Ultrasensitive Small RNA Sequencing Uncovers HuR Binding Landscapes
HuR (ELAVL1) is a key RNA-binding protein that regulates mRNA stability, localization, and translation by interacting with AU-rich elements in target transcripts. Although HuR has a well-established role in endothelial biology and cardiovascular disease, a comprehensive and high-resolution map of its RNA interactions remains incomplete, largely because conventional RNA immunoprecipitation (RIP) and CLIP-based approaches often require substantial input material and show limited sensitivity for low-abundance transcripts.  In this study, we combined formaldehyde-crosslinked RNA immunoprecipitation with ultrasensitive small RNA sequencing to profile HuR-associated RNAs in human umbilical vein endothelial cells (HUVECs). Using a semi-ligation (CATL) library preparation strategy, we generated high-complexity sequencing libraries from minimal RNA input and identified a broad repertoire of HuR-associated transcripts. In addition to protein-coding mRNAs, the HuR targetome included multiple classes of non-coding RNAs, such as long non-coding RNAs, microRNAs, and small nucleolar RNAs, revealing a broader spectrum of HuR interactions than previously appreciated.  To achieve precise mapping of HuR binding sites, we further developed Direct Crosslinking and Immunoprecipitation (DiCLIP), a modified CLIP workflow that incorporates formaldehyde fixation, endogenous RNase trimming, antibody-targeted protease elution, and ultrasensitive library preparation. DiCLIP enabled reproducible transcriptome-wide identification of HuR binding sites from sub-nanogram RNA inputs with high positional accuracy. Notably, DiCLIP peak summits aligned directly with canonical AU-rich HuR recognition motifs, whereas conventional iCLIP datasets showed a consistent downstream shift. In addition, DiCLIP identified previously unrecognized HuR binding sites, demonstrating improved sensitivity relative to classical approaches.  Together, these findings establish ultrasensitive small RNA sequencing combined with RIP and DiCLIP as a robust and scalable framework for mapping RNA–protein interactions from limited biological material. This work expands the known landscape of HuR-mediated post-transcriptional regulation in endothelial cells and provides a versatile platform for studying other RNA-binding proteins in low-input experimental settings.