The carboxyl-terminal domain (CTD) of the large subunit of mammalian RNA polymerase II contains 52 repeats of a heptapeptide that is the target of a variety of kinases. The hyperphosphorylated CTD recruits important factors for mRNA capping, splicing, and 3-processing. The role of the CTD for the transcription process in vivo, however, is not yet clear. We have conditionally expressed an ␣-amanitin-resistant large subunit with an almost entirely deleted CTD (LS*⌬5) in B-cells. These cells have a defect in global transcription of cellular genes in the presence of ␣-amanitin. Moreover, pol II harboring LS*⌬5 failed to transcribe up to the promoterproximal pause sites in the hsp70A and c-fos gene promoters. The results indicate that the CTD is already required for steps that occur before promoter-proximal pausing and maturation of mRNA.Eukaryotic mRNA synthesis is catalyzed by the multisubunit RNA polymerase II (pol II).1 The large subunit of pol II (LS) is highly conserved among eukaryotic RNA polymerases and also shows striking homology to the large subunit of Escherichia coli RNA polymerase (1). The LS has evolved a particularly structured carboxyl-terminal domain (CTD) that is not present in other RNA polymerases (2). This CTD comprises multiple copies of a heptapeptide repeat with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. The number of repeats varies from 26/27 in yeast to 52 in mouse and human cells (3). Deletion of more than half of the repeats in yeast and mouse interferes with cell viability (4 -6). Mice homozygous for a deletion of 13 repeats are smaller than wild-type littermates and have a high rate of neonatal lethality (7), suggesting that CTD is important in regulating growth during mammalian development. In cells, two forms of pol II are detectable containing either a hypophosphorylated (pol IIA) or hyperphosphorylated CTD (pol II0). Although pol IIA is consistently found in the initiation complex, pol II0 is associated with elongating complexes.An increasing number of genes have been shown to be regulated by promoter-proximal pausing of pol II. These genes include Drosophila hsp70 and hsp26 genes, as well as the mammalian c-myc, c-fos, and immunoglobulin genes (8 -15). The passage of the paused pol II into a processive mode coincides in vivo with hyperphosphorylation of the CTD (11, 16).Recent studies suggest that the hyperphosphorylated CTD functions as a platform for the assembly of complexes that cap, splice, cleave, and polyadenylate pre-mRNA (2, 17, 18). Capping of mRNA occurs shortly after transcription initiation (19), preceding other mRNA processing events such as mRNA splicing and polyadenylation. The capping enzyme is not stably associated with basal transcription factors or the RNA pol II holoenzyme but is directly and specifically recruited to the hyperphosphorylated form of CTD (20 -22, 24). Similarly, several components of the splicing machinery (25, 26) and related factors such as SR proteins and SR-like proteins (27-29) are recruited to pol II by the hyperphosphorylated CT...
Idiopathic pulmonary fibrosis (IPF) is a rare and devastating chronic lung disease of unknown etiology. Despite the approved treatment options nintedanib and pirfenidone, the medical need for a safe and well-tolerated antifibrotic treatment of IPF remains high. The human prostaglandin F receptor (hFP-R) is widely expressed in the lung tissue and constitutes an attractive target for the treatment of fibrotic lung diseases. Herein, we present our research toward novel quinoline-based hFP-R antagonists, including synthesis and detailed structure–activity relationship (SAR). Starting from a high-throughput screening (HTS) hit of our corporate compound library, multiple parameter improvementsincluding increase of the relative oral bioavailability F rel from 3 to ≥100%led to a highly potent and selective hFP-R antagonist with complete oral absorption from suspension. BAY-6672 (46) representsto the best of our knowledgethe first reported FP-R antagonist to demonstrate in vivo efficacy in a preclinical animal model of lung fibrosis, thus paving the way for a new treatment option in IPF.
Gq proteins are universally important for signal transduction in mammalian cells. The underlying kinetics and transformation from extracellular stimuli into intracellular signaling, however could not be investigated in detail so far. Here we present the human Neuropsin (hOPN5) for specific and repetitive manipulation of Gq signaling in vitro and in vivo with high spatio-temporal resolution. Properties and G protein specificity of hOPN5 are characterized by UV light induced IP3 generation, Ca2+ transients and inhibition of GIRK channel activity in HEK cells. In adult hearts from a transgenic animal model, light increases the spontaneous beating rate. In addition, we demonstrate light induced contractions in the small intestine, which are not detectable after pharmacological Gq protein block. All-optical high-throughput screening for TRPC6 inhibitors is more specific and sensitive than conventional pharmacological screening. Thus, we demonstrate specific Gq signaling of hOPN5 and unveil its potential for optogenetic applications.
Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next‐generation drugs. Separate synthesis of multiple peptide fragments and tedious chain‐to‐chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre‐made diaminodiacid bridge at A‐B chain terminal disulfide can be easily and rapidly synthesized by a single‐shot automated solid‐phase synthesis and expedient one‐step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.
The carboxy-terminal domain of the large subunit of mouse and human RNA polymerase II contains 52 repeats of a heptapeptide which are the targets for a variety of kinases. We have used an K K-amanitin resistant form of the large subunit of pol II to study the role of the carboxy-terminal domain in the expression of chromosomal genes. The large subunit of RNA polymerase II and deletion mutants thereof, which contain only 31 (LSv v31) and 5 (LSv v5) repeats, were expressed in 293 cells. Subsequently, the endogenous large subunit of RNA polymerase II was inhibited by K K-amanitin and the induction of chromosomal c-fos and hsp70A genes was determined. Cells expressing the large subunit of RNA polymerase II and LSv v31 were able to transcribe the c-fos and hsp70A genes after treatment with the phorbolester TPA and after heat-shock, respectively. In contrast, cells expressing LSv v5 failed to induce expression of both genes.z 1999 Federation of European Biochemical Societies.
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