Membrane transporters and protein traffic networks differentially affecting metal tolerance: a genomic phenotyping study in yeast

Genomic phenotyping was used to assess the role of all non-essential S. cerevisiae proteins in modulating cell viability after exposure to cadmium, nickel and other metals

Intragenic promoter adaptation and facilitated RNA polymerase III recycling in the transcription of SCR1, the 7SL RNA gene of Saccharomyces cerevisiae

The SCR1 gene, coding for the 7SL RNA of the signal recognition particle, is the last known class III gene of Saccharomyces cerevisiae that remains to be characterized with respect to its mode of transcription and promoter organization. We show here that SCR1 represents a unique case of a non-tRNA class III gene in which intragenic promoter elements (the TFIIIC-binding A- and B-blocks), corresponding to the D and TpsiC arms of mature tRNAs, have been adapted to a structurally different small RNA without losing their transcriptional function. In fact, despite the presence of an upstream canonical TATA box, SCR1 transcription strictly depends on the presence of functional, albeit quite unusual, A- and B-blocks and requires all the basal components of the RNA polymerase III transcription apparatus, including TFIIIC. Accordingly, TFIIIC was found to protect from DNase I digestion an 80-bp region comprising the A- and B-blocks. B-block inactivation completely compromised TFIIIC binding and transcription capacity in vitro and in vivo. An inactivating mutation in the A-block selectively affected TFIIIC binding to this promoter element but resulted in much more dramatic impairment of in vivo than in vitro transcription. Transcriptional competition and nucleosome disruption experiments showed that this stronger in vivo defect is due to a reduced ability of A-block-mutated SCR1 to compete with other genes for TFIIIC binding and to counteract the assembly of repressive chromatin structures through TFIIIC recruitment. A kinetic analysis further revealed that facilitated RNA polymerase III recycling, far from being restricted to typical small sized class III templates, also takes place on the 522-bp-long SCR1 gene, the longest known class III transcriptional unit

A Nick-sensing DNA 3′-Repair Enzyme fromArabidopsis

DNA single-strand breaks, a major cause of genome instability, often produce unconventional end groups that must be processed to restore terminal moieties suitable for reparative DNA gap filling or ligation. Here, we describe a bifunctional repair enzyme from Arabidopsis (named AtZDP) that recognizes DNA strand breaks and catalyzes the removal of 3'-end-blocking lesions. The isolated C-terminal domain of AtZDP is by itself competent for 3'-end processing, but not for strand break recognition. The N-terminal domain instead contains three Cys(3)-His zinc fingers and recognizes various kinds of damaged double-stranded DNA. Gapped DNA molecules are preferential targets of AtZDP, which bends them by approximately 73 degrees upon binding, as measured by atomic force microscopy. Potential partners of AtZDP were identified in the Arabidopsis genome using the human single-strand break repairosome as a reference. These data identify a novel pathway for single-strand break repair in which a DNA-binding 3'-phosphoesterase acts as a "nick sensor" for damage recognition, as the catalyst of one repair step, and possibly as a nucleation center for the assembly of a fully competent repair complex

A plant 3'-phosphoesterase involved in the repair of DNA strand breaks generated by oxidative damage.

Two novel, structurally and functionally distinct phosphatases have been identified through the functional complementation, by maize cDNAs, of an Escherichia coli diphosphonucleoside phosphatase mutant strain. The first, ZmDP1, is a classical Mg(2+)-dependent and Li(+)-sensitive diphosphonucleoside phosphatase that dephosphorylates both 3'-phosphoadenosine 5'-phosphate (3'-PAP) and 2'-PAP without any discrimination between the 3'- and 2'-positions. The other, ZmDP2, is a distinct phosphatase that also catalyzes diphosphonucleoside dephosphorylation, but with a 12-fold lower Li(+) sensitivity, a strong preference for 3'-PAP, and the unique ability to utilize double-stranded DNA molecules with 3'-phosphate- or 3'-phosphoglycolate-blocking groups as substrates. Importantly, ZmDP2, but not ZmDP1, conferred resistance to a DNA repairdeficient E. coli strain against oxidative DNA-damaging agents generating 3'-phosphate- or 3'-phosphoglycolate-blocked single strand breaks. ZmDP2 shares a partial amino acid sequence similarity with a recently identified human polynucleotide kinase 3'-phosphatase that is thought to be involved in DNA repair, but is devoid of 5'-kinase activity. ZmDP2 is the first DNA 3'-phosphoesterase thus far identified in plants capable of converting 3'-blocked termini into priming sites for reparative DNA polymerization

A novel RNA polymerase III transcription factor fraction that is not required for template commitment.

