An Investigation of a Role for U2 snRNP Spliceosomal Components in Regulating Transcription

There is mounting evidence to suggest that the synthesis of pre-mRNA transcripts and their subsequent splicing are coordinated events. Previous studies have implicated the mammalian spliceosomal U2 snRNP as having a novel role in stimulating transcriptional elongation in vitro through interactions with the elongation factors P-TEFb and Tat-SF1; however, the mechanism remains unknown [1]. These factors are conserved in Saccharomyces cerevisiae, a fact that suggests that a similar interaction may occur in yeast to stimulate transcriptional elongation in vivo. To address this possibility we have looked for evidence of a role for the yeast Tat-SF1 homolog, Cus2, and the U2 snRNA in regulating transcription. Specifically, we have performed a genetic analysis to look for functional interactions between Cus2 or U2 snRNA and the P-TEFb yeast homologs, the Bur1/2 and Ctk1/2/3 complexes. In addition, we have analyzed Cus2-deleted or -overexpressing cells and U2 snRNA mutant cells to determine if they show transcription-related phenotypes similar to those displayed by the P-TEFb homolog mutants. In no case have we been able to observe phenotypes consistent with a role for either spliceosomal factor in transcription elongation. Furthermore, we did not find evidence for physical interactions between the yeast U2 snRNP factors and the P-TEFb homologs. These results suggest that in vivo, S. cerevisiae do not exhibit functional or physical interactions similar to those exhibited by their mammalian counterparts in vitro. The significance of the difference between our in vivo findings and the previously published in vitro results remains unclear; however, we discuss the potential importance of other factors, including viral proteins, in mediating the mammalian interactions

An investigation of a role for U2 snRNP spliceosomal components in regulating transcription.

There is mounting evidence to suggest that the synthesis of pre-mRNA transcripts and their subsequent splicing are coordinated events. Previous studies have implicated the mammalian spliceosomal U2 snRNP as having a novel role in stimulating transcriptional elongation in vitro through interactions with the elongation factors P-TEFb and Tat-SF1; however, the mechanism remains unknown [1]. These factors are conserved in Saccharomyces cerevisiae, a fact that suggests that a similar interaction may occur in yeast to stimulate transcriptional elongation in vivo. To address this possibility we have looked for evidence of a role for the yeast Tat-SF1 homolog, Cus2, and the U2 snRNA in regulating transcription. Specifically, we have performed a genetic analysis to look for functional interactions between Cus2 or U2 snRNA and the P-TEFb yeast homologs, the Bur1/2 and Ctk1/2/3 complexes. In addition, we have analyzed Cus2-deleted or -overexpressing cells and U2 snRNA mutant cells to determine if they show transcription-related phenotypes similar to those displayed by the P-TEFb homolog mutants. In no case have we been able to observe phenotypes consistent with a role for either spliceosomal factor in transcription elongation. Furthermore, we did not find evidence for physical interactions between the yeast U2 snRNP factors and the P-TEFb homologs. These results suggest that in vivo, S. cerevisiae do not exhibit functional or physical interactions similar to those exhibited by their mammalian counterparts in vitro. The significance of the difference between our in vivo findings and the previously published in vitro results remains unclear; however, we discuss the potential importance of other factors, including viral proteins, in mediating the mammalian interactions

Ribosome hibernation facilitates tolerance of stationary-phase bacteria to aminoglycosides.

