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Updated: Oct 1, 2025
A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells
Published on: June 16, 2022
CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis
Souvik Ghosh1, Meric Ataman1,2, Maciej Bak1,2
1Computational and Systems Biology, Biozentrum, University of Basel, Spitalstrasse 41, 4056 Basel, Switzerland.
Abstract:
The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3' UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities.
Insights
Mammalian cleavage factor I (CFIm) regulates alternative polyadenylation (APA) in over 800 genes, impacting protein metabolism and miRNA pathways. CFIm levels correlate with ERK pathway activity, influencing cell growth and metabolism.
Area of Science:
- Molecular Biology
- RNA Biology
- Cellular Metabolism
Background:
- The mammalian cleavage factor I (CFIm) complex is crucial for alternative polyadenylation (APA).
- CFIm dysregulation is linked to various conditions, including cancer and neurological deficits.
- Understanding CFIm's role in translating expression levels into cellular phenotypes is essential.
Purpose of the Study:
- To systematically analyze the impact of CFIm subunit (CFIm25/CFIm68) levels on gene expression and APA.
- To identify core APA targets regulated by CFIm and elucidate downstream signaling pathways.
- To investigate the relationship between CFIm, ERK pathway activity, and cellular metabolism.
Main Methods:
- Multi-omics analysis of cell lines with manipulated CFIm25 or CFIm68 expression (siRNA knockdown and overexpression).
- Identification of genes with coherent APA in response to CFIm level changes.
- Analysis of signaling pathways, including the ERK pathway, and miRNA pathway components.
Main Results:
- >800 genes exhibited coherent APA in response to altered CFIm levels, clustering in protein metabolism-related functional classes.
- ERK pathway activity was found to correlate with CFIm concentration, explaining observed changes in cell growth and metabolism.
- CFIm-dependent APA affects transcripts in the miRNA pathway, increasing miRNA biogenesis and activity while shortening some 3' UTRs.
Conclusions:
- This study provides the first systematic assessment of core APA targets responding to CFIm subunit level changes.
- Identified downstream signaling pathways clarify CFIm's role in integrating RNA processing with cellular activities.
- CFIm acts as a key regulator connecting RNA processing, miRNA pathways, and cellular metabolism.
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