Ribonucleases J1 and J2: two novel endoribonucleases in B.subtilis with functional homology to E.coli RNase E

Many prokaryotic organisms lack an equivalent of RNase E, which plays a key role in mRNA degradation in Escherichia coli. In this paper, we report the purification and identification by mass spectrometry in Bacillus subtilis of two paralogous endoribonucleases, here named RNases J1 and J2, which share functional homologies with RNase E but no sequence similarity. Both enzymes are able to cleave the B.subtilis thrS leader at a site that can also be cleaved by E.coli RNase E. We have previously shown that cleavage at this site increases the stability of the downstream messenger. Moreover, RNases J1/J2 are sensitive to the 5′ phosphorylation state of the substrate in a site-specific manner. Orthologues of RNases J1/J2, which belong to the metallo-β-lactamase family, are evolutionarily conserved in many prokaryotic organisms, representing a new family of endoribonucleases. RNases J1/J2 appear to be implicated in regulatory processing/maturation of specific mRNAs, such as the T-box family members thrS and thrZ, but may also contribute to global mRNA degradation

Polyadenylation of a functional mRNA controls gene expression in Escherichia coli

Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine–6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5′ monophosphate and a 3′ stable hairpin is highly susceptible to poly(A)-dependent degradation

T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry

Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted β-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP206-214. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results

Characterization of E. coli ribosomal particles : combined analysis of whole proteins by mass spectrometry and of proteolytic digests by liquid chromatography-tandem mass spectrometry.

This chapter describes the purification of ribosomal particles from a mutant strain of Escherichia coli using sucrose gradients and the characterization of their protein composition by a combination of mass spectrometry (MS) techniques. The main objective is to identify the ribosomal proteins that are missing in an aberrant ribosomal particle corresponding to a defective large subunit. To address this question, the tryptic digests of the purified ribosomal particles are analyzed by the coupling between liquid chromatography and tandem MS. The presence or absence of a given ribosomal protein in the defective particle is determined by comparing the MS intensities of its identified tryptic peptides with that of the mature large subunit. These analyses also allow identification of proteins copurifying with the ribosomal particles. To detect low-mass proteins escaping identification by the above method, intact proteins are also analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) and nano-ESI-QqTOF MS

Chimeric Fusions of Subunit IV and PetL in the b 6 f Complex of Chlamydomonas reinhardtii

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Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules in Paramecium

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A thiol peroxidase is an H2O2 receptor and redox-transducer in gene activation.

International audienceThe Yap1 transcription factor regulates hydroperoxide homeostasis in S. cerevisiae. Yap1 is activated by oxidation when hydroperoxide levels increase. We show that Yap1 is not directly oxidized by hydroperoxide. We identified the glutathione peroxidase (GPx)-like enzyme Gpx3 as a second component of the pathway, serving the role of sensor and transducer of the hydroperoxide signal to Yap1. When oxidized by H2O2, Gpx3 Cys36 bridges Yap1 Cys598 by a disulfide bond. This intermolecular disulfide bond is then resolved into a Yap1 intramolecular disulfide bond, the activated form of the regulator. Thioredoxin turns off the pathway by reducing both sensor and regulator. These data reveal a redox-signaling function for a GPx-like enzyme and elucidate a eukaryotic hydroperoxide-sensing mechanism. Gpx3 is thus a hydroperoxide receptor and redox-transducer

Glycoproteomics: a challenge (balance) to mass spectrometry acquisition and interpretation

International audienceGlycopeptide-based tandem-mass-spectrometry enables site-specific glycosylation analysis, allowing us to explore the glycomes and potential functions. While performing glycopeptides MS/MS, fragmentation can be complicated by the heterogeneous glycans and amino-acid combination, which is the first bottleneck. Several fragmentation modes, such as stepped-collision-energy (sce), and a hybrid of both electron transfer and collision-induced dissociation, were reported to have more potential to generate an informative spectrum(1, 2). Furthermore, the superimposition of fragments from peptides and glycans on a single spectrum is a major limitation for annotation and identification accuracy(3, 4). Hence, obtaining unambiguous interrogation depends on the quality of both acquisition and interpretation.We have performed MS/MS of tryptic peptides separated prior by reversed-phase chromatography on a Tribrid system(ThermoFisher Eclipse) with HCD followed by the aforementioned two fragmentation modes triggered by product-dependent (pd) ion of glycan. MS/MS spectra were searched against the UniProt database using Proteome Discoverer (PD) with the Byonic searching node, and the results were manually verified. Further validation was conducted by releasing glycans with a homemade glycosidase and mapping with a MALDI-TOF/TOF(Sciex 5800) system. Our analysis of N-glycosylated samples demonstrated that pd-EthcD didn’t improve the identifications compared to other methods.Moreover, several aspects require improvements during annotation and interrogation, regardless of the fragmentation types. Firstly, glycopeptide MS1-peaking can fail when performing a match-between-run chromatogram and spectrum alignment. Secondly, MS/MS peaks-fragments annotation may be misleading when one isotopic-cluster is simultaneously assigned to two fragments. Thirdly, filtering results by scores as a common proteomics workflow might not lead to confident identifications. For example, scores> 300 and DeltaMod scores> 10 are Byonic-defined thresholds for confident peptide-sequence-match and modification localization. However, manual reviewing of the identifications showed insufficient justifications.Eventually, we proposed a workflow combining glycan MALDI-MS-MS/MS analyses to improve the robustness of glycopeptide nanoLC-MS/MS spectrum annotation and identification. The complementary profiling was applied to the porcine Luteinizing hormone study