The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach

Yasuyuki Ohkawa,Taro Tachibana,Kazumitsu Maehara,Tomohiko Yoshimi,Akihito Harada,Seiji Okada,Jun Odawara,Koichi Akashi

doi:10.1186/1471-2164-12-516

Yasuyuki Ohkawa, Taro Tachibana + Show 6 more

Open Access

https://doi.org/10.1186/1471-2164-12-516

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Journal: BMC Genomics	Publication Date: Oct 20, 2011
Citations: 57	License type: cc-by

Affiliation: Kyushu University, Osaka City University

Abstract

BackgroundCellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII), which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq.ResultsWe first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII.ConclusionsWe demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII.

Highlights

Cellular function is regulated by the balance of stringently regulated amounts of mRNA
The accuracy of RNASeq is improved by permitting a small number of ‘multihits’ To understand the transcriptional regulation mechanisms mediated by RNA polymerase II (RNAPII), it was necessary to evaluate mRNA expression as accurately and quantitatively as possible
We studied the association between mRNA expression level and RNAPII phosphorylation state in Hela cells using a deep sequencer for RNAseq and ChIPseq analysis

Summary

Introduction

Cellular function is regulated by the balance of stringently regulated amounts of mRNA. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. In mouse ES cells, Ser phosphorylated and Ser unphosphorylated RNAPII accumulates around the TSSs in bivalent genes [11] These genes, as differentiation markers, can be detected at low levels, despite their association with pluripotency [12]. High throughput sequencing technology and cDNA analysis have emerged as revolutionary tools in recent years, but whether these sequencing data come from active transcription or pausing state genes, and the genome-wide phosphorylation status of RNAPII in vivo, have not been studied. Evaluation of the relationship between the phosphorylation state of RNAPII and mRNA expression level will permit the identification of those genes that are actively transcribed and those that are pausing

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Conclusion