Abstract
Purpose :
RNA-binding proteins (RBPs) establish cell type-specific transcriptional programs by regulating all aspects of RNA metabolism including RNA splicing, stability, transport, and translation. In addition to binding RNA directly, RBPs interact with RNA polymerases and epigenetic factors to regulate transcription. Not surprisingly, mutations in genes encoding ubiquitous RBPs cause a variety of human disorders including retinitis pigmentosa. However, the mechanisms by which RBPs regulate their target genes and in a cell type-specific manner remains poorly understood. We hypothesize that RBPs can interact with cell type-specific transcription factors to exert distinct cellular functions. The objective of this study is to identify RBPs that interact with the rod-specific transcription factor NRL and to elucidate the biological significance of these interactions.
Methods :
We performed glutathione-S-transferase (GST) pull-downs and Yeast two-hybrid screenings followed by mass spectrometry. Identified RBPs were studied by affinity purifications under different conditions and proximity ligation assays (PLA). The role of NRL in R-loop formation was assessed in mouse retinas and HEK293 cells overexpressing NRL by dot blot analysis of genomic DNA. R-loop levels were studied in retinas from mice at different postnatal ages and in adult mice during the regular light-dark cycle. The binding of NRL to R-loops was studied by performing pull downs of RNA/DNA hybrids.
Results :
We identified several RBPs that interact with NRL including the RNA helicases DHX9 and DDX5. Intriguingly, most of the RBPs identified were part of the R-loop proteome. R-loops are non-B DNA structures comprised of RNA-DNA hybrids with displaced single-stranded DNA. R-loops play key roles in transcriptional regulation, and their accumulation causes alterations in gene expression, genomic instability, and inflammation. We show that the interaction between NRL and the R-loop resolvases DHX9 and DDX5 occurs in euchromatin and is negatively regulated by RNA. We also show that retinal R-loops are dynamic during development and influenced by light. In agreement with NRL interaction with R-loop proteins, we found that R-loops are negatively regulated by NRL in retina and HEK293 cells.
Conclusions :
This study uncovers a new mechanism of action of NRL and underscores the importance of R-loops in maintaining retinal homeostasis.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.