Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

J Vis Exp. 2019 Jan 17:(143). doi: 10.3791/58790.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Video-Audio Media

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Cell Survival / drug effects
  • Cells, Cultured
  • Clone Cells
  • Drug Resistance, Microbial / drug effects
  • Humans
  • Mice
  • Neomycin / pharmacology
  • RNA / genetics*
  • RNA, Long Noncoding / genetics*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Telomere / genetics*
  • Telomere / metabolism

Substances

  • RNA, Long Noncoding
  • RNA, Messenger
  • RNA
  • Neomycin