端粒会发生转录，产生包含端粒重复序列的长链非编码RNA（TERRA），有人提出TERRA在端粒生物学中发挥重要作用，包括异染色质形成和端粒长度稳态。最近的研究发现表明，TERRA分子还与染色体内部区域相互作用，以调节小鼠胚胎干细胞（ES细胞）中的基因表达。基于这一证据，RNA荧光原位杂交（RNA-FISH）分析表明，只有一部分TERRA转录本定位于染色体末端。更好地了解TERRA分子的动态变化将有助于明确其功能和作用机制。在此，我们描述了一种使用MS2-GFP系统在癌细胞中标记并可视化单端粒TERRA转录本的方法。为此，我们提供了一个方案，利用AGS人胃癌细胞系生成稳定克隆，该细胞系在单个亚端粒处整合了MS2序列。从带有MS2标签的端粒转录TERRA会导致表达带有MS2标签的TERRA分子，当与绿色荧光蛋白（GFP）融合的MS2 RNA结合蛋白共表达时，这些分子可通过活细胞荧光显微镜观察到。这种方法使研究人员能够研究癌细胞中单端粒TERRA分子的动态变化，并可应用于其他细胞系。
