SUMMARY RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ~60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.

The active sites of multisubunit RNA polymerases have a “trigger loop” (TL) that multitasks in substrate selection, catalysis, and translocation. To dissect the Saccharomyces cerevisiae RNA polymerase II TL at individual-residue resolution, we quantitatively phenotyped nearly all TL single variants en masse. Three mutant classes, revealed by phenotypes linked to transcription defects or various stresses, have distinct distributions among TL residues. We find that mutations disrupting an intra-TL hydrophobic pocket, proposed to provide a mechanism for substrate-triggered TL folding through destabilization of a catalytically inactive TL state, confer phenotypes consistent with pocket disruption and increased catalysis. Furthermore, allele-specific genetic interactions among TL and TL-proximal domain residues support the contribution of the funnel and bridge helices (BH) to TL dynamics. Our structural genetics approach incorporates structural and phenotypic data for high-resolution dissection of transcription mechanisms and their evolution, and is readily applicable to other essential yeast proteins.

Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that β8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. β8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor β (TGFβ) ligands and stimulates TGFβ receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFβ activation and signaling by forming intercellular protein complexes with β8 integrin. Cell type-specific ablation of β8 integrin, Nrp1, or canonical TGFβ receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFβ signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFβ signaling during cerebral angiogenesis.

Replication of chloroplast in plant cells is an essential process that requires co-assembly of the tubulin-like plastid division proteins FtsZ1 and FtsZ2 at mid-chloroplast to form a ring structure called the Z-ring. The Z-ring is stabilized via its interaction with the transmembrane protein ARC6 on the inner envelope membrane of chloroplasts. Plants lacking ARC6 are defective in plastid division and contain only one or two enlarged chloroplasts per cell with abnormal localization of FtsZ: instead of a single Z-ring, many short FtsZ filaments are distributed throughout the chloroplast. ARC6 is thought to be the anchoring point for FtsZ assemblies. To investigate the role of ARC6 in FtsZ anchoring, the mobility of green fluorescent protein-tagged FtsZ assemblies was assessed by single particle tracking in mutant plants lacking the ARC6 protein. Mean square displacement analysis showed that the mobility of FtsZ assemblies is to a large extent characterized by anomalous diffusion behavior (indicative of intermittent binding) and restricted diffusion suggesting that besides ARC6-mediated anchoring, an additional FtsZ-anchoring mechanism is present in chloroplasts.

Nck promotes breast carcinoma progression and metastasis by directing the polarized interaction of carcinoma cells with collagen fibrils, decreasing actin turnover, and enhancing the localization and activity of MMP14 at the cell surface through modulation of the spatiotemporal activation of Cdc42 and RhoA.