E2-regulated transcriptome complexity revealed by long-read direct RNA sequencing: from isoform discovery to truncated proteins

Oestrogen receptor alpha (ERα)-positive (ER+) breast cancers are driven by the binding of 17β-oestradiol (E2) to ERα, which transcriptionally regulates target genes. Although microarrays and conventional RNA sequencing have identified E2 target genes, pre-designed probes and short read lengths are limited in their ability to accurately capture complex transcript structures. Long-read 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 modelling to explore the landscape of the 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, which assists ERα-chromatin interactions for a subset of E2 targets. The IPA isoform produces a C-terminus truncated protein that lacks the WDR interaction domain but retains dimerization/tetramerization capacity through its intact N-terminal Q-domain. Structural modelling and protein-based assays confirmed the dimerization potential and nuclear localization of the truncated protein. Functional assays showed that the overexpression of truncated TLE1 reduced the E2-induced upregulation of <i>TFF1</i> and <i>GREB1</i>, E2-responsive genes, thereby disrupting transcriptional regulation. Importantly, a lower IPA isoform ratio is associated with worse survival in ER+ breast cancers, highlighting clinical relevance. Our study revealed 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.