Protein Degradation of RNA Polymerase II-Association Factor 1(PAF1) Is Controlled by CNOT4 and 26S Proteasome.

Hwa-Young Sun,Nari Kim,Joo-Yeon Yoo,Cheol-Sang Hwang,Tae-Young Roh

doi:10.1371/journal.pone.0125599

Abstract

The PAF complex (PAFc) participates in various steps of the transcriptional process, from initiation to termination, by interacting with and recruiting various proteins to the proper locus for each step. PAFc is an evolutionarily conserved, multi-protein complex comprising PAF1, CDC73, CTR9, LEO1, yRTF1 and, in humans, hSKI8. These components of PAFc work together, and their protein levels are closely interrelated. In the present study, we investigated the mechanism of PAF1 protein degradation. We found that PAF1 protein levels are negatively regulated by the expression of CNOT4, an ortholog of yNOT4 and a member of the CCR4-NOT complex. CNOT4 specifically controls PAF1 but not other components of PAFc at the protein level by regulating the polyubiquitination of PAF1 and its subsequent degradation by the 26S proteasome. The degradation of PAF1 was found to require nuclear localization, as no PAF1 degradation by CNOT4 and the 26S proteasome was observed with NLS (nucleus localization signal)-deficient PAF1 mutants. However, chromatin binding by PAF1 was not necessary for 26S proteasome- or CNOT4-mediated degradation. Our results suggest that CNOT4 controls the degradation of chromatin-unbound PAF1 via the 26S proteasome.

Highlights

Transcription is a highly complicated biochemical process which RNA is synthesized from a DNA template by RNA polymerase
Because the recruitment of PAF complex (PAFc) to chromatin is impaired in Not4-defecient S. cerevisiae [10], we first examined the potential regulatory effect of the CCR4-NOT complex on the PAF1 protein
The RING domain of yNOT4 was previously reported to be responsible for its E3 ligase enzymatic activity and was required to mediate the stability of target proteins [9]; we examined the contribution of the RING domain of CNOT4 using the CNOT4 (ΔRING) mutant

Summary

Introduction

Transcription is a highly complicated biochemical process which RNA is synthesized from a DNA template by RNA polymerase. To properly respond and adapt to internal and external environmental changes, the production, assembly, sub-cellular localization, and degradation of transcription-associated complexes are tightly controlled [1, 2]. Post-translational modifications of histones or the transcriptional machinery can adjust the association of these molecules with RNA polymerase in the chromatin [3, 4]. CCR4-NOT is an evolutionarily conserved multi-functional protein complex that functions in many aspects of transcriptional regulation, primarily controlling mRNA and protein life cycles from synthesis to degradation [5, 6]. PLOS ONE | DOI:10.1371/journal.pone.0125599 May 1, 2015

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