Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage.

Jonathan Liu,Hernan G Garcia,Donald Hansen,Elizabeth Eck,Meghan Turner,Yang Joon Kim,Simon Alamos,James R Faeder

doi:10.1371/journal.pcbi.1008999

Jonathan Liu, Hernan G Garcia + Show 6 more

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https://doi.org/10.1371/journal.pcbi.1008999

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Abstract

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

Highlights

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript (Fig 1A; [1])
Elongation rates contribute to determining mRNA cleavage and RNA polymerase (RNAP) termination efficiency [5,6,7,8], and functional linkages have been demonstrated between transcription initiation and termination [9, 10]
To quantitatively dissect the transcription cycle in its entirety from live imaging data, we developed a simple model (Fig 1A) in which RNAP molecules are loaded at the promoter of a gene of total length L with a time-dependent loading rate R(t)

Summary

Introduction

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript (Fig 1A; [1]). Each of these three steps can be controlled to regulate transcriptional activity. Binding of transcription factors to enhancers dictates initiation rates [2], modulation of elongation rates helps determine splicing efficiency [3], and regulation of cleavage controls aspects of 3’ processing such as alternative polyadenylation [4]. Elongation rates contribute to determining mRNA cleavage and RNA polymerase (RNAP) termination efficiency [5,6,7,8], and functional linkages have been demonstrated between transcription initiation and termination [9, 10]. In order to dissect the entire transcription cycle, it is necessary to develop a holistic approach that makes it possible to understand how the regulation of each step dictates mRNA production and to unearth potential couplings among these steps

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