The Non-Canonical CTD of RNAP-II Is Essential for Productive RNA Synthesis in Trypanosoma brucei

Anish Das,Vivian Bellofatto,Laurent Rénia

doi:10.1371/journal.pone.0006959

Abstract

The carboxy-terminal domain (CTD) of the largest subunit (RPB1) of RNA polymerase II (RNAP-II) is essential for gene expression in metazoa and yeast. The canonical CTD is characterized by heptapeptide repeats. Differential phosphorylation of canonical CTD orchestrates transcriptional and co-transcriptional maturation of mRNA and snRNA. Many organisms, including trypanosomes, lack a canonical CTD. In these organisms, the CTD is called a non-canonical CTD or pseudo-CTD (ΨCTD. In the African trypanosome, Trypanosoma brucei, the ΨCTD is ∼285 amino acids long, rich in serines and prolines, and phosphorylated. We report that T. brucei RNAP-II lacking the entire ΨCTD or containing only a 95-amino-acid-long ΨCTD failed to support cell viability. In contrast, RNAP-II with a 186-amino-acid-long ΨCTD maintained cellular growth. RNAP-II with ΨCTD truncations resulted in abortive initiation of transcription. These data establish that non-canonical CTDs play an important role in gene expression.

Highlights

RNA polymerase II (RNAP-II) is the eukaryotic enzyme responsible for synthesis of mRNA
T. brucei YCTD undergoes phosphorylation It has been shown previously that T. brucei RNAP-II is modified by phosphorylation, despite the lack of consensus heptapeptide repeat sequences [20]
To determine whether the phosphorylation occurs within the YCTD, we directly visualized RNAP-II labeled with 32P-orthophosphate

Summary

Introduction

RNA polymerase II (RNAP-II) is the eukaryotic enzyme responsible for synthesis of mRNA. This enzyme is an ,550 kDa complex consisting of twelve subunits, RPB1-12 [1]. The carboxy-terminal domain (CTD) of the largest subunit, RPB1, is essential for cell survival. In well-studied organisms, such as metazoa and yeast, the CTD consists of consensus heptapeptide repeats having the sequence Y1S2P3T4S5P6S7 ([2,3] and reviewed in [4,5]). Non-canonical CTDs, which lack the heptapeptide repeats and are pseudo CTDs (YCTDs), exist in a wide variety of eukaryotic organisms [10,11]. We sought to determine whether a YCTD is essential for gene expression

Methods

Results

Conclusion