The ribosome quality control factor Asc1 determines the fate of HSP70 mRNA on and off the ribosome
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: 
Lokha Ranjani Alagar Boopathy
LAB HEAD: Maria Vera Ugalde
PROVIDER: PXD037545 | Pride | 2023-04-19
REPOSITORIES: pride
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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
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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.
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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.
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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.
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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.
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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.
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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.
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