Van De Velde, J. | Search bioRxiv

bioRxiv2025

TurboID proximity interactome mapping reveals NR2E3 association with AP-1 and retinal developmental complexes

The photoreceptor-specific nuclear receptor NR2E3 is a key transcription factor in the retinal transcriptional network, essential in photoreceptor cell fate and maintenance. Various biallelic loss-of-function variants lead to autosomal recessive inherited retinal disease (IRD), while a single missense variant G56R has been identified to cause autosomal dominant retinitis pigmentosa (adRP). However, a comprehensive understanding of the interactome of NR2E3 remains elusive. Here, we aimed to map the interactome of this nuclear receptor and the G56R pathogenic variant through proximity labelling followed by mass spectrometry. We used the TurboID "T2A split/link design" to identify the proximity interactome of wild-type NR2E3 and mutant G56R-NR2E3 in ARPE-19 cells. Several protein complexes involved in retinal development, including the AP-1 complex, were enriched. The results suggest similar interactomes for NR2E3 and G56R-NR2E3 in ARPE-19. However, BCOR was exclusively enriched in the G56R-NR2E3 network, strengthening the connection with the IRD phenotype. In conclusion, our approach has mapped the proximity interactome of NR2E3 and G56R-NR2E3, revealing interaction with protein complexes involved in (retinal) development.

systems biology↗

bioRxiv2025

A laser capture microdissection-based method for high-sensitivity transcriptomics from archived FFPE tissue slides with single-cell resolution using LCM-FFPEseq

Understanding gene expression within its spatial context is essential for unravelling biological processes. Laser Capture Microdissection (LCM) has emerged as a transformative technology, enabling targeted isolation of individual cells or regions from tissue sections while preserving spatial context. However, its application to formalin-fixed, paraffin-embedded (FFPE) tissues has been limited by RNA degradation, leaving the vast repository of clinical FFPE samples underutilized. To address this, we introduce LCM-FFPEseq, a novel method combining LCM with the advanced Smart-seq3xpress protocol and FFPE-specific adaptations for spatial transcriptomics of FFPE sections. Unlike traditional protocols requiring thousands of cells to generate high-quality libraries, LCM-FFPEseq achieves high sensitivity, reproducibility, and transcript coverage. With as few as 30 FFPE-embedded K562 cells and Sertoli cells, we detected over 14,000 protein-coding genes per sample, with no substantial gains when a higher number of cells were isolated. Even individual LCM-isolated cells yielded an average of 7,353 or 6,490 protein-coding genes per K562 or Sertoli single cell, respectively. To demonstrate its clinical utility, we applied LCM-FFPEseq to archived testicular FFPE samples from transgender females receiving gender-affirming hormone therapy. Transcriptomic profiling of isolated seminiferous tubules revealed tubular hyalinization to be associated with greater upregulation of extracellular matrix remodelling and inflammatory pathways, alongside stronger downregulation of spermatogenesis-associated pathways. These findings suggest that testicular fibrosis and/or tubular hyalinization may contribute to germ cell loss following inappropriate hormonal exposure. By enabling high-resolution transcriptomics in archived FFPE samples, LCM-FFPEseq unlocks new possibilities for investigating rare cell types, spatial heterogeneity, and therapy-induced tissue remodelling in vast FFPE repositories.

genomics↗