Transcription elongation is finely tuned by dozens of regulatory factors.

Understanding the complex network that regulates transcription elongation requires the quantitative analysis of RNA polymerase II (Pol II) activity in a wide variety of regulatory environments. We performed native elongating transcript sequencing (NET-seq) in 41 strains of lacking known elongation regulators, including RNA processing factors, transcription elongation factors, chromatin modifiers, and remodelers. We found that the opposing effects of these factors balance transcription elongation and antisense transcription.

Live-Cell Assays for Cell Stress Responses Reveal New Patterns of Cell Signaling Caused by Mutations in Rhodopsin, α-Synuclein and TDP-43.

Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling, we created live-cell assays for endoplasmic reticulum (ER)-mediated cell stress and second messenger signaling. We first examined neurodegenerative mutations associated with direct ER stress by exploring the effect of rhodopsin mutations on ER stress and Ca signaling.

Cell-Cycle Modulation of Transcription Termination Factor Sen1.

Many non-coding transcripts (ncRNA) generated by RNA polymerase II in S. cerevisiae are terminated by the Nrd1-Nab3-Sen1 complex. However, Sen1 helicase levels are surprisingly low compared with Nrd1 and Nab3, raising questions regarding how ncRNA can be terminated in an efficient and timely manner.

The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain.

The carboxy-terminal domain (CTD) extends from the largest subunit of RNA polymerase II (Pol II) as a long, repetitive and largely unstructured polypeptide chain. Throughout the transcription process, the CTD is dynamically modified by post-translational modifications, many of which facilitate or hinder the recruitment of key regulatory factors of Pol II that collectively constitute the 'CTD code'. Recent studies have revealed how the physicochemical properties of the CTD promote phase separation in the presence of other low-complexity domains.

Subgenic Pol II interactomes identify region-specific transcription elongation regulators.

Transcription, RNA processing, and chromatin-related factors all interact with RNA polymerase II (Pol II) to ensure proper timing and coordination of transcription and co-transcriptional processes. Many transcription elongation regulators must function simultaneously to coordinate these processes, yet few strategies exist to explore the complement of factors regulating specific stages of transcription. To this end, we developed a strategy to purify Pol II elongation complexes from subgenic regions of a single gene, namely the 5' and 3' regions, using sequences in the nascent RNA.

Comprehensive RNA Polymerase II Interactomes Reveal Distinct and Varied Roles for Each Phospho-CTD Residue.

Transcription controls splicing and other gene regulatory processes, yet mechanisms remain obscure due to our fragmented knowledge of the molecular connections between the dynamically phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD) and regulatory factors. By systematically isolating phosphorylation states of the CTD heptapeptide repeat (Y1S2P3T4S5P6S7), we identify hundreds of protein factors that are differentially enriched, revealing unappreciated connections between the Pol II CTD and co-transcriptional processes. These data uncover a role for threonine-4 in 3' end processing through control of the transition between cleavage and termination.