Strand-specific, high-resolution mapping of modified RNA polymerase II.

Laura Milligan,Elisabeth Petfalski,Guido Sanguinetti,Rodrigo Lombraña,David Tollervey,Alex Tuck,Grzegorz Kudla,Vân A Huynh‐Thu,Clémentine Delan‐Forino

doi:10.15252/msb.20166869

Abstract

Reversible modification of the RNAPII C‐terminal domain links transcription with RNA processing and surveillance activities. To better understand this, we mapped the location of RNAPII carrying the five types of CTD phosphorylation on the RNA transcript, providing strand‐specific, nucleotide‐resolution information, and we used a machine learning‐based approach to define RNAPII states. This revealed enrichment of Ser5P, and depletion of Tyr1P, Ser2P, Thr4P, and Ser7P in the transcription start site (TSS) proximal ~150 nt of most genes, with depletion of all modifications close to the poly(A) site. The TSS region also showed elevated RNAPII relative to regions further 3′, with high recruitment of RNA surveillance and termination factors, and correlated with the previously mapped 3′ ends of short, unstable ncRNA transcripts. A hidden Markov model identified distinct modification states associated with initiating, early elongating and later elongating RNAPII. The initiation state was enriched near the TSS of protein‐coding genes and persisted throughout exon 1 of intron‐containing genes. Notably, unstable ncRNAs apparently failed to transition into the elongation states seen on protein‐coding genes.

Highlights

Eukaryotic RNA polymerase II (RNAPII) is a large, multisubunit complex
The RNAPII C-terminal domain (CTD) shows dynamic changes in post-translational modification (PTM) as the polymerase traverses protein-coding genes in yeast, which can be detected using a set of antibodies that each recognize a specific phosphorylation
After quality filtering and removal of PCR duplicates, more than 90% of these sequences were mapped to genes transcribed by RNAPII, including mRNAs, several lncRNAs classes (CUTs, SUTs, NUTs, XUTs, and antisense transcripts), small nucleolar RNAs, and small nuclear RNAs, indicating specific recovery of RNAPII transcripts (Fig 1B)

Summary

Introduction

Eukaryotic RNA polymerase II (RNAPII) is a large, multisubunit complex. The largest RNAPII subunit, termed Rpo in yeast, has a catalytic N-terminal domain (NTD) that performs nucleotide polymerization, and a regulatory C-terminal domain (CTD), consisting of multiple heptad (consensus YSPTSPS) repeats (26 in yeast, 52 in humans). The RNAPII CTD shows dynamic changes in PTMs as the polymerase traverses protein-coding genes in yeast (see, e.g., Mayer et al, 2010), which can be detected using a set of antibodies that each recognize a specific phosphorylation (reviewed in Eick & Geyer, 2013; Heidemann et al, 2013) Such antibodies have been used to assess the distribution of the different modifications across individual transcription units in ChIP analyses, or across many transcription units in ChIP-Seq. Such antibodies have been used to assess the distribution of the different modifications across individual transcription units in ChIP analyses, or across many transcription units in ChIP-Seq These analyses have been highly informative and have played important roles in our current understanding of eukaryotic gene expression.

Methods

Results

Conclusion