Novak Richard, Hart Kristina, Mathies Richard A
UCSF/UC Berkeley Graduate Program in Bioengineering, University of California, Berkeley, CA 94720, USA.
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Nucleic Acids Res. 2015 Sep 18;43(16):e104. doi: 10.1093/nar/gkv477. Epub 2015 Jul 21.
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.
端粒酶是一种逆转录酶，可维持染色体末端的端粒，使快速分裂的细胞得以增殖，同时避免衰老和凋亡。在单细胞水平上了解端粒酶基因的表达和剪接，有助于深入了解端粒酶在正常细胞生长以及癌症发展过程中的作用。在此，我们采用基于微滴的单细胞培养，随后进行单细胞或集落转录本丰度分析，以研究编码RNA组分（hTR）和蛋白质组分（hTERT）的端粒酶基因的转录本丰度与细胞生长之间的关系，以及hTERT的剪接情况。在正常细胞培养条件下以及暴露于姜黄素（一种具有抗癌和降低端粒酶活性特性的天然化合物）期间，对Jurkat和K562细胞进行了检测。单个细胞主要表达单一的hTERT剪接变体，α+/β-变体表现出显著的转录本丰度双峰性，且这种双峰性在细胞分裂过程中持续存在。亚致死剂量的姜黄素暴露会导致所有hTERT剪接变体的双峰性降低，以及α剪接的显著上调，这表明其在细胞应激反应中可能发挥作用。本文介绍的单细胞培养和转录本丰度分析方法提供了多参数单细胞分析所需的工具，这对于理解异质细胞群体、疾病细胞群体及其药物反应的表型至关重要。
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