Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair

Hélène Gaillard,Alfonso Rodríguez-Gil,Sebastián Chávez,Cristina González-Aguilera,Cristina Tous,Joaquín Ariño,María L García-Rubio,Maria José Quintero,Javier Botet,José Luis Revuelta,Laia Viladevall,Antonio Marín,Andrés Aguilera

doi:10.1371/journal.pgen.1000364

Abstract

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation.

Highlights

Synthesis of an RNA transcript by RNA polymerase II (RNAPII) requires the successful completion of at least four steps in the transcription cycle: promoter binding and initiation, promoter clearance, elongation, and termination
MRNA levels that were at least 2-fold above or below mock treated cells were found for 376 genes in cells treated with 4-NQO and for 295 genes in cells treated with mycophenolic acid (MPA) (Table S1)
Of the genes affected by either treatment, very few were coincident, which is consistent with the fact that 4-NQO and MPA affect different cellular processes (Figure 1A)

Summary

Introduction

Synthesis of an RNA transcript by RNA polymerase II (RNAPII) requires the successful completion of at least four steps in the transcription cycle: promoter binding and initiation, promoter clearance, elongation, and termination. While many studies have focused on the regulation of initiation, more recent studies have demonstrated that transcription elongation is a dynamic and highly regulated stage of the transcription cycle capable of coordinating downstream events. Numerous factors have been identified that target elongation [1]. It appears that distinct factors act in specific transcriptional contexts; the requirements of these factors are largely unknown and highlight the need to improve our understanding of elongation in vivo. Several lines of evidence indicate that transcript elongation by RNAPII involves frequent pausing and stalling, and an important role of the many accessory factors may be to minimize the negative impact of such events on transcription [4]

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Discussion

Conclusion