RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination

Abstract

RNA Polymerase II (RNAPII) transcription termination is regulated by the phosphorylation status of the C-terminal domain (CTD). The phosphatase Rtr1 has been shown to regulate serine 5 phosphorylation on the CTD; however, its role in the regulation of RNAPII termination has not been explored. As a consequence of RTR1 deletion, interactions within the termination machinery and between the termination machinery and RNAPII were altered as quantified by Disruption-Compensation (DisCo) network analysis. Of note, interactions between RNAPII and the cleavage factor IA (CF1A) subunit Pcf11 were reduced in rtr1Δ, whereas interactions with the CTD and RNA-binding termination factor Nrd1 were increased. Globally, rtr1Δ leads to decreases in numerous noncoding RNAs that are linked to the Nrd1, Nab3 and Sen1 (NNS)-dependent RNAPII termination pathway. Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 leads to increased termination at noncoding genes. Additionally, premature RNAPII termination increases globally at protein-coding genes with a decrease in RNAPII occupancy occurring just after the peak of Nrd1 recruitment during early elongation. The effects of rtr1Δ on RNA expression levels were lost following deletion of the exosome subunit Rrp6, which works with the NNS complex to rapidly degrade a number of noncoding RNAs following termination. Overall, these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT cells to prevent premature termination of mRNAs and ncRNAs. Rtr1 facilitates low-level elongation of noncoding transcripts that impact RNAPII interference thereby shaping the transcriptome.

Biogenesis of RNA Polymerases in Yeast

“…Rtr1 ("regulator of transcription" 1) has been described as a phosphorylated RNA pol II interactor by acting as an S5-P CTD phosphatase during the transition from the initiation of the transcription to elongation in vivo (Gibney et al, 2008;Mosley et al, 2009Mosley et al, , 2013Hsu et al, 2014;Smith-Kinnaman et al, 2014;Hunter et al, 2016). Additional roles have been proposed for Rtr1 in transcription and mRNA stability (Mosley et al, 2013;Hsu et al, 2014;Hodko et al, 2016;Victorino et al, 2020) (our unpublished data).…”

Boosting detection of low abundance proteins in thermal proteome profiling experiments by addition of an isobaric trigger channel to TMT multiplexes

“…Previously performed experiments found that the entire CPF complex copurifies with FLAG-tagged Pta1 35 . In theory, addition of an affinity purified CPF sample to one channel of the TMT multiplex would increase the MS1 ion intensity of CPF subunits and would “trigger” the mass spectrometer to pick peptides from CPF complex subunits more often in a DDA analysis than in samples that lack an isobaric trigger.…”

“…To boost detection of the native CPF subunits, subsequent mTPP replicates of WT and ssu72-2 included the addition of a trigger channel consisting of an affinity-purified CPF complexes. Affinity purification of CPF via Pta1-FLAG was performed as described previously for Ssu72-FLAG purifications 35 . The Pta1-FLAG affinity purified sample was added at a ratio of 6.25 ug trigger to 50 ug of the lowest heat-treated sample (1:8 ratio) for the initial study.…”

“…As a proof-of-concept, we investigated the ability of this approach to enhance detection of the relatively low abundance protein complex cleavage and polyadenylation factor (CPF) complex in a mTPP workflow. Affinity purified CPF that we have previously characterized [35][36][37][38][39] was incorporated as an isobaric trigger channel into our mTPP workflow at a ratio to the lowest heat-treated mTPP sample of ~1:8 and ~1:50. Using this approach, a significant increase in the abundance of CPF complex members was observed, including those that were not readily identified without the isobaric trigger channel.…”

Boosting Detection of Low-Abundance Proteins in Thermal Proteome Profiling Experiments by Addition of an Isobaric Trigger Channel to TMT Multiplexes

“…Affinity purification of native CPF via Pta1-FLAG was performed as described previously for Ssu72-FLAG purifications. 37 The Pta1-FLAG affinity-purified sample was added at a ratio of 6.25 μg trigger to 50 μg of the lowest heat-treated sample (1:8 ratio) for the biological replicates. The untreated samples were removed from the multiplex from no trigger samples to accommodate for the isobaric trigger channel to be labeled with TMT126.…”