Biology subjects

Selbach, M.

Publications and source records attributed to Selbach, M..

5 recordsLinked to original sources

bioRxiv2018

The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant

A century ago, influenza A virus (IAV) infection caused the 1918 flu pandemic and killed an estimated 20-40 million people. Pandemic IAV outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular details of this species barrier are incompletely understood. We combined metabolic pulse labeling and quantitative shotgun proteomics to globally monitor protein synthesis upon infection of human cells with a human-and a bird-adapted IAV strain. While production of host proteins was remarkably similar, we observed striking differences in the kinetics of viral protein synthesis over the course of infection. Most importantly, the matrix protein M1 was inefficiently produced by the bird-adapted strain at later stages. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Experiments with reporter constructs and recombinant influenza viruses revealed that strain-specific M segment splicing is controlled by the 3 splice site and functionally important for permissive infection. Independent in silico evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, our data identifies M segment RNA splicing as a viral determinant of host range.

systems biology↗

bioRxiv2018

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

molecular biology↗

bioRxiv2017

Evolutionary plasticity in the innate immune function of Akirin

Eukaryotic gene expression requires the coordinated action of transcription factors, chromatin remodelling complexes and RNA polymerase. The conserved nuclear protein Akirin plays a central role in immune gene expression in insects and mammals, linking the SWI/SNF chromatin-remodelling complex with the transcription factor NF{kappa}B. Although nematodes lack NF{kappa}B, Akirin is also indispensable for the expression of defence genes in the epidermis of Caenorhabditis elegans following natural fungal infection. Through a combination of reverse genetics and biochemistry, we discovered that in C. elegans Akirin has conserved its role of bridging chromatin-remodellers and transcription factors, but that the identity of its functional partners is different since it forms a physical complex with NuRD proteins and the POU-class transcription factor CEH-18. In addition to providing a substantial step forward in our understanding of innate immune gene regulation in C. elegans, our results give insight into the molecular evolution of lineage-specific signalling pathways.

immunology↗

bioRxiv2017

Picky: a simple method designer for targeted proteomics

Targeted proteomic approaches like selected reaction monitoring (SRM) and parallel reaction monitoring (PRM) are increasingly popular because they enable sensitive and rapid analysis of a preselected set of proteins1-3. However, developing targeted assays is tedious and requires the selection, synthesis and mass spectrometric analysis of candidate peptides before the actual samples can be analyzed. The SRMatlas and ProteomeTools projects recently published fragmentation spectra of synthetic peptides covering the entire human proteome4,5. These datasets provide very valuable resources. However, extracting the relevant data for selected proteins of interest is not straightforward. For example, developing scheduled acquisition methods (i.e. analyzing specific peptides in defined elution time windows) is complicated and requires adjustments to specific chromatographic conditions employed. Moreover, the number of peptide candidates to be targeted in parallel often exceeds the analytical abilities of the mass spectrometer. In this case, the key question is which peptides can be omitted without losing too much information. Ideally, a method design tool would automatically select the most informative peptides in each retention time window. Until now, none of the available tools automatically generates such optimized scheduled SRM and PRM methods (Figure S1).\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC=\"FIGDIR/small/163154_figS1.gif\" ALT=\"Figure 1\">\nView larger version (13K):\norg.highwire.dtl.DTLVardef@828org.highwire.dtl.DTLVardef@17a73aorg.highwire.dtl.DTLVardef@e40740org.highwire.dtl.DTLVardef@9816e7_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. S1:C_FLOATNO Comparison between different available SRM or PRM method generators.\n\nC_FIG

biochemistry↗

bioRxiv2017

Mutations In Disordered Regions Cause Disease By Creating Endocytosis Motifs

Mutations in intrinsically disordered regions (IDRs) of proteins can cause a wide spectrum of diseases. Since IDRs lack a fixed three-dimensional structure, the mechanism by which such mutations cause disease is often unknown. Here, we employ a proteomic screen to investigate the impact of mutations in IDRs on protein-protein interactions. We find that mutations in disordered cytosolic regions of three transmembrane proteins (GLUT1, ITPR1 and CACNA1H) lead to an increased binding of clathrins. In all three cases, the mutation creates a dileucine motif known to mediate clathrin-dependent trafficking. Follow-up experiments on GLUT1 (SLC2A1), a glucose transporter involved in GLUT1 deficiency syndrome, revealed that the mutated protein mislocalizes to intracellular compartments. A systematic analysis of other known disease-causing variants revealed a significant and specific overrepresentation of gained dileucine motifs in cytosolic tails of transmembrane proteins. Dileucine motif gains thus appear to be a recurrent cause of disease.

biochemistry↗