Simultaneous Measurement of Transcriptional and Post-transcriptional Parameters by 3′ End RNA-Seq

Manfred Schmid,Agnieszka Tudek,Torben Heick Jensen

doi:10.1016/j.celrep.2018.07.104

Abstract

SummaryCellular RNA levels are determined by transcription and decay rates, which are fundamental in understanding gene expression regulation. Measurement of these two parameters is usually performed independently, complicating analysis as well as introducing methodological biases and batch effects that hamper direct comparison. Here, we present a simple approach of concurrent sequencing of S. cerevisiae poly(A)+ and poly(A)− RNA 3′ ends to simultaneously estimate total RNA levels, transcription, and decay rates from the same RNA sample. The transcription data generated correlate well with reported estimates and also reveal local RNA polymerase stalling and termination sites with high precision. Although the method by design uses brief metabolic labeling of newly synthesized RNA with 4-thiouracil, the results demonstrate that transcription estimates can also be gained from unlabeled RNA samples. These findings underscore the potential of the approach, which should be generally applicable to study a range of biological questions in diverse organisms.

Highlights

RNA transcription and decay determine cellular RNA levels, and their changes are important contributors to gene expression responses during cellular transition
Cellular RNA levels are determined by transcription and decay rates, which are fundamental in understanding gene expression regulation
We present a simple approach of concurrent sequencing of S. cerevisiae poly(A)+ and poly(A)À RNA 30 ends to simultaneously estimate total RNA levels, transcription, and decay rates from the same RNA sample

Summary

Introduction

RNA transcription and decay determine cellular RNA levels, and their changes are important contributors to gene expression responses during cellular transition. Owing to the dense organization of the S. cerevisiae genome, many of the mentioned transcription units (TUs) are in immediate proximity and frequently overlap on opposite strands. Analysis of their transcription and RNA turnover relies critically on strand-specific high-resolution methodologies. This consideration applies to most biological systems given the generality of pervasive transcription (Jensen et al, 2013)

Methods

Results

Discussion

Conclusion