Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
Contributed by Thomas R. Cech, July 19, 2019 (sent for review May 16, 2019; reviewed by Steven Artandi and Karen L. Beemon)
Significance
Telomerase, which extends DNA at chromosome ends, is composed of an RNA template and a catalytic protein subunit, telomerase reverse transcriptase (TERT). TERT gene expression has been of great interest because it is required for proliferation of most cancers, but expression investigations have been limited due to low endogenous mRNA levels. Here, we employ single-molecule RNA fluorescent in situ hybridization (FISH) in 10 human cancer cell lines and make findings that are unanticipated based upon bulk TERT mRNA measurements. For example, there is great cell-to-cell variation in the number of transcription sites, and spliced TERT mRNA has greater nuclear localization than cytoplasmic. Overall, our findings provide insights regarding TERT expression, localization patterns, and variability in cancer on a single-cell level.
Abstract
Telomerase is pathologically reactivated in most human cancers, where it maintains chromosomal telomeres and allows immortalization. Because telomerase reverse transcriptase (TERT) is usually the limiting component for telomerase activation, numerous studies have measured TERT mRNA levels in populations of cells or in tissues. In comparison, little is known about TERT expression at the single-cell and single-molecule level. To address this, we analyzed TERT expression across 10 human cancer lines using single-molecule RNA fluorescent in situ hybridization (FISH) and made several unexpected findings. First, there was substantial cell-to-cell variation in number of transcription sites and ratio of transcription sites to gene copies. Second, previous classification of lines as having monoallelic or biallelic TERT expression was found to be inadequate for capturing the TERT gene expression patterns. Finally, spliced TERT mRNA had primarily nuclear localization in cancer cells and induced pluripotent stem cells (iPSCs), in stark contrast to the expectation that spliced mRNA should be predominantly cytoplasmic. These data reveal unappreciated heterogeneity, complexity, and unconventionality in TERT expression across human cancer cells.
Footnotes
↵1T.J.R. and G.D. contributed equally to this work.
- ↵2To whom correspondence may be addressed. Email: thomas.cech{at}colorado.edu.
Author contributions: T.J.R., G.D., J.L.R., and T.R.C. designed research; T.J.R., G.D., and E.P.H. performed research; T.J.R., G.D., E.P.H., J.L.R., and T.R.C. analyzed data; and T.J.R., G.D., E.P.H., J.L.R., and T.R.C. wrote the paper.
Reviewers: S.A., Stanford University; and K.L.B., Johns Hopkins University.
Conflict of interest statement: T.R.C. is on the board of directors of Merck, Inc., and a consultant for Storm Therapeutics, neither of which provided funding for this study.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1908275116/-/DCSupplemental.
Published under the PNAS license.
Single-cell imaging reveals unexpected heterogeneity of telomerase reverse transcriptase expression across human cancer cell lines
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