The Role of Ctk1 Kinase in Termination of Small Non-Coding RNAs

Tineke L Lenstra,Dik Van Leenen,Sandra Clauder,Frank C P Holstege,Patrick Kemmeren,Domenico Libri,Lars M Steinmetz,Zhenyu Xu,Agnieszka Tudek,Spyridon T Pachis,Grzegorz Kudla

doi:10.1371/journal.pone.0080495

Tineke L Lenstra, Dik Van Leenen + Show 9 more

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https://doi.org/10.1371/journal.pone.0080495

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Journal: PloS one	Publication Date: Dec 4, 2013
Citations: 16	License type: CC BY 3.0

Affiliation: University Medical Center Utrecht

Abstract

Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) via the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.

Highlights

Transcription termination is essential for generating normal transcripts and for the release of RNA Polymerase II (Pol II) from the DNA template, which prevents interference between adjacent transcription units
The results show that removal of the carboxy-terminal domain (CTD)-Ser2 phosphorylation (Ser2P) kinase Ctk1 results in readthrough at many snoRNAs, cryptic unstable transcripts (CUTs), stable uncharacterized transcripts (SUTs) and some mRNAs
The possibility that this is caused by residual Ser2P has been ruled out, since the rpb1-S2A mutant shows similar expression changes as ctk1D, lacking extensive additional changes compared to ctk1D that would be expected upon a global termination defect

Summary

Introduction

Transcription termination is essential for generating normal transcripts and for the release of RNA Polymerase II (Pol II) from the DNA template, which prevents interference between adjacent transcription units. Transcription of mRNA coding genes is terminated by the Cleavage and Polyadenylation Factor/Cleavage factor I and II (CPF/CFI-II) that cleaves the nascent RNA and polyadenylates the released transcript. The Nrd1p-Nab3p-Sen1p (NNS) complex terminates transcription of sn-/snoRNAs and cryptic unstable transcripts (CUTs), one of the main products of hidden transcription. NNS-dependent termination is tightly coupled to nuclear RNA processing by the Rrp6p/exosome complex [1,2], which leads to trimming of primary sn-/snoRNA transcripts and full degradation of CUTs. the choice of termination pathway is essential to determine the fate of transcripts, which are stable and can be exported for translation in the case of the CPF/CFI-II pathway but are generally unstable when generated by the NNS pathway [1,2,3,4]

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