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.
Highlights
The nuclear organization of eukaryotic cells can play important roles in the regulation of genome function (Pederson, 2002)
Our data reveal a previously uncharacterized, direct correlation between Polymerase II (Pol II) cluster lifetime and the number of nascent mRNA molecules subsequently synthesized. We find that this correlation between Pol II cluster lifetime and nascent mRNA output is predictive in nature, and may be utilized by an experimenter to stall or induce a burst of transcription, at will using a drug treatment
We set out to elucidate the spatiotemporal dynamics of Pol II in live mouse embryonic fibroblasts (MEF) using single-molecule based super-resolution microscopy (Hess et al, 2006; Betzig et al, 2006; Rust et al, 2006)
Summary
The nuclear organization of eukaryotic cells can play important roles in the regulation of genome function (Pederson, 2002). Enzymatic clusters have been reported in many genetic processes in the cell (Carmo-Fonseca, 2002; Cremer et al, 2006; Schneider and Grosschedl, 2007), including replication (Kennedy et al, 2000), DNA repair (Misteli and Soutoglou, 2009), transcription (Cook, 2010; Verschure et al, 1999) and RNA processing (Kumaran et al, 2008) These clusters can form de novo and their stability can be dynamically regulated in vivo making it difficult to capture them and to study their function with mechanistic detail (Sutherland and Bickmore, 2009; Fraser and Bickmore, 2007; Buckley and Lis, 2014). From these fixed cells studies emerged theories interpreting the Pol II clusters as static pre-assemblies termed “transcription factories.” attempts to directly visualize Pol II clusters in living cells had been initially unsuccessful (Sugaya et al, 2000; Kimura et al, 2002), raising a debate over their existence in vivo (Carter et al, 2008; Sutherland and Bickmore, 2009)
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