Dataset Information

Exon inclusion signatures enable accurate estimation of splicing factor activity

ABSTRACT: Splicing factors control exon inclusion in messenger RNA, shaping transcriptome and proteome diversity. Their catalytic activity is regulated by multiple layers, making single-omic measurements on their own fall short in identifying which splicing factors underlie a phenotype. Here, we propose that splicing factor activity can be estimated by interpreting changes in exon inclusion. To see if this is the case we benchmarked methods to construct splicing factor→exon networks and calculate splicing factor activity. Combining RNA-seq perturbation-based networks with VIPER (virtual inference of protein activity by enriched regulon analysis) accurately captures splicing factor activation modulated by different regulatory layers. This approach consolidates splicing factor regulation into a single score derived solely from exon inclusion signatures, allowing functional interpretation of heterogeneous conditions. As a proof of concept, we identify recurrent cancer splicing programs, revealing oncogenic- and tumor suppressor-like splicing factors missed by conventional methods. These programs correlate with patient survival and key cancer hallmarks: initiation, proliferation, and immune evasion. Altogether, we show splicing factor activity can be accurately estimated from exon inclusion changes, enabling comprehensive analyses of splicing regulation with minimal data requirements. In this dataset, we share our results from analyzing the BJ fibroblast-based model for carcinogenesis through proteomics and phosphoproteomics.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Fibroblast

DISEASE(S): Carcinoma

SUBMITTER: Miquel Anglada Girotto

LAB HEAD: Luis Serrano

PROVIDER: PXD066623 | Pride | 2025-07-27

REPOSITORIES: Pride

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			Action	DRS
	2025NK023_CASE_001_02_500ng.raw	Raw
	2025NK023_CASE_002_02_500ng.raw	Raw
	2025NK023_CASE_003_01_500ng.raw	Raw
	2025NK023_CASE_004_01_500ng.raw	Raw
	2025NK023_CASE_005_01_500ng.raw	Raw

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Publications

Exon inclusion signatures enable accurate estimation of splicing factor activity.

Anglada-Girotto Miquel M Segura-Morales Carolina C Moakley Daniel F DF Zhang Chaolin C Miravet-Verde Samuel S Califano Andrea A Serrano Luis L

bioRxiv : the preprint server for biology 20250729

Splicing factors control exon inclusion in messenger RNAs, shaping transcriptome and proteome diversity. Their catalytic activity is regulated by multiple layers, making single-omic measurements on their own fall short in identifying which splicing factors underlie a phenotype. Here, we posit that splicing factor activity can be estimated from changes in exon inclusion. To test this hypothesis, we benchmarked methods for constructing splicing factor→exon networks and estimating splicing factor a ...[more]

PMID: 38979366

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Project description:Alternative RNA splicing analysis in Hep3B cell cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen Hypoxia is a common characteristic of many solid tumors. The hypoxic microenvironment stabilizes hypoxia-inducible transcription factor 1? (HIF1?) and 2? (HIF2?) to activate gene transcription, which promotes tumor cell survival. 95% of human genes are alternatively spliced, producing RNA isoforms that code functionally distinct proteins. Thus, effective hypoxia response requires increased HIF target gene transcription as well as proper RNA splicing of these HIF target genes. However, it is unclear if and how hypoxia regulates RNA splicing of HIF target genes. This study determined the effects of hypoxia on alternative splicing (AS) of HIF and non-HIF target genes in Hep3B cells and characterized the role of HIF in regulating AS of HIF induced genes. The results indicated that hypoxia generally promotes exon inclusion for hypoxia-induced, but reduces exon inclusion for hypoxia reduced genes. Mechanistically, HIF activity, but not hypoxia per se is found to be necessary and sufficient to increase exon inclusion of several HIF target genes including pyruvate dehydrogenase kinase 1 (PDK1). PDK1 splicing reporters confirmed that transcriptional activation by HIF is sufficient to increase exon inclusion of PDK1 splicing reporter. In contrast, transcriptional activation of the PDK1 minigene by other transcription factor in the absence of endogenous HIF target gene activation fails to alter PDK1 RNA splicing, demonstrating a novel role of HIF target gene(s) in regulating RNA splicing of HIF target genes. Implications:This study demonstrates a novel function of HIF in regulating RNA splicing of HIF target genes. We analyzed total RNA from Hep3B cells cultured under 21% (N1,3,5) or 1.2% (H2,4,6) oxygen using the Affymetrix Human Exon 1.0 ST platform. Array data was processed by Altanalyze software version 2.0.7. Techinical replicates were performed for Nx and Hx treated Hep3B cells