The landscape of alternative polyadenylation during EMT and its regulation by the RNA-binding protein Quaking

ABSTRACT

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3’ end anchored RNA sequencing, we mapped the alternative polyadenylation (APA) landscape following Transforming Growth Factor (TGF)-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3’UTR lengthening during this cell state transition. Investigation of potential mediators of APA indicated the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, regulates a subset of events including the length of its own transcript. Analysis of QKI crosslinked immunoprecipitation (CLIP)-sequencing data identified the binding of QKI within 3’ untranslated regions (UTRs) was enriched near cleavage and polyadenylation sites. Following QKI knockdown, APA of many transcripts is altered to produce predominantly shorter 3’UTRs associated with reduced gene expression. These findings reveal the changes in APA that occur during EMT and identify a potential role for QKI in this process.

KEYWORDS:

• Quaking
• epithelial-mesenchymal transition
• alternative polyadenylation
• Crosslinked immunopreciptation (CLIP) sequencing
• 3’ untranslated region (3’UTR)
• RNA binding protein (RBP)

Acknowledgments

We acknowledge the assistance of Angavai Swaminathan for PAT-seq library preparation, and Paul Harrison and the Monash Bioinformatics Platform for PAT-seq data management. This work was supported by grants from the National Health and Medical Research Council of Australia to P.A.G. and G.J.G. (1128479, 1164669), the National Breast Cancer Foundation (IIRS-18-147) and the Hospital Research Foundation. T.H.B. is supported by the ARC (FT180100049). G.J.G is supported by a National Health and Medical Research Council fellowship (1118170). P.A.G. and L.A.S. are supported by Principal Cancer Research Fellowships awarded by Cancer Council’s Beat Cancer project on behalf of its donors, the state Government through the Department of Health, and the Australian Government through the Medical Research Future Fund.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

D.P.N and P.A.G conceived the project. D.P.N., B.K.D., A.G.B., C.A.P., R.L., Y.R., T.H.B. and P.A.G. performed experiments. D.P.N. and K.A.P. performed the bioinformatic analysis. C.P.B, B.G.H., L.A.S., G.J.G., and P.A.G. supervised the project. D.P.N. and P.A.G. wrote the manuscript with input from the supervisory team. All authors read and approved the final manuscript.

Data deposition

The PAT-seq data and QKI HITS-CLIP data have been deposited into the Gene Expression Omnibus (GEO) with accession number GSE228932.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15476286.2023.2294222

Additional information

Funding

The work was supported by the Australian Research Council [FT180100049]; Cancer Council South Australia [Principal Cancer Research Fellowship]; National Breast Cancer Foundation [IIRS-18-147]; National Health and Medical Research Council [1128479, 1164669, 1118170]; National Health and Medical Research Council [1128479,1164669].