Abstract
Telomerase is a reverse transcriptase that maintains telomeres on the ends of chromosomes, allowing rapidly dividing cells to proliferate while avoiding senescence and apoptosis. Understanding telomerase gene expression and splicing at the single cell level could yield insights into the roles of telomerase during normal cell growth as well as cancer development. Here we use droplet-based single cell culture followed by single cell or colony transcript abundance analysis to investigate the relationship between cell growth and transcript abundance of the telomerase genes encoding the RNA component (hTR) and protein component (hTERT) as well as hTERT splicing. Jurkat and K562 cells were examined under normal cell culture conditions and during exposure to curcumin, a natural compound with anti-carcinogenic and telomerase activity-reducing properties. Individual cells predominantly express single hTERT splice variants, with the α+/β− variant exhibiting significant transcript abundance bimodality that is sustained through cell division. Sub-lethal curcumin exposure results in reduced bimodality of all hTERT splice variants and significant upregulation of alpha splicing, suggesting a possible role in cellular stress response. The single cell culture and transcript abundance analysis method presented here provides the tools necessary for multiparameter single cell analysis which will be critical for understanding phenotypes of heterogeneous cell populations, disease cell populations and their drug response.
Highlights
Telomerase is a ribonucleoprotein complex that maintains telomeres at the ends of chromosomes through reverse transcription [1,2]
Telomeres shorten with each cell division, and their maintenance is a key requirement for avoiding apoptosis
To simultaneously measure targets with a wide range of abundance levels in single cells, we developed and optimized a multiplex RT-polymerase chain reaction (PCR) assay followed by hemi-nested PCR reamplification
Summary
Telomerase is a ribonucleoprotein complex that maintains telomeres at the ends of chromosomes through reverse transcription [1,2]. The human enzyme consists of an RNA component, hTR, and a protein component, hTERT, in addition to other factors. HTR acts as a template for reverse transcription, and hTERT provides the catalytic activity as well as various binding sites for other proteins involved in telomere maintenance [2,6]. In addition to regulating telomerase activity via hTERT levels, hTERT mRNA can be subject to alternative splicing that results in catalytically inactive protein [9]. Several studies have shown non-enzymatic roles for telomerase, including some of the alternative splice variants [9]. Alpha and beta splice variants are the most frequently observed as well as the best studied, and both have been shown to inhibit telomerase activity [9,11,12]. This study examines only these most common four splice variants
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