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

Area of Science:

• Molecular Biology
• Genomics
• Cancer Research

Background:

• Estrogen receptor alpha (ERα)-positive (ER+) breast cancers are driven by estrogen signaling, which regulates gene expression.
• Traditional sequencing methods have limitations in capturing the full complexity of transcript structures, hindering a complete understanding of estrogen-responsive genes.

Purpose of the Study:

• To explore the landscape of the estrogen (E2)-responsive transcriptome and its protein-level implications using advanced sequencing techniques.
• To identify and characterize novel E2-responsive isoforms, including intronically polyadenylated (IPA) mRNAs, and their functional consequences.

Main Methods:

• Nanopore long-read direct RNA sequencing (DRS) coupled with 3'-end sequencing.
• In vitro experiments, deep learning-based protein modeling, structural modeling, and protein-based assays.
• Functional assays to assess the impact of identified isoforms on gene regulation and patient survival data analysis.

Main Results:

• Discovery of diverse E2-responsive coding and non-coding RNA isoforms, including IPA mRNAs.
• Identification of an IPA isoform of TLE1 that produces a C-terminus truncated protein, retaining dimerization but lacking the WDR interaction domain.
• Demonstration that truncated TLE1 disrupts the E2-induced upregulation of key genes (TFF1, GREB1) and that a lower IPA isoform ratio correlates with worse survival in ER+ breast cancer.

Conclusions:

• Long-read sequencing provides a comprehensive view of the E2-responsive transcriptome, revealing novel regulatory mechanisms.
• Truncated TLE1 protein isoforms represent a new layer of complexity in ER+ breast cancer, impacting gene regulation and clinical outcomes.
• These findings offer insights into gene regulation and potential avenues for novel therapeutic strategies in ER+ breast cancer.