Histone acetylation dynamics play a critical role in co- transcriptional spliceosome assembly and spliceosomal rearrangements

In the last several years, a number of studies have shown that spliceosome assembly and splicing catalysis can occur co-transcriptionally. However, it has been unclear which specific transcription factors play key roles in coupling splicing to transcription and the mechanisms through which they act. Here we report the discovery that Gcn5, which encodes the histone acetyltransferase (HAT) activity of the SAGA complex, has HAT-dependent genetic interactions with the genes encoding the heterodimeric U2 snRNP proteins Msl1 and Lea1, suggesting a functional relationship between Gcn5 HAT activity and Msl1/Lea1 function. To understand this relationship, we carried out an analysis of Gcn5's role in co-transcriptional recruitment of Msl1 and Lea1 to pre-mRNA and find that Gcn5 HAT activity is required for co-transcriptional recruitment of the U2 snRNP (and subsequent snRNP) components to the branchpoint. Although previous studies suggested that transcription elongation can alter co- transcriptional pre-mRNA splicing, we do not observe evidence of defective transcription elongation for these genes in the absence of Gcn5, while Gcn5-dependent histone acetylation is enriched in the promoter regions. While all these data suggest a role for histone acetylation in co- transcriptional spliceosome assembly. A closer examination of the functional interactions between histone mutants and the U2 snRNP and the effects of histone mutants and histone deacetylation on spliceosome assembly provide convincing evidence of the functional coordination of histone deaceylation and splicing. Mutations in histone residues targeted by Gcn5 show genetic interactions with the U2 snRNP and splicing defects that mirror GCN5 deletion. Furthermore, not only is Gcn5 associated throughout intron-containing genes, but deletion of multiple HDACs reveals peaks in acetylation in these regions, and this results in defects in spliceosome assembly. Finally, we present data that support a model whereby the Gcn5-dependent U2 snRNP recruitment facilitates HDAC recruitment, suggesting that splicing factors can, in fact, affect histone acetylation. These studies show that co-transcriptional spliceosome rearrangements are driven by dynamic changes in the acetylation state of histones and provide a model whereby spliceosome assembly is tightly coupled to histone modificatio

Intracellular expression of <i>phhA</i> and growth of <i>L. pneumophila</i> in U937 cell macrophages.

<p>Intracellular expression of <i>phhA</i> and <i>letA</i> transcripts in macrophages, which were infected with wild-type (WT) 130b for 24 h, 48 and 72 h and then RT-PCR was done using primers that amplify the specific transcripts. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate reverse transcriptase (- RT). The results are representative of two independent experiments.</p

Phenylalanine Hydroxylase from <em>Legionella pneumophila</em> Is a Thermostable Enzyme with a Major Functional Role in Pyomelanin Synthesis

<div><h3>Background</h3><p><em>Legionella pneumophila</em> is a pathogenic bacterium that can cause Legionnaires’ disease and other non-pneumonic infections in humans. This bacterium produces a pyomelanin pigment, a potential virulence factor with ferric reductase activity. In this work, we have investigated the role of phenylalanine hydroxylase from <em>L. pneumophila</em> (lpPAH), the product of the <em>phhA</em> gene, in the synthesis of the pyomelanin pigment and the growth of the bacterium in defined compositions.</p> <h3>Methodology/Principal Findings</h3><p>Comparative studies of wild-type and <em>phhA</em> mutant corroborate that lpPAH provides the excess tyrosine for pigment synthesis. <em>phhA</em> and <em>letA</em> (<em>gacA</em>) appear transcriptionally linked when bacteria were grown in buffered yeast extract medium at 37°C. <em>phhA</em> is expressed in <em>L. pneumophila</em> growing in macrophages. We also cloned and characterized lpPAH, which showed many characteristics of other PAHs studied so far, including Fe(II) requirement for activity. However, it also showed many particular properties such as dimerization, a high conformational thermal stability, with a midpoint denaturation temperature (<em>T</em>_m) = 79±0.5°C, a high specific activity at 37°C (10.2±0.3 µmol L-Tyr/mg/min) and low affinity for the substrate (<em>K</em>_m (L-Phe) = 735±50 µM.</p> <h3>Conclusions/Significance</h3><p>lpPAH has a major functional role in the synthesis of pyomelanin and promotes growth in low-tyrosine media. The high thermal stability of lpPAH might reflect the adaptation of the enzyme to withstand relatively high survival temperatures.</p> </div

Location and expression of <i>phhA</i>.

<p>(A) Depiction of the region of the <i>L. pneumophila</i> chromosome containing <i>phhA</i>. The white horizontal arrows denote the relative size and orientation of <i>phhA</i> and its neighboring genes. The thin horizontal line below the gene map signifies the approximate size and location of the <i>phhA</i>-specific transcript identified by RT-PCR analysis. (B) Expression of <i>phhA</i> transcripts. Wild-type strain 130b was grown in BYE or CDM broth at the indicated temperatures, and then RNA was analyzed by RT-PCR utilizing primers specific to <i>phhA</i>. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate RT. PCR products obtained from genomic DNA appear in the left-most lane, indicating that the mRNAs observed are full-length. RT-PCR analysis of 16S rRNA served as a positive control. The results presented are representative of at least three independent experiments.</p

Pigmentation of wild-type and <i>phhA</i> mutant <i>L</i>. <b><i>pneumophila</i></b>.

