Discovery and characterization of isoform level complexity in estrogen-induced MCF7 cells using long-read RNA sequencing

Date

2025-8-26

Author

Özgül, İbrahim

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Estrogen (E2) signaling is a central driver of estrogen receptor-positive (ER+) breast cancer, yet our understanding of its transcriptomic impact is constrained by the limitations of short-read sequencing, which masks the complexity of transcript isoform regulation. This thesis aimed to overcome this gap by employing long-read direct RNA sequencing (DRS) to create a isoform-resolved map of the E2 response in the MCF7 cells. Our analysis revealed that E2 stimulation triggers widespread post-transcriptional remodeling of both the coding and non-coding transcriptome. For protein-coding genes, we detected E2 induced upregulation of isoforms generated through alternative splicing (AS) and alternative polyadenylation (APA). Notably, E2-induced intronic polyadenylation (IPA) generated truncated isoforms of ER pathway regulators, exemplified by TLE1 and GREB1, lacking critical C-terminus functional domains. The non-coding transcriptome analysis also identified a variety of isoform structures. We focused on several long non-coding RNAs (lncRNAs) and confirmed the DRS results, exemplified by the E2-regulated SNHG family. DRS analysis revealed that the SNHG9 isoform in MCF7 cells is a spliced transcript. Based on this isoform, we proposed a sponge function using reporter assays. Functionally, overexpression of this SNHG9 isoform, inhibited E2-driven proliferation, and clinically, high SNHG9 expression correlates with improved survival in ER+ patients, suggesting a role in a feedback loop that limits proliferation. In conclusion, we deciphered a complex architecture of isoforms regulated by E2. Hence, the use of long read sequencing techniques is likely to provide a more accurate understanding of gene expression dynamics, uncovering isoform structures that are overlooked using conventional techniques.

Subject Keywords

Breast cancer, Estrogen Receptor, Direct RNA sequencing, Transcript isoform, Noncoding RNA

URI

https://hdl.handle.net/11511/115564

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Graduate School of Natural and Applied Sciences, Thesis