Proteomics

Dataset Information

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Proximity labelling and LCMSMS analysis of spliceosome complexes using SUGP1-miniTurbo

ABSTRACT: The purpose of the project is to identify proteins binding differentially to the human specific spliceosomal protein SUGP1 in the context of point mutations to its G-patch domain. SUGP1 wt and mutant (G603N or L570E) C-terminally tagged with mini-Turbo-NLS was overexpressed for 48 h in HEK293T cells prior to a 2 min labelling pulse with 200 µM biotin. A non-transfection control was included and all samples were prepared in four independent biological replicates. Biotinylated proteins were captured by incubating total cell lysates for 30 min at 4 °C with streptavidin beads. The enrichment was then performed exactly as described in Cho et al. Nat Protoc 15, 3971–3999 (2020) pmid: 33139955

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Juan Oses-Prieto  

LAB HEAD: Hiten D.

PROVIDER: PXD038067 | Pride | 2023-10-05

REPOSITORIES: pride

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Dataset's files

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Action DRS
SUGP1_BioID-TMT16-protein_list_FDR-1percent.xlsx Xlsx
Y20220502-20_FTMSms2hcd.raw Raw
checksum.txt Txt
list_of_samples_and_TMT_channel_IDs.xlsx Xlsx
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Publications

Targeted high-throughput mutagenesis of the human spliceosome reveals its in vivo operating principles.

Beusch Irene I   Rao Beiduo B   Studer Michael K MK   Luhovska Tetiana T   Šukytė Viktorija V   Lei Susan S   Oses-Prieto Juan J   SeGraves Em E   Burlingame Alma A   Jonas Stefanie S   Madhani Hiten D HD  

Molecular cell 20230703 14

The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pl  ...[more]

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