Mechanism for the initiation of spliceosome disassembly.

Pre-mRNA splicing requires the assembly, remodeling, and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome1. Recent studies have shed light on spliceosome assembly and remodeling for catalysis2-6, but the mechanism of disassembly remains unclear. Here, we report 2.6 to 3.2 Å resolution cryo-electron microscopy structures of nematode and human terminal intron-lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 thus controls both the start5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes.

Imbalance of peptidoglycan biosynthesis alters the cell surface charge of Listeria monocytogenes.

The bacterial cell wall is composed of a thick layer of peptidoglycan and cell wall polymers, which are either embedded in the membrane or linked to the peptidoglycan backbone and referred to as lipoteichoic acid (LTA) and wall teichoic acid (WTA), respectively. Modifications of the peptidoglycan or WTA backbone can alter the susceptibility of the bacterial cell towards cationic antimicrobials and lysozyme. The human pathogen Listeria monocytogenes is intrinsically resistant towards lysozyme, mainly due to deacetylation and O-acetylation of the peptidoglycan backbone via PgdA and OatA. Recent studies identified additional factors, which contribute to the lysozyme resistance of this pathogen. One of these is the predicted ABC transporter, EslABC. An eslB mutant is hyper-sensitive towards lysozyme, likely due to the production of thinner and less O-acetylated peptidoglycan. Using a suppressor screen, we show here that suppression of eslB phenotypes could be achieved by enhancing peptidoglycan biosynthesis, reducing peptidoglycan hydrolysis or alterations in WTA biosynthesis and modification. The lack of EslB also leads to a higher negative surface charge, which likely stimulates the activity of peptidoglycan hydrolases and lysozyme. Based on our results, we hypothesize that the portion of cell surface exposed WTA is increased in the eslB mutant due to the thinner peptidoglycan layer and that latter one could be caused by an impairment in UDP-N-acetylglucosamine (UDP-GlcNAc) production or distribution.

Repression and 3D-restructuring resolves regulatory conflicts in evolutionarily rearranged genomes.

Regulatory landscapes drive complex developmental gene expression, but it remains unclear how their integrity is maintained when incorporating novel genes and functions during evolution. Here, we investigated how a placental mammal-specific gene, Zfp42, emerged in an ancient vertebrate topologically associated domain (TAD) without adopting or disrupting the conserved expression of its gene, Fat1. In ESCs, physical TAD partitioning separates Zfp42 and Fat1 with distinct local enhancers that drive their independent expression. This separation is driven by chromatin activity and not CTCF/cohesin. In contrast, in embryonic limbs, inactive Zfp42 shares Fat1's intact TAD without responding to active Fat1 enhancers. However, neither Fat1 enhancer-incompatibility nor nuclear envelope-attachment account for Zfp42's unresponsiveness. Rather, Zfp42's promoter is rendered inert to enhancers by context-dependent DNA methylation. Thus, diverse mechanisms enabled the integration of independent Zfp42 regulation in the Fat1 locus. Critically, such regulatory complexity appears common in evolution as, genome wide, most TADs contain multiple independently expressed genes.

Listeria monocytogenes genes supporting growth under standard laboratory cultivation conditions and during macrophage infection.

The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth inside and outside the host. Here, we have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach identified 394 open reading frames that are required for growth under standard laboratory conditions and 42 further genes, which become necessary during intracellular growth in macrophages. Most of these genes encode components of the translation machinery and act in chromosome-related processes, cell division, and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions are also required for growth, suggesting novel options for the development of antilisterial drugs. Among the genes specifically required during intracellular growth are known virulence factors, genes compensating intracellular auxotrophies, and several cell division genes. Our experiments also highlight the importance of PASTA kinase signaling for general viability and of glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

MurA escape mutations uncouple peptidoglycan biosynthesis from PrkA signaling.

Gram-positive bacteria are protected by a thick mesh of peptidoglycan (PG) completely engulfing their cells. This PG network is the main component of the bacterial cell wall, it provides rigidity and acts as foundation for the attachment of other surface molecules. Biosynthesis of PG consumes a high amount of cellular resources and therefore requires careful adjustments to environmental conditions. An important switch in the control of PG biosynthesis of Listeria monocytogenes, a Gram-positive pathogen with a high infection fatality rate, is the serine/threonine protein kinase PrkA. A key substrate of this kinase is the small cytosolic protein ReoM. We have shown previously that ReoM phosphorylation regulates PG formation through control of MurA stability. MurA catalyzes the first step in PG biosynthesis and the current model suggests that phosphorylated ReoM prevents MurA degradation by the ClpCP protease. In contrast, conditions leading to ReoM dephosphorylation stimulate MurA degradation. How ReoM controls degradation of MurA and potential other substrates is not understood. Also, the individual contribution of the ~20 other known PrkA targets to PG biosynthesis regulation is unknown. We here present murA mutants which escape proteolytic degradation. The release of MurA from ClpCP-dependent proteolysis was able to activate PG biosynthesis and further enhanced the intrinsic cephalosporin resistance of L. monocytogenes. This latter effect required the RodA3/PBP B3 transglycosylase/transpeptidase pair. One murA escape mutation not only fully rescued an otherwise non-viable prkA mutant during growth in batch culture and inside macrophages but also overcompensated cephalosporin hypersensitivity. Our data collectively indicate that the main purpose of PrkA-mediated signaling in L. monocytogenes is control of MurA stability during standard laboratory growth conditions and intracellular growth in macrophages. These findings have important implications for the understanding of PG biosynthesis regulation and ß-lactam resistance of L. monocytogenes and related Gram-positive bacteria.

Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis.

The highly oxygen-sensitive hydrogen uptake (Hup) hydrogenase from Dehalococcoides mccartyi forms part of a protein-based respiratory chain coupling hydrogen oxidation with organohalide reduction on the outside of the cell. The HupXSL proteins were previously shown to be synthesized and enzymatically active in Escherichia coli. Here we examined the growth conditions that deliver active Hup enzyme that couples H2 oxidation to benzyl viologen (BV) reduction, and identified host factors important for this process. In a genetic background lacking the three main hydrogenases of E. coli we could show that additional deletion of genes necessary for selenocysteine biosynthesis resulted in inactive Hup enzyme, suggesting requirement of a formate dehydrogenase for Hup activity. Hup activity proved to be dependent on the presence of formate dehydrogenase (Fdh-H), which is typically associated with the H2-evolving formate hydrogenlyase (FHL) complex in the cytoplasm. Further analyses revealed that heterologous Hup activity could be recovered if the genes encoding the ferredoxin-like electron-transfer protein HupX, as well as the related HycB small subunit of Fdh-H were also deleted. These findings indicated that the catalytic HupL and electron-transferring HupS subunits were sufficient for enzyme activity with BV. The presence of the HupX or HycB proteins in the absence of Fdh-H therefore appears to cause inactivation of the HupSL enzyme. This is possibly because HupX or HycB aided transfer of electrons to the quinone pool or other oxidoreductase complexes, thus maintaining the HupSL heterodimer in a continuously oxidized state causing its inactivation. This proposal was supported by the observation that growth under either aerobic or anaerobic respiratory conditions did not yield an active HupSL. These studies thus provide a system to understand the redox sensitivity of this heterologously synthesized hydrogenase.