Erson-Bensan, A. E. | bioRxiv Science

bioRxiv2025

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

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.

cancer biology↗

bioRxiv2023

Differential translation of mRNA isoforms underlies oncogenic activation of cell cycle kinase Aurora A

Aurora Kinase A (AURKA) is an oncogenic kinase with major roles in mitosis, but also exerts cell cycle- and kinase-independent functions linked to cancer. Therefore control of its expression, as well as its activity, is crucial. A short and a long 3UTR isoform exist for AURKA mRNA, resulting from alternative polyadenylation (APA). We initially observed that in Triple Negative Breast Cancer, where AURKA is typically overexpressed, the short isoform is predominant and this correlates with faster relapse times of patients. The short isoform is characterized by higher translational efficiency since translation and decay rate of the long isoform are targeted by hsa-let-7a tumor-suppressor miRNA. Additionally, hsa-let-7a regulates the cell cycle periodicity of translation of the long isoform, whereas the short isoform is translated highly and constantly throughout interphase. Finally, disrupted production of the long isoform led to an increase in proliferation and migration rates of cells. In sum, we uncovered a new mechanism dependent on the cooperation between APA and miRNA targeting likely to be a route of oncogenic activation of human AURKA.

cell biology↗