Depletion of Trypanosome CTR9 Leads to Gene Expression Defects

The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1

Genome-Wide Analysis of Factors Affecting Transcription Elongation and DNA Repair: A New Role for PAF and Ccr4-Not in Transcription-Coupled Repair

RNA polymerases frequently deal with a number of obstacles during transcription elongation that need to be removed for transcription resumption. One important type of hindrance consists of DNA lesions, which are removed by transcription-coupled repair (TC-NER), a specific sub-pathway of nucleotide excision repair. To improve our knowledge of transcription elongation and its coupling to TC-NER, we used the yeast library of non-essential knock-out mutations to screen for genes conferring resistance to the transcription-elongation inhibitor mycophenolic acid and the DNA-damaging agent 4-nitroquinoline-N-oxide. Our data provide evidence that subunits of the SAGA and Ccr4-Not complexes, Mediator, Bre1, Bur2, and Fun12 affect transcription elongation to different extents. Given the dependency of TC-NER on RNA Polymerase II transcription and the fact that the few proteins known to be involved in TC-NER are related to transcription, we performed an in-depth TC-NER analysis of a selection of mutants. We found that mutants of the PAF and Ccr4-Not complexes are impaired in TC-NER. This study provides evidence that PAF and Ccr4-Not are required for efficient TC-NER in yeast, unraveling a novel function for these transcription complexes and opening new perspectives for the understanding of TC-NER and its functional interconnection with transcription elongation

Expanding the Klebsiella pneumoniae volatile metabolome using advanced analytical instrumentation for the detection of novel metabolites

Aims: The purpose of this study was to identify the volatile molecules produced by the pathogenic Gram-negative bacterium Klebsiella pneumoniae (ATCC 13883) during in vitro growth using comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC-TOFMS). Methods and Results: Klebsiella pneumoniae ATCC 13883 was incubated in lysogeny broth to mid-exponential and stationary growth phases. Headspace volatile molecules from culture supernatants were concentrated using solid-phase microextraction (SPME) and analysed via GC×GC-TOFMS. Ninety-two K. pneumoniae-associated volatile molecules were detected, of which 78 (85%) were detected at both phases of growth and 14 (15%) were detected at either mid-exponential or stationary growth phases. Conclusions: This study has increased the total number of reported K. pneumoniae-associated volatile molecules from 77 to 150, demonstrating the sensitivity and resolution achieved by employing GC×GC-TOFMS for the analysis of bacterial headspace volatiles. Significance and Impact of the Study: This study represents an early-stage comprehensive volatile metabolomic analysis of an opportunistic bacterial pathogen. Characterizing the volatile molecules produced by K. pneumoniae during in vitro growth could provide us with a better understanding of this organisms’ metabolism, an area that has not been extensively studied to date

Volatile metabolic diversity of Klebsiella pneumoniae in nutrient-replete conditions

Introduction: Microorganisms catabolize carbon-containing compounds in their environment during growth, releasing a subset of metabolic byproducts as volatile compounds. However, the relationship between growth media and the production of volatile compounds has been largely unexplored to-date. Objectives: To assess the core and media-specific components of the Klebsiella pneumoniae volatile metabolome via growth in four in vitro culture media. Methods: Headspace volatiles produced by cultures of K. pneumoniae after growth to stationary phase in four rich media (brain heart infusion broth, lysogeny broth, Mueller-Hinton broth, and tryptic soy broth) were analyzed using comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC×GC-TOFMS). Differences in the composition of headspace volatiles as a function of growth media were assessed using hierarchical clustering analysis (HCA) and principal component analysis (PCA). Results: A total of 365 volatile compounds were associated with the growth of K. pneumoniae across all media, of which 36 (10%) were common to all growth media, and 148 (41%) were specific to a single medium. In addition, utilizing all K. pneumoniae-associated volatile compounds, strains clustered as a function of growth media, demonstrating the importance of media in determining the metabolic profile of this organism. Conclusion: K. pneumoniae produces a core suite of volatile compounds across all growth media studied, although the volatile metabolic signature of this organism is fundamentally media-dependent

Regulation of transcriptional elongation in pluripotency and cell differentiation by the PHD-finger protein Phf5a

Pluripotent embryonic stem cells (ESCs) self-renew or differentiate into all tissues of the developing embryo and cell-specification factors are necessary to balance gene expression. Here we delineate the function of the PHD-finger protein 5a (Phf5a) in ESC self-renewal and ascribe its role in regulating pluripotency, cellular reprogramming, and myoblast specification. We demonstrate that Phf5a is essential for maintaining pluripotency, since depleted ESCs exhibit hallmarks of differentiation. Mechanistically, we attribute Phf5a function to the stabilization of the Paf1 transcriptional complex and control of RNA polymerase II elongation on pluripotency loci. Apart from an ESC-specific factor, we demonstrate that Phf5a controls differentiation of adult myoblasts. Our findings suggest a potent mode of regulation by the Phf5a in stem cells, which directs their transcriptional program ultimately regulating maintenance of pluripotency and cellular reprogramming

Mutants of the Paf1 Complex Alter Phenotypic Expression of the Yeast Prion [PSI+]

The yeast [PSI+] prion is an epigenetic modifier of translation termination fidelity that causes nonsense suppression. The prion [PSI+] forms when the translation termination factor Sup35p adopts a self-propagating conformation. The presence of the [PSI+] prion modulates survivability in a variety of growth conditions. Nonsense suppression is essential for many [PSI+]-mediated phenotypes, but many do not appear to be due to read-through of a single stop codon, but instead are multigenic traits. We hypothesized that other global mechanisms act in concert with [PSI+] to influence [PSI+]-mediated phenotypes. We have identified one such global regulator, the Paf1 complex (Paf1C). Paf1C is conserved in eukaryotes and has been implicated in several aspects of transcriptional and posttranscriptional regulation. Mutations in Ctr9p and other Paf1C components reduced [PSI+]-mediated nonsense suppression. The CTR9 deletion also alters nonsense suppression afforded by other genetic mutations but not always to the same extent as the effects on [PSI+]-mediated read-through. Our data suggest that the Paf1 complex influences mRNA translatability but not solely through changes in transcript stability or abundance. Finally, we demonstrate that the CTR9 deletion alters several [PSI+]-dependent phenotypes. This provides one example of how [PSI+] and genetic modifiers can interact to uncover and regulate phenotypic variability