RESUMO
In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs.
Assuntos
Imagem Molecular/métodos , RNA Fúngico/genética , RNA não Traduzido/genética , RNA/genética , Saccharomyces cerevisiae/genética , Telomerase/genética , Sequências Repetitivas de Ácido Nucleico , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Telomerase, the enzyme that elongates telomeres in most eukaryotes, is a ribonucleoprotein complex composed of a reverse transcriptase catalytic subunit (TERT in human, Est2 in the budding yeast S. cerevisiae), regulatory factors and a noncoding RNA called hTERC (in human) or TLC1 (in budding yeast). Telomerase trafficking is a major process in the biogenesis and regulation of telomerase action at telomeres. Due to its higher signal-to-noise ratio, imaging of the telomerase RNA moiety is frequently used to determine telomerase intracellular localization. Here we describe how to image telomerase RNA in human and yeast cells using fluorescence in situ hybridization.