Abstract We have identified and partially characterized a novel class III transcription factor fraction (TFIIIE) from yeast nuclear extracts. TFIIIE is functionally distinct from the standard yeast transcription factor fractions, TFIIIB and TFIIIC. It is also different from either of the TFIIIB subfractions, B' and B". TFIIIE is essential for specific transcription of both tRNA and 5 S RNA genes, its activity is sensitive to proteinase K, and it exhibits an apparent sedimentation coefficient of 4.0 S when analyzed on glycerol gradients. In the case of a tRNA gene, TFIIIE does not play a role in the formation of stable preinitiation complexes containing TFIIIB and TFIIIC. It is required for single as well as multiple rounds of transcription, however. Thus, TFIIIE is involved in the utilization of stable transcription complexes, but its action is not restricted to reinitiation events

Secretory Phospholipases A2 Induce Neurite Outgrowth in PC12 Cells through Lysophosphatidylcholine Generation and Activation of G2A Receptor

We previously demonstrated that secretory phospholipase A2 (sPLA2) and lysophosphatidylcholine (LPC) exhibit neurotrophin-like neuritogenic activity in the rat pheochromocytoma cell line PC12. In this study, we further analyzed the mechanism whereby sPLA2 displays neurite-inducing activity. Exogenously added mammalian group X sPLA2 (sPLA2-X), but not group IB and IIA sPLA2s, induced neuritogenesis, which correlated with the ability of sPLA2-X to liberate LPC into the culture media. In accordance, blocking the effect of LPC by supplementation of bovine serum albumin or phospholipase B attenuated neuritogenesis by sPLA2 or LPC. Overproduction or suppression of G2A, a G-protein-coupled receptor involved in LPC signaling, resulted in the enhancement or reduction of neuritogenesis induced by sPLA2 treatment. These results indicate that the neuritogenic effect of sPLA2 is mediated by generation of LPC and subsequent activation of G2A

Non-exhaustive DNA methylation-mediated transposon silencing in the black truffle genome, a complex fungal genome with massive repeat element content

Background: We investigated how an extremely transposon element (TE)-rich organism such as the plant-symbiotic ascomycete truffle Tuber melanosporum exploits DNA methylation to cope with the more than 45,000 repeated elements that populate its genome. Results: Whole-genome bisulfite sequencing performed on different developmental stages reveals a high fraction of methylated cytosines with a strong preference for CpG sites. The methylation pattern is highly similar among samples and selectively targets TEs rather than genes. A marked trend toward hypomethylation is observed for TEs located within a 1 kb distance from expressed genes, rather than segregated in TE-rich regions of the genome. Approximately 300 hypomethylated or unmethylated TEs are transcriptionally active, with higher expression levels in free-living mycelium compared to fruitbody. Indeed, multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs are found almost exclusively in free-living mycelium. A reduction of DNA methylation, restricted to non-CpG sites and accompanied by an increase in TE expression, is observed upon treatment of free-living mycelia with 5-azacytidine. Conclusions: Evidence derived from analysis of the T. melanosporum methylome indicates that a non-exhaustive, partly reversible, methylation process operates in truffles. This allows for the existence of hypomethylated, transcriptionally active TEs that are associated with copy number variant regions of the genome. Non-exhaustive TE methylation may reflect a role of active TEs in promoting genome plasticity and the ability to adapt to sudden environmental changes

A comprehensive resource of genomic, epigenomic and transcriptomic sequencing data for the black truffle Tuber melanosporum

Abstract Background: Tuber melanosporum, also known in the gastronomic community as "truffle", features one of the largest fungal genomes (125 Mb) with an exceptionally high transposable element (TE) and repetitive DNA content (>58%). The main purpose of DNA methylation in fungi is TE silencing. As obligate outcrossing organisms, truffles are bound to a sexual mode of propagation, which together with TEs is thought to represent a major force driving the evolution of DNA methylation. Thus, it was of interest to examine if and how T. melanosporum exploits DNA methylation to maintain genome integrity. Findings: We performed whole-genome DNA bisulfite sequencing and mRNA sequencing on different developmental stages of T. melanosporum; namely, fruitbody ("truffle"), free-living mycelium and ectomycorrhiza. The data revealed a high rate of cytosine methylation (>44%), selectively targeting TEs rather than genes with a strong preference for CpG sites. Whole genome DNA sequencing uncovered multiple TE-enriched, copy number variant regions bearing a significant fraction of hypomethylated and expressed TEs, almost exclusively in free-living mycelium propagated in vitro. Treatment of mycelia with 5-azacytidine partially reduced DNA methylation and increased TE transcription. Our transcriptome assembly also resulted in the identification of a set of novel transcripts from 614 genes

An Optimized Workflow for the Discovery of New Antimicrobial Compounds Targeting Bacterial RNA Polymerase Complex Formation

Bacterial resistance represents a major health problem worldwide and there is an urgent need to develop first-in-class compounds directed against new therapeutic targets. We previously developed a drug-discovery platform to identify new antimicrobials able to disrupt the protein-protein interaction between the beta' subunit and the sigma(70) initiation factor of bacterial RNA polymerase, which is essential for transcription. As a follow-up to such work, we have improved the discovery strategy to make it less time-consuming and more cost-effective. This involves three sequential assays, easily scalable to a high-throughput format, and a subsequent in-depth characterization only limited to hits that passed the three tests. This optimized workflow, applied to the screening of 5360 small molecules from three synthetic and natural compound libraries, led to the identification of six compounds interfering with the beta'-sigma(70) interaction, and thus was capable of inhibiting promoter-specific RNA transcription and bacterial growth. Upon supplementation with a permeability adjuvant, the two most potent transcription-inhibiting compounds displayed a strong antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) values among the lowest (0.87-1.56 mu M) thus far reported for beta'-sigma PPI inhibitors. The newly identified hit compounds share structural feature similarities with those of a pharmacophore model previously developed from known inhibitors