Upon entry into stationary phase, bacteria dimerize 70S ribosomes into translationally inactive 100S particles by a process called ribosome hibernation. Previously, we reported that the hibernation-promoting factor (HPF) of Listeria monocytogenes is required for 100S particle formation and facilitates adaptation to a number of stresses. Here, we demonstrate that HPF is required for the high tolerance of stationary-phase cultures to aminoglycosides but not to beta-lactam or quinolone antibiotics. The sensitivity of a Δhpf mutant to gentamicin was suppressed by the bacteriostatic antibiotics chloramphenicol and rifampin, which inhibit translation and transcription, respectively. Disruption of the proton motive force by the ionophore carbonyl cyanide m-chlorophenylhydrazone or mutation of genes involved in respiration also suppressed the sensitivity of the Δhpf mutant. Accordingly, Δhpf mutants had aberrantly high levels of ATP and reducing equivalents during prolonged stationary phase. Analysis of bacterial uptake of fluorescently labeled gentamicin demonstrated that the Δhpf mutant harbored increased intracellular levels of the drug. Finally, deletion of the main ribosome hibernation factor of Escherichia coli, ribosome modulation factor (rmf), rendered these bacteria susceptible to gentamicin. Taken together, these data suggest that HPF-mediated ribosome hibernation results in repression of the metabolic activity that underlies aminoglycoside tolerance. HPF is conserved in nearly every bacterial pathogen, and the role of ribosome hibernation in antibiotic tolerance may have clinical implications

Development of a single-gene, signature-tag-based approach in combination with alanine mutagenesis to identify listeriolysin O residues critical for the in vivo survival of Listeria monocytogenes.

Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin (CDC) family and a primary virulence factor of the intracellular pathogen Listeria monocytogenes. LLO mediates rupture of phagosomal membranes, thereby releasing bacteria into the growth-permissive host cell cytosol. Several unique features of LLO allow its activity to be precisely regulated in order to facilitate phagosomal escape, intracellular growth, and cell-to-cell spread. To improve our understanding of the multifaceted contribution of LLO to the pathogenesis of L. monocytogenes, we developed a screen that combined saturation mutagenesis and signature tags, termed in vivo analysis by saturation mutagenesis and signature tags (IVASS). We generated a library of LLO mutant strains, each harboring a single amino acid substitution and a signature tag, by using the previously described pPL2 integration vector. The signature tags acted as molecular barcodes, enabling high-throughput, parallel analysis of 40 mutants in a single animal and identification of attenuated mutants by negative selection. Using the IVASS technique we were able to screen over 90% of the 505 amino acids present in LLO and identified 60 attenuated mutants. Of these, 39 LLO residues were previously uncharacterized and potentially revealed novel functions of the toxin during infection. The mutants that were subsequently analyzed in vivo each conferred a 2- to 4-orders of magnitude loss in virulence compared to wild type, thereby validating the screening methods. Phenotypic analysis of the LLO mutant library using common in vitro techniques suggested that the functional contributions of some residues could only have been revealed through in vivo analysis

Development of a single-gene, signature-tag-based approach in combination with alanine mutagenesis to identify listeriolysin O residues critical for the in vivo survival of Listeria monocytogenes.

Listeriolysin O (LLO) is a pore-forming toxin of the cholesterol-dependent cytolysin (CDC) family and a primary virulence factor of the intracellular pathogen Listeria monocytogenes. LLO mediates rupture of phagosomal membranes, thereby releasing bacteria into the growth-permissive host cell cytosol. Several unique features of LLO allow its activity to be precisely regulated in order to facilitate phagosomal escape, intracellular growth, and cell-to-cell spread. To improve our understanding of the multifaceted contribution of LLO to the pathogenesis of L. monocytogenes, we developed a screen that combined saturation mutagenesis and signature tags, termed in vivo analysis by saturation mutagenesis and signature tags (IVASS). We generated a library of LLO mutant strains, each harboring a single amino acid substitution and a signature tag, by using the previously described pPL2 integration vector. The signature tags acted as molecular barcodes, enabling high-throughput, parallel analysis of 40 mutants in a single animal and identification of attenuated mutants by negative selection. Using the IVASS technique we were able to screen over 90% of the 505 amino acids present in LLO and identified 60 attenuated mutants. Of these, 39 LLO residues were previously uncharacterized and potentially revealed novel functions of the toxin during infection. The mutants that were subsequently analyzed in vivo each conferred a 2- to 4-orders of magnitude loss in virulence compared to wild type, thereby validating the screening methods. Phenotypic analysis of the LLO mutant library using common in vitro techniques suggested that the functional contributions of some residues could only have been revealed through in vivo analysis