<p>(A) Wild-type (WT) <i>L. pneumophila</i> 130b was inoculated into either standard CDM, CDM lacking tyrosine (CDMˆno Tyr), or CDM containing twice the normal amount of tyrosine (CDMˆ2X Tyr), and then at 24 and 48 h post-inoculation the levels of pigment in the cultures were determined by measuring the OD₄₀₀ of the cultures supernatants. (B) <i>L. pneumophila</i> 130b WT, <i>phhA</i> mutants NU406 and NU407, and <i>lly</i> mutant NU408 were inoculated into standard CDM containing tyrosine, and then at 24 and 48 h, the amount of pigment produced was determined by assessing the OD₄₀₀ of culture supernatants. (C) WT and mutant NU406 carrying the vector pMMB2002 and NU406 carrying the <i>phhA</i> gene cloned into pMMB2002 (i.e., pPhhA) were cultured in either standard CDM containing tyrosine (gray bars) or CDM containing twice the normal amount of tyrosine (black bars), and then at 24 h post-inoculation the OD₄₀₀ of the cultures were determined. (D) Photographs of cell-free supernatants from 24-h CDM cultures (with the standard amount of tyrosine) of WT and <i>phhA</i> mutant strain NU406 carrying vector and NU406 carrying the cloned <i>phhA</i>. In panels (A – C), the asterisks indicate a <i>P</i> value of <0.05 compared to the WT control (Student’s <i>t</i>-test). All experiments are representative of three independent trials.</p

Conformational stability of lpPAH.

<p>(A) Far-UV CD spectrum of lpPAH (6 µM in 50 mM Na-phosphate buffer, pH 6.5) at 37°C (<b>^____</b>), at 85°C (––) and at 37°C after heating the sample to 100°C (⋅⋅⋅⋅⋅). [θ], mean residual ellipticity. (B) CD-monitored (at 222 nm) thermal denaturation lpPAH (6 µM in 20 mM Na-Hepes, 200 mM NaCl, pH 7.0) without (•) or with (○) 6 µM Fe(II) (added as ferrous ammonium sulphate) and 6 µM Fe(II) and 5 mM L-Phe (▾). The lines show a fitting of the data to a two-state unfolding equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046209#pone.0046209-Swint1" target="_blank">[79]</a> and points are averaged over ten data points after conversion to fraction unfolded <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046209#pone.0046209-Agashe1" target="_blank">[80]</a>. (C) DSC-monitored thermal denaturation of lpPAH (30 µM) in 20 mM Na-Hepes, pH 7.0. The scan rate was 1°C/min.</p

Transcriptional linkage between <i>phhA</i> and <i>letA</i>.

<p>(A) The gray horizontal arrows denote the <i>phhA</i> and <i>letA</i> genes. The thin horizontal lines below the genes signify the approximate size and location of transcripts identified by RT-PCR analysis, including an intergenic transcript. (B) Wild-type strain 130b was grown in BYE at 37°C, and then RNA was analyzed by RT-PCR utilizing primer pairs specific to either <i>phhA, letA,</i> or the intergenic region spanning <i>phhA</i> and <i>letA</i>. That the PCR products obtained resulted from mRNA templates was confirmed by the lack of product obtained when the PCR did not incorporate RT (-RT). PCR products obtained from genomic DNA appear in the left-most lanes, indicating that the mRNAs observed are full-length. The results presented are representative of at least three independent experiments.</p

Growth of wild-type and <i>phhA</i> mutant <i>L. pneumophila</i> in CDM containing different amounts of tyrosine.

<p><i>L. pneumophila</i> 130b wild-type (WT) and <i>phhA</i> mutant strains NU406 and NU407 were inoculated into either standard CDM containing tyrosine (A) or CDM lacking tyrosine (B) and at the indicated time points, the extent of bacterial growth was determined by recording the optical density (OD) of the cultures. In panel (B), the ODs of the mutant cultures were significantly less than that of the WT cultures at 18, 24, and 40 h post-inoculation, as indicated by the asterisks (<i>P</i><0.05, Student’s <i>t</i>-test). (C) WT and mutant NU406 carrying the empty vector (pMMB2002) and NU406 carrying the <i>phhA</i> gene cloned into pMMB2002 (i.e., pPhhA) were cultured in CDM without the tyrosine supplement and then at 24 h post-inoculation the OD of the cultures were determined. The OD of the NU406 (pMMB2002) culture was significantly less than that of both the WT and the complemented mutant (<i>P</i><0.05, Student’s <i>t</i>-test). All experiments are representative of three independent trials.</p