PXD034016

PXD034016 is an original dataset announced via ProteomeXchange.

Title	High throughput screen of 100,000 small molecules in C9orf72 ALS neurons
Description	Background. An intronic G4C2 repeat expansion in the C9orf72 gene is the major known cause for Amyotrophic Lateral Sclerosis. There is evidence forthree possible disease mechanisms: a pathological gain of function of nuclear repeat RNA foci, repeat associated noncanonical (RAN) translation into toxic dipeptide repeat (DPR) polyproteins as well as C9orf72 haploinsufficiency. Which of these defects need to be restored to prevent or halt disease progression remains a fundamental question for the development of ALS therapeutics. Methods. Here we developed a high content imaging assay for G4C2 RNA foci detection in C9orf72 patient iPS derived neurons miniaturised to the 1536well format. 96’200 small molecules were screened for RNA foci modulation, with counterscreens for cell toxicity and general gene expression inhibition. Hits were validated in relevant cell models from different C9orf72 donors and selected for further characterization of their cellular and molecular mode of action. Results. Among the validated hits we identified analogues of known modulators of SF3B1, a major component of the U2 snRNP spliceosomal subunit. Structure activity relationship and genome wide splice analysis confirmed that compounds to clear nuclear G4C2 RNA foci by targeting SF3B1. A combination of genetically engineered G4C2 reporter systems and RNA-protein interactome studies reveals that pharmacological SF3B1 modulation promotes recruitment of SRSF1 to nuclear G4C2 RNA, mobilizing it from RNA foci into nucleocytoplasmic export. Thisleadsto increased RAN translation and, ultimately, enhanced DPR cell toxicity. Preventing SRSF1 phosphorylation by small molecule inhibition of SR protein kinase (SRPK) conversely results in a build up of nuclear RNA foci, antagonistic to SF3B1 modulation. Conclusion. Together, our data provide a new pharmacological entry point with publically available, antagonistic tool compoundsfor modulating G4C2 repeat expansion pathology in C9orf72 ALS models. This will facilitate the dissection of C9orf72 pathobiology by uncoupling RNA foci from RAN translation. Based on our data we propose that therapeutic RNA foci elimination strategies warrant caution due to a potential storage function, counteracting translation into toxic dipeptide repeat polyproteins. Instead, our data support modulation of nuclear export via SRSF1 or SR protein kinases as possible targets for future pharmacological drug discovery
HostingRepository	PRIDE
AnnounceDate	2025-03-07
AnnouncementXML	Submission_2025-03-07_03:42:06.488.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hans Voshol
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	No PTMs are included in the dataset
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2022-05-20 10:44:21	ID requested
⏵ 1	2025-03-07 03:42:06	announced

Dataset with its publication pending

submitter keyword: ALS, SRSF1,C9orf72

Hans Voshol
contact affiliation	Novartis Inst. for BioMedical Research
contact email	johannes.voshol@novartis.com
lab head
Hans Voshol
contact affiliation	Novartis Inst. for BioMedical Research
contact email	johannes.voshol@novartis.com
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2025/03/PXD034016

PRIDE project URI

• PRIDE
  1. PXD034016
     1. Label: PRIDE project
     2. Name: High throughput screen of 100,000 small molecules in C9orf72 ALS neurons