RNA Polymerase II cluster dynamics predict mRNA output in living cells

Creators: Cho, Won-Ki; Jayanth, Namrata; English, Brian P.; Inoue, Takuma; Andrews, J. Owen; Conway, William; Grimm, Jonathan B.; Spille, Jan-Hendrik; Lavis, Luke D.; Lionnet, Timothée; Cisse, Ibrahim I.

Abstract

Protein clustering is a hallmark of genome regulation in mammalian cells. However, the dynamic molecular processes involved make it difficult to correlate clustering with functional consequences in vivo. We developed a live-cell super-resolution approach to uncover the correlation between mRNA synthesis and the dynamics of RNA Polymerase II (Pol II) clusters at a gene locus. For endogenous β-actin genes in mouse embryonic fibroblasts, we observe that short-lived (~8 s) Pol II clusters correlate with basal mRNA output. During serum stimulation, a stereotyped increase in Pol II cluster lifetime correlates with a proportionate increase in the number of mRNAs synthesized. Our findings suggest that transient clustering of Pol II may constitute a pre-transcriptional regulatory event that predictably modulates nascent mRNA output.

Additional Information

© 2016, Cho et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited. Received: December 8, 2015. Accepted: May 2, 2016. Accepted Manuscript published: May 3, 2016 (version 1). Version of Record published: June 30, 2016 (version 2). We thank Jeff Gore (MIT), Arjun Narayanan (MIT), Jeremy England (MIT), Sebastian Lourido (MIT), Hervé Rouault (Janelia Research Campus) and Arjun Raj (UPenn) for helpful comments. K McGowan, M Ramirez and X Zhang (Janelia Molecular Biology Shared Resource) provided assistance in cloning; H White and Z Ma (Janelia Molecular and Cell Culture Resource) assisted with cell culture and Flow Cytometry sorting. Research reported in this publication was supported by the National Cancer Institute of the National Institutes of Health under Award Number DP2CA195769 to IIC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by funds from the MIT Department of Physics and the Howard Hughes Medical Institute. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Author contributions. W-KC, Acquired and analyzed all data in the main manuscript, Conceived the time dependent mRNA output and cluster lifetime experiments, Conceived the drug experiments, Drafting or revising the article, Contributed unpublished essential data or reagents; NJ, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents; BPE, Conceived the project, Conceived and acquired H2B control data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents; TI, Co-built the original microscopes used for imaging, Acquisition of data, Contributed unpublished essential data or reagents; JOA, Developed the analyses software used, Co-developed the molecular counting analysis, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents; WC, Acquisition of data, Analysis and interpretation of data, Contributed unpublished essential data or reagents; JBG, LDL, Developed the JF dyes, Contributed unpublished essential data or reagents; J-HS, Co-developed the molecular counting analyses, Analysis and interpretation of data, Drafting or revising the article; TL, Conceived the project, Supervised all aspects of the cell engineering and the theoretical modeling, Analysis and interpretation of data, Drafting or revising the article; IIC, Supervised all aspects of the project, Conceived the project, Analysis and interpretation of data, Drafting or revising the article. The author declares that no competing interests exist.

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