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.

Using affinity purification MS approaches, we have identified a novel role for casein kinase II (CKII) in the modification of the polymerase associated factor complex (PAF-C). Our data indicate that the facilitates chromatin transcription complex (FACT) interacts with CKII and may facilitate PAF complex phosphorylation. Posttranslational modification analysis of affinity-isolated PAF-C shows extensive CKII phosphorylation of all five subunits of PAF-C, although CKII subunits were not detected as interacting partners. Consistent with this, recombinant CKII or FACT-associated CKII isolated from cells can phosphorylate PAF-C in vitro, whereas no intrinsic kinase activity was detected in PAF-C samples. Significantly, PAF-C purifications combined with stable isotope labeling in cells (SILAC) quantitation for PAF-C phosphorylation from wild-type and CKII temperature-sensitive strains (cka1⌬ cka2-8) showed that PAF-C phosphorylation at consensus CKII sites is significantly reduced in cka1⌬ cka2-8 strains. Consistent with a role of CKII in FACT and PAF-C function, we show that decreased CKII function in vivo results in decreased levels of histone H2B lysine 123 monoubiquitylation, a modification dependent on FACT and PAF-C. Taken together, our results define a coordinated role of CKII and FACT in the regulation of RNA polymerase II transcription through chromatin via phosphorylation of PAF-C.

The main goal of this project is to identify how cells respond and adapt to genetic changes that alter the process of RNA synthesis. Using baker's yeast as the study system, experimental approaches provide undergraduate students with early exposure to cutting‐edge research technologies that are broadly applicable in both academia and industry. The project introduces students at all levels of training to the importance of quantitative methods to interrogate biology. The specific focus of the research is to understand the consequences of genetic perturbations that knock out or reduce expression of non‐essential elongation factors on RNA polymerase II‐catalyzed RNA synthesis (transcription). Preliminary data suggest that such genetic perturbations lead to significant adaptations within the RNA polymerase II protein‐protein interaction networks. These adaptations may include (but are not limited to) disruption of interactions with RNA polymerase II, compensation leading to novel protein recruitment, and activation of rescue pathways to dispose of arrested RNA polymerase II. The laboratory portion of an undergraduate course in molecular biology was redesigned to generate elongation factor knock‐out yeast strains with a focus on key molecular biology concepts and techniques including: genomic DNA isolation, oligonucleotide design, PCR, genotyping, protein isolation, and western blotting. In addition to these key concepts, we introduced students to the theory and practice of proteomics analysis through mass spectrometry including discussions of bioinformatics analysis of proteomics data, calculation of false discovery rates, and affinity purification mass spectrometry approaches. Thus far in this study, 9 elongation factor knock out strains have been made and verified by DNA sequencing and are in the process of being analyzed by quantitative mass spectrometry. Data from pre‐ and post‐instructional tests will be presented which demonstrate increased student understanding of mass spectrometry. Additionally, students participated in an 8 week summer internship held at the IU School of Medicine where they extended the project from the classroom lab to performing quantitative mass spectrometry analysis of their elongation factor knock‐outs.Support or Funding InformationDePauw University Student Faculty Research Fund, Showalter Fund (IU), NSF Award Number:1515748 PI Amber Mosley, PhD

1 2 RNA Polymerase II (RNAPII) transcription termination is regulated by the 3 phosphorylation status of the C-terminal domain (CTD). Using disruption-compensation 4 (DisCo) protein-protein interaction network analysis, interaction changes were observed 5 within the termination machinery as a consequence of deletion of the serine 5 RNAPII 6 CTD phosphatase Rtr1. Interactions between RNAPII and the cleavage factor IA 7 (CF1A) subunit Pcf11 were reduced in rtr1∆, whereas interactions with the CTD and 8 RNA-binding termination factor Nrd1 were increased. These changes could be the 9 result of altered interactions between the termination machinery and/or increased levels 10 of premature termination of RNAPII. Transcriptome analysis in rtr1∆ cells found 11 decreased pervasive transcription and a shift in balance of expression of sense and 12 antisense transcripts. Globally, rtr1∆ leads to decreases in noncoding RNAs that are 13 linked to the Nrd1, Nab3 and Sen1 (NNS) -dependent RNAPII termination pathway. 14 Genome-wide analysis of RNAPII and Nrd1 occupancy suggests that loss of RTR1 15 leads to increased termination at noncoding genes and increased efficiency of snRNA 16 termination. Additionally, premature termination increases globally at protein-coding 17 genes where NNS is recruited during early elongation. The effects of rtr1∆ on RNA 18 expression levels were erased following deletion of the exosome subunit Rrp6, which 19 works with the NNS complex to rapidly degrade terminated noncoding RNAs. Overall, 20 these data suggest that Rtr1 restricts the NNS-dependent termination pathway in WT 21 cells to prevent premature RNAPII termination of mRNAs and ncRNAs. Additionally, 22Rtr1 phosphatase activity facilitates low-level elongation of noncoding transcripts that 23 impact the transcriptome through RNAPII interference. AUTHOR SUMMARY 1Many cellular RNAs including those that encode for proteins are produced by the 2 enzyme RNA Polymerase II. In this work, we have defined a new role for the 3 phosphatase Rtr1 in the regulation of RNA Polymerase II progression from the start of 4 transcription to the 3' end of the gene where the nascent RNA from protein-coding 5 genes is typically cleaved and polyadenylated. Deletion of the gene that encodes RTR1 6 leads to changes in the interactions between RNA polymerase II and the termination 7 machinery. Rtr1 loss also causes early termination of RNA Polymerase II at many of its 8 target gene types including protein coding genes and noncoding RNAs. Evidence 9 suggests that the premature termination observed in RTR1 knockout cells occurs 10 through the termination factor and RNA binding protein Nrd1 and its binding partner 11 Nab3. Additionally, many of the prematurely terminated noncoding RNA transcripts are 12 degraded by the Rrp6-containing nuclear exosome, a known component of the Nrd1-13 Nab3 termination coupled RNA degradation pathway. These findings suggest that Rtr1 14 normally promotes elongation of RNA Polymerase II transcripts through preventation of 15 Nrd1-directed term...