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 Saccharomyces cerevisiae 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. Different sets of factors tightly regulate Pol II progression across gene bodies so that Pol II density peaks at key points of RNA processing. These regulators control where Pol II pauses with each obscuring large numbers of potential pause sites that are primarily determined by DNA sequence and shape. Antisense transcription varies highly across the regulatory landscapes analyzed, but antisense transcription in itself does not affect sense transcription at the same locus. Our findings collectively show that a diverse array of factors regulate transcription elongation by precisely balancing Pol II activity.

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 Ca2+ signaling. The rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa (RP), produced increased ER stress levels compared to wild type (WT) rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca2+ signaling in a stress-dependent manner. Analysis of single-cell Ca2+ signaling profiles revealed unique Ca2+ signaling responses exist in cells expressing WT or P23H rhodopsin, consistent with the idea that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in neurodegenerative diseases that may not have a direct effect on ER-mediated cell stress, we examined how various mutants of α-synuclein and TDP-43 affected ER stress. Mutants of both α-synuclein and TDP-43 associated with Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS) demonstrated increased ER stress compared to WT proteins. To examine the effect of α-synuclein and TDP-43 mutants on cellular signaling, we created a second live-cell assay to monitor changes in cAMP signaling during expression of various forms of α-synuclein and TDP-43. The increased cell stress caused by expression of the mutant proteins was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors for cellular stress and signaling can provide nuanced, new views of neurodegenerative disease processes.

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. We show that Sen1 levels increase during the S and G2 phases of the cell cycle, leading to increased termination activity of NNS. Overexpression of Sen1 or failure to modulate its abundance by ubiquitin-proteasome-mediated degradation greatly decreases cell fitness. Sen1 toxicity is suppressed by mutations in other termination factors, and NET-seq analysis shows that its overexpression leads to a decrease in ncRNA production and altered mRNA termination. We conclude that Sen1 levels are carefully regulated to prevent aberrant termination. We suggest that ncRNA levels and coding gene transcription termination are modulated by Sen1 to fulfill critical cell cycle-specific functions.

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. Here, we discuss the intricacies of the CTD code and how the newly characterized physicochemical properties of the CTD expand the function of the CTD beyond the code.

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. Applying this strategy to Saccharomyces cerevisiae, we determined the specific set of factors that interact with Pol II at precise stages during transcription. We identify many known region-specific factors as well as determine unappreciated associations of regulatory factors during early and late stages of transcription. These data reveal a role for the transcription termination factor, Rai1, in regulating the early stages of transcription genome-wide and support the role of Bye1 as a negative regulator of early elongation. We also demonstrate a role for the ubiquitin ligase, Bre1, in regulating Pol II dynamics during the latter stages of transcription. These data and our approach to analyze subgenic transcription elongation complexes will shed new light on the myriad factors that regulate the different stages of transcription and coordinate co-transcriptional processes.

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. Furthermore, serine-5 phosphorylation seeds spliceosomal assembly immediately downstream of 3' splice sites through a direct interaction with spliceosomal subcomplex U1. Strikingly, threonine-4 phosphorylation also impacts splicing by serving as a mark of co-transcriptional spliceosome release and ensuring efficient post-transcriptional splicing genome-wide. Thus, comprehensive Pol II interactomes identify the complex and functional connections between transcription machinery and other gene regulatory complexes.

Mechanism of membrane permeation induced by synthetic ß-hairpin peptides.

We have investigated the membrane destabilizing properties of synthetic amphiphilic cationic peptides, MAX1 and MAX35, which have the propensity to form ß-hairpin structures under certain conditions, and a control non-ß-hairpin-forming peptide MAX8V16E. All three peptides bind to liposomes containing a mixture of zwitterionic POPC and negatively charged POPS lipids as determined by Zeta potential measurements. Circular dichroism measurements indicated folding of MAX1 and MAX35 in the presence of the POPC/POPS liposomes, whereas no such folding was observed with MAX8V16E. There was no binding or folding of these peptides to liposomes containing only POPC. MAX1 and MAX35 induced release of contents from negatively charged liposomes, whereas MAX8V16E failed to promote solute release under identical conditions. Thus, MAX1 and MAX35 bind to, and fold at the surface of negatively charged liposomes adopting a lytic conformation. We ruled out leaky fusion as a mechanism of release by including 2 mol % PEG-PE in the liposomes, which inhibits aggregation/fusion but not folding of MAX or MAX-induced leakage. Using a concentration-dependent quenching probe (calcein), we determined that MAX-induced leakage of liposome contents was an all-or-none process. At MAX1 concentrations, which cause release of ~50% of the liposomes that contain small (R(h) <1.5 nm) markers, only ~15% of those liposomes release a fluorescent dextran of 40 kDa. A multimeric model of the pore is presented based on these results. Atomistic molecular dynamics simulations show that barrels consisting of 10 ß-hairpin MAX1 and MAX35 peptides are relatively more stable than MAX8V16E barrels in the bilayer, suggesting that barrels of this size are responsible for the peptides lytic action.

The ferritin superfamily: Supramolecular templates for materials synthesis.

Members of the ferritin superfamily are multi-subunit cage-like proteins with a hollow interior cavity. These proteins possess three distinct surfaces, i.e. interior and exterior surfaces of the cages and interface between subunits. The interior cavity provides a unique reaction environment in which the interior reaction is separated from the external environment. In biology the cavity is utilized for sequestration of irons and biomineralization as a mechanism to render Fe inert and sequester it from the external environment. Material scientists have been inspired by this system and exploited a range of ferritin superfamily proteins as supramolecular templates to encapsulate nanoparticles and/or as well-defined building blocks for fabrication of higher order assembly. Besides the interior cavity, the exterior surface of the protein cages can be modified without altering the interior characteristics. This allows us to deliver the protein cages to a targeted tissue in vivo or to achieve controlled assembly on a solid substrate to fabricate higher order structures. Furthermore, the interface between subunits is utilized for manipulating chimeric self-assembly of the protein cages and in the generation of symmetry-broken Janus particles. Utilizing these ideas, the ferritin superfamily has been exploited for development of a broad range of materials with applications from biomedicine to electronics.