50 research outputs found
Nucleic Acids Research / Inosine induces context-dependent recoding and translational stalling
No full textRNA modifications are present in all classes of RNAs. They control the fate of mRNAs by affecting their processing, translation, or stability. Inosine is a particularly widespread modification in metazoan mRNA arising from deamination of adenosine catalyzed by the RNA-targeting adenosine deaminases ADAR1 or ADAR2. Inosine is commonly thought to be interpreted as guanosine by cellular machines and during translation. Here, we systematically test ribosomal decoding using mass spectrometry. We show that while inosine is primarily interpreted as guanosine it can also be decoded as adenosine, and rarely even as uracil. Decoding of inosine as adenosine and uracil is context-dependent. In addition, mass spectrometry analysis indicates that inosine causes ribosome stalling especially when multiple inosines are present in the codon. Indeed, ribosome profiling data from human tissues confirm inosine-dependent ribosome stalling in vivo. To our knowledge this is the first study where decoding of inosine is tested in a comprehensive and unbiased way. Thus, our study shows novel, unanticipated functions for inosines in mRNAs, further expanding coding potential and affecting translational efficiency
Identifying protein kinase–specific effectors of the osmostress response in yeast
No full textPhosphoproteomics identifies direct and indirect targets of the kinase Hog1 during the yeast osmostress response.</jats:p
Weak Organic Acid Stress Triggers Hyperphosphorylation of the Yeast Zinc-Finger Transcription Factor War1 and Dampens Stress Adaptation
No full textIdentification of a conserved phosphorylation motif within RNA metabolic phosphoproteins
No full text<p><b>Copyright information:</b></p><p>Taken from "Phosphoproteomics reveals extensive phosphorylation of Arabidopsis proteins involved in RNA metabolism"</p><p>Nucleic Acids Research 2006;34(11):3267-3278.</p><p>Published online 17 Jul 2006</p><p>PMCID:PMC1904105.</p><p>© 2006 The Author(s)</p> The phosphorylated Ser residue is indicated in boldface. Identity and homology of amino acids are marked by black and gray shades, respectively
Crosstalk between casein kinase II and Ste20-related kinase Nak1
No full textAustrian Science Fund [P23609, P21437, F3403]; HFSP [RGY0069/2010]; Slovak Research and Development Agency [APVV-0111-12, APVV-0334-12]; European Community [PERG07-GA-2010-268167, PCIG11-GA-2012-322300]; National Institutes of Health [GM058406-14]Although the sterile 20 (Ste20) serine/threonine protein kinase was originally identified as a component of the S. cerevisiae mating pathway, it has homologs in higher eukaryotes and is part of a larger family of Ste20-like kinases. Ste20-like kinases are involved in multiple cellular processes, such as cell growth, morphogenesis, apoptosis and immune response. Carrying out such a diverse array of biological functions requires numerous regulatory inputs and outputs in the form of protein-protein interactions and post-translational modifications. Hence, a thorough knowledge of Ste20-like kinase binding partners and phosphorylation sites will be essential for understanding the various roles of these kinases. Our recent study revealed that Schizosaccharomyces pombe Nak1 (a conserved member of the GC-kinase sub-family of Ste20-like kinases) is in a complex with the leucine-rich repeat-containing protein Sog2. Here, we show a novel and unexpected interaction between the Nak1-Sog2 kinase complex and Casein kinase 2 (Cka1, Ckb1 and Ckb2) using tandem-affinity purification followed by mass spectrometric analysis. In addition, we identify unique phosphosites on Nak1, Sog2 and the catalytic subunit of casein kinase 2, Cka1. Given the conserved nature of these kinases, we expect this work will shed light on the functions of these proteins both in yeast and higher eukaryotes
Hyperphosphorylation of Human Osteopontin and Its Impact on Structural Dynamics and Molecular Recognition
No full textCrosstalk between casein kinase II and Ste20-related kinase Nak1
No full textAlthough the sterile 20 (Ste20) serine/threonine protein kinase was originally identified as a component of the S. cerevisiae mating pathway, it has homologs in higher eukaryotes and is part of a larger family of Ste20-like kinases. Ste20-like kinases are involved in multiple cellular processes, such as cell growth, morphogenesis, apoptosis and immune response. Carrying out such a diverse array of biological functions requires numerous regulatory inputs and outputs in the form of protein-protein interactions and post-translational modifications. Hence, a thorough knowledge of Ste20-like kinase binding partners and phosphorylation sites will be essential for understanding the various roles of these kinases. Our recent study revealed that Schizosaccharomyces pombe Nak1 (a conserved member of the GC-kinase sub-family of Ste20-like kinases) is in a complex with the leucine-rich repeat-containing protein Sog2. Here, we show a novel and unexpected interaction between the Nak1-Sog2 kinase complex and Casein kinase 2 (Cka1, Ckb1 and Ckb2) using tandem-affinity purification followed by mass spectrometric analysis. In addition, we identify unique phosphosites on Nak1, Sog2 and the catalytic subunit of casein kinase 2, Cka1. Given the conserved nature of these kinases, we expect this work will shed light on the functions of these proteins both in yeast and higher eukaryotes
Chemical acetylation of ligands and two-step digestion protocol for reducing codigestion in affinity purification–mass spectrometry
No full textWe present an effective, fast, and user-friendly method to reduce codigestion of bead-bound ligands, such as antibodies or streptavidin, in affinity purification-mass spectrometry experiments. A short preincubation of beads with Sulfo-NHS-Acetate leads to chemical acetylation of lysine residues, making ligands insusceptible to Lys-C-mediated proteolysis. In contrast to similar approaches, our procedure offers the advantage of exclusively using nontoxic chemicals and employing mild chemical reaction conditions. After binding of bait proteins to Sulfo-NHS-Acetate treated beads, we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion of the released proteins with trypsin. The implementation of this protocol results in a strong reduction of contaminating ligand peptides, which allows significantly higher amounts of sample to be subjected to LC–MS analysis, improving sensitivity and quantitative accuracy
Hyperphosphorylation of Human Osteopontin and Its Impact on Structural Dynamics and Molecular Recognition
No full text60final_published17AT_PUBLICATIONPublikacja bezkosztow
