E2-Regulated Transcriptome Complexity Revealed by Long-Read Direct RNA Sequencing: From Isoform Discovery to Truncated Proteins

Dioken, D. N.; Ozgul, I.; Yilmazbilek, I.; Almeric, E.; Eroglu, I. C.; CICEK, M.; YILMAZ, U. C.; KARAGOZOGLU, D.; CICEK, E.; Karaca, E.; CAN, T.; Erson-Bensan, A. E.

doi:10.1101/2025.07.31.667732

bioRxiv · 10.1101/2025.07.31.667732

E2-Regulated Transcriptome Complexity Revealed by Long-Read Direct RNA Sequencing: From Isoform Discovery to Truncated Proteins

Dioken, D. N.·Ozgul, I.·Yilmazbilek, I.·Almeric, E.·Eroglu, I. C.·CICEK, M.·YILMAZ, U. C.·KARAGOZOGLU, D.·CICEK, E.·Karaca, E.·CAN, T.·Erson-Bensan, A. E.

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Abstract

Estrogen receptor alpha (ER)-positive (ER+) breast cancers are driven by 17{beta}-estradiol (E2) binding to ER, which transcriptionally regulates downstream target genes. Although microarrays and conventional RNA sequencing have identified E2 target genes, pre-designed probes and short read lengths are limited in accurately capturing complex transcript structures. Long-read RNA sequencing offers a solution by spanning entire transcripts, providing a more complete view of the transcriptome. Here, we employed nanopore long-read direct RNA sequencing (DRS) complemented with 3-end sequencing, in vitro experiments, and deep learning-based protein modeling to explore the intricate landscape of E2-responsive transcriptome and protein level implications. Our analysis revealed a range of E2-responsive non-coding and coding isoforms, including intronically polyadenylated (IPA) mRNAs. One of these IPA isoforms was detected for TLE1 (Transducin-like enhancer protein 1), which positively assists ER-chromatin interactions for a subset of E2 target genes. The IPA isoform produces a C-terminus truncated protein, lacking the WDR interaction domain, but retains dimerization/tetramerization capacity through its intact N-terminus Q-domain. Structural modeling and protein-based assays confirmed the truncated proteins dimerization potential and nuclear localization. Functional assays showed that overexpression of truncated TLE1 reduces the E2-induced upregulation of GREB1, an E2-responsive gene, thereby disrupting transcriptional regulation. Importantly, a lower IPA isoform ratio is associated with worse survival in ER+ patients, highlighting clinical relevance. Our study uncovers new layers of complexity in the E2-regulated transcriptome, providing insights into truncated proteins. These findings contribute to a deeper understanding of gene regulation and may help the development of new therapeutic strategies.

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Dioken, D. N., Ozgul, I., Yilmazbilek, I., Almeric, E., Eroglu, I. C., CICEK, M., YILMAZ, U. C., KARAGOZOGLU, D., CICEK, E., Karaca, E., CAN, T., Erson-Bensan, A. E.. 2025-08-02. E2-Regulated Transcriptome Complexity Revealed by Long-Read Direct RNA Sequencing: From Isoform Discovery to Truncated Proteins. https://doi.org/10.1101/2025.07.31.667732

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