Project description:In a previous study we applied a quantitative proximity-labeling technique called Biotin Identification (BioID, Roux et al., 2012) to analyze the microenvironment of the Gβ-like scaffold protein Asc1 (Opitz et al., 2017). The WD40-repeat protein Asc1 binds to the head region of the small 40S ribosomal (hr40S) subunit. In this study, we analyzed the hr40S from the perspective of Rps2, a ribosomal neighbor of Asc1. We intended to confirm proteins of the Asc1 proxiOME at the hr40S and to observe changes when Asc1 was absent. References: Roux K.J., Kim D.I., Raida M., Burke B. 2012. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol 196:801-810 Opitz N., Schmitt K., Hofer-Pretz V., Neumann B., Krebber H., Braus G.H., Valerius O. 2017. Capturing the Asc1p/RACK1 microenvironment at the head region of the 40S ribosome with quantitative BioID in yeast. Mol Cell Proteomics 16:2199-2218
Project description:To understand the influence of phosphorylation on the protein-protein interactions, we performed an AP-MS of SHP-1(FLAG) in HEK293T/17 cells co-transfected with TAOK3 and/or SRC kinase. Whilst TAOK3 phosphorylates Thr-394, SRC phosphorylates Tyr536 and Tyr564.
Project description:The in vivo labeling technique Biotin identification (BioID, Roux et al., 2012) was used to capture proteins in the microenvironments of the ribosomal proteins Rps3 (uS3) and Rps20 (us10) in Saccharomyces cerevisiae. Biotinylated proteins were captured from cell lysates using affinity purification and subjected to SDS-PAGE followed by in-gel digestion with trypsin. Liquid chromatography-mass spectrometry (LC-MS) was performed to identify and relatively quantify peptides and thus proteins. Relative quantification of proteins was based on stable isotope labeling with amino acids in cell culture (SILAC). To account for putative differences in the expression levels of the enriched proteins, an input control was analyzed as well. To this end, an aliquot of the cell lysate was taken prior to protein enrichment and analyzed as well. Beyond the known and expected interaction partners of Rps3 and Rps20, we identified proteins not known to co-localize with them. Moreover, we could also observe changes in their environment when the WD40-repeat protein Asc1/RACK1 was absent from the head region.
Project description:Ribosome profiling performed in Mycobacterium smegmatis MC2 155 wild-type cells versus cells with deletions of genes encoding small ORFs MSMEG_0945 and MSMEG_1916.
Project description:Activating signal co-integrator complex (ASCC) supports diverse genome maintenance and gene expression processes. Its ASCC3 subunit is an unconventional nucleic acid helicase, harboring tandem Ski2-like NTPase/helicase cassettes crucial for ASCC functions. Presently, the molecular mechanisms underlying ASCC3 helicase activity and regulation remain unresolved. Here, we present cryogenic electron microscopy, DNA-protein cross-linking/mass spectrometry as well as in vitro and cellular functional analyses of the ASCC3-ASC1/TRIP4 sub-module of ASCC. Unlike the related spliceosomal SNRNP200 RNA helicase, ASCC3 can thread substrates through both helicase cassettes. ASC1 docks on ASCC3 via a zinc finger domain and stimulates the helicase by positioning a C-terminal ASC1-homology domain next to the C-terminal helicase cassette of ASCC3, likely assisting the DNA exit. ASC1 binds ASCC3 mutually exclusively with the dealkylase, ALKBH3, directing ASCC for specific ASCC-dependent processes. Our findings define ASCC3-ASC1/TRIP4 as a tunable motor module of ASCC that encompasses two cooperating ATPase/helicase units functionally expanded by ASC1/TRIP4.
Project description:Ribosome-associated quality control (RQC) pathways monitor and respond to stalling of translating ribosomes. Using a newly developed technique based on in vivo UV crosslinking and mass spectrometry, we identify a C-terminal region in Hel2/Rqt1 as an RNA binding domain, with amino acids L501/K502 directly interacting with RNA. In vivo crosslinking of Hel2 revealed binding to 18S rRNA and translating mRNAs. Consistent with the 18S binding site located between mRNA entrance and exit channels, Hel2 preferentially bound mRNA both upstream and downstream of the termination codon. A C-terminal truncation that deleted L501/K502, abolished crosslinking to 18S rRNA, altered mRNA binding patterns, and reduced Hel2 function comparable to hel2∆. Asc1, also participates in RQC and ASC1 deletion impaired Hel2 18S and mRNA binding. We conclude that Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. Ribosome-bound Hel2 interacts with mRNA, predominately during translation termination.
