RNA Polymerase II Transcription Attenuation at the Yeast DNA Repair Gene, DEF1, Involves Sen1-Dependent and Polyadenylation Site-Dependent Termination

Abstract

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5′-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.

Transcriptional Control by Premature Termination: A Forgotten Mechanism

“…The prematurely terminated transcripts sometimes initiate at a TSS upstream of the protein-coding gene [102] . Interestingly, it was recently shown that the DNA repair gene DEF1 is attenuated by Sen1 and CPA factors, without Nrd1 and Nab3 involvement [106] ; therefore, PTT in S. cerevisiae might not be limited to the NNS pathway. There are no Nrd1/Nab3 homologues known in plants.…”

Transcriptional control by premature termination: a forgotten mechanism

“…Interestingly, it was recently shown that the DNA repair gene DEF1 is attenuated by Sen1 and CPA factors, without Nrd1 and Nab3 involvement [95], therefore PTT in S. cerevisiae might not be limited to the NNS pathway.…”

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif

“…snRNAs, snoRNAs, cryptic unstable transcripts) ( Arndt and Reines 2015 ). Our laboratory more recently characterized an attenuator in the DNA repair gene DEF1 that relies on CF, CPF, and Sen1 without apparent involvement of Nrd1/Nab3, suggesting that yeast attenuation is not constrained to NNS ( Whalen et al 2018 ). We referred to the DEF1 attenuation mechanism as “hybrid” termination, recognizing that others have documented similar CPF-CF and NNS crosstalk in 3′-end gene regions as part of a failsafe mechanism ( Lemay and Bachand 2015 ).…”

“…We initially identified the yeast DNA repair gene DEF1 as an attenuation target based on its usage of both promoter-proximal and promoter-distal pA sites ( Graber et al 2013 ; Whalen et al 2018 ). DEF1 encodes a protein essential for the degradation of Pol II stalled at UV-induced DNA lesions, which provides DNA repair factors with access to their substrate ( Akinniyi and Reese 2021 ).…”