Dataset Information

Label-free LC-MS/MS analysis of proteins associated with specific RNAs in HeLa cells

ABSTRACT: Proteins co-localizing with RNA targets of interest in HeLa cells were identified using our recently optimized hybridization-proximity labeling approach (HyPro2) combined with label-free mass spectrometry. Briefly, fixed and permeabilized cells were hybridized with digoxigenin-labeled antisense oligonucleotide probes targeting the long noncoding RNA PNCTR or pre-mRNAs encoding ACTB and FUS proteins. A no-probe negative control was also included. The purified HyPro2 enzyme, which contains a digoxigenin-binding domain and a modified ascorbate peroxidase domain, was then recruited to the RNA targets, enabling in vitro biotinylation of target-proximal proteins. Following streptavidin pull-down, biotinylated proteins were fragmented using a Trypsin/Lys-C protease mix and identified by label-free LC-MS/MS analysis. All experiments were performed in triplicate.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cervical Adenocarcinoma Cell

SUBMITTER: Steven Lynham

LAB HEAD: Eugene Makeyev

PROVIDER: PXD063191 | Pride | 2025-08-04

REPOSITORIES: Pride

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Publications

M6A-dependent RNA condensation underlies FUS autoregulation and can be harnessed for ALS therapy development.

Huang Wan-Ping WP Kumar Vedanth V Yap Karen K An Haiyan H John Sabin J SJ Hodgson Rachel E RE Avila Anna Sanchez AS Day Emily E Ellis Brittany C S BCS Chung Tek Hong TH Lord Jenny J Müller-McNicoll Michaela M Makeyev Eugene V EV Shelkovnikova Tatyana A TA

Science advances 20250723 30

Mutations in the <i>FUS</i> gene cause aggressive amyotrophic lateral sclerosis (ALS-FUS). Beyond mRNA, <i>FUS</i> generates partially processed transcripts retaining introns 6 and 7. We demonstrate that these FUSint6&7-RNA molecules form nuclear condensates, scaffolded by the highly structured intron 7 and associated with nuclear speckles. Using hybridization-proximity labeling proteomics, we show that the FUSint6&7-RNA condensates are enriched for splicing factors and the N6-methyladenosine (m ...[more]

PMID: 40700505

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			Action	DRS
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	PR702_KY1_10_F1.mzid_PR702_KY1_10_F1.MGF	Mzid
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