Project description:Ribosome-associated quality control (RQC) pathways monitor and respond to stalling of translating ribosomes. Using a newly developed technique based on in vivo UV crosslinking and mass spectrometry, we identify a C-terminal region in Hel2/Rqt1 as an RNA binding domain, with amino acids L501/K502 directly interacting with RNA. In vivo crosslinking of Hel2 revealed binding to 18S rRNA and translating mRNAs. Consistent with the 18S binding site located between mRNA entrance and exit channels, Hel2 preferentially bound mRNA both upstream and downstream of the termination codon. A C-terminal truncation that deleted L501/K502, abolished crosslinking to 18S rRNA, altered mRNA binding patterns, and reduced Hel2 function comparable to hel2∆. Asc1, also participates in RQC and ASC1 deletion impaired Hel2 18S and mRNA binding. We conclude that Hel2 is recruited or stabilized on translating 40S ribosomal subunits by interactions with 18S rRNA and Asc1. Ribosome-bound Hel2 interacts with mRNA, predominately during translation termination.
Project description:The phosphorylation of Bre5p was analyzed in ASC1 wild-type and Δasc1 cells. Bre5p was co-purified from cell extracts with its GFP-tagged interaction partner Ubp3p in GFP-trap experiments. Trapped proteins were in-gel digested with trypsin and analyzed with the Q Exactive HF (Thermo Scientific). Protein and phospho-peptide identification and calculation of LFQ intensities were done with the MaxQuant software. For validation of phospho-peptides/-sites single ion monitoring by tSIM analysis was performed.
Project description:Our computational analyses of sequencing depth and the discovery rates of sequence elements in the 3M-bM-^@M-^YUTR strongly demonstrate that interrogation of the 3M-bM-^@M-^YUTRome in specific tissues and cell types across development will greatly expand the identification of new 3M-bM-^@M-^YUTR isoforms and the sequence elements therein. Therefore, we will generate and sequence the 3M-bM-^@M-^YUTRs from the cell types isolated from the developing germline, mature germ cells, and early embryogenesis. In addition, we will identify the 3M-bM-^@M-^YUTRs for the transcripts isolated from the major somatic tissues during late- and post-embryonic development. Based on our sequencing estimates, we anticipate that the tissue-specific interrogation of the 3M-bM-^@M-^YUTRome will reveal a far greater diversity of novel 3M-bM-^@M-^YUTR isoform expression that is masked in the whole-worm 3M-bM-^@M-^YUTRome sequence data. Using the transgenic myo-3::PABP strain, we have generated polyA-captured libraries for late embryos and across the major stages of post-embryonic development for the muscle transcriptome. We propose to generate these polyA-captured libraries for transcripts expressed in the other major tissues across development. PolyA-captured libraries will be generated for deep sequencing following the tissue-specific isolation of mRNAs by PABP pulldowns (the PABP immunoprecipitations will take advantage of a FLAG epitope which is fused to PABP in all of the transgenic strains). We propose to validate the expression of tissue- specific transcripts by qPCR for select transcripts known to be expressed in those particular tissues. In addition, following the strategy we have employed for the large-scale polyA-captured libraries from muscle, we will perform quality checks for 3M-JM-< end capture by manually sequencing ~60 clones isolated from each library. Once we have obtained the deep sequencing data, we will analyze all of the sequence reads using the bioinformatics pipeline that we established for the whole-worm polyA- captured sequences (Mangone et al., 2010). Four samples representing gravid, L2, L3, and L4 developmental stages were analyzed.
Project description:Phosphorylation of Asc1p and Asc1p-dependent phospho-proteome We determined 1) the phosphorylation sites within the Asc1 protein purified from Saccharomyces cerevisiae via its Strep-tag and 2) the Asc1p-dependent phospho-proteome. 1) Phospho-sites within tryptic peptides of Asc1p were identified using the Proteome Discoverer 1.4 software with the SequestHT and Mascot search engines and phosphorylation site localization was evaluated with the phosphoRS tool. 2) Quantitative triple SILAC-based phospho-proteome comparison of an ASC1 wild-type strain with asc1 mutant strains. MS data were analyzed with MaxQuant and Perseus.