pyRBDome: A comprehensive computational platform for enhancing and interpreting RNA-binding proteome data

High-throughput proteomics approaches have revolutionised the identification of RNA-binding proteins (RBPome) and RNA-binding sequences (RBDome) across organisms. Yet the extent of noise, including false-positives, associated with these methodologies, is difficult to quantify as experimental approaches for validating the results are generally low throughput. To address this, we introduce pyRBDome, a pipeline for enhancing RNA-binding proteome data in silico. It aligns the experimental results with RNA-binding site (RBS) predictions from distinct machine learning tools and integrates high-resolution structural data when available. Its statistical evaluation of RBDome data enables quick identification of likely genuine RNA-binders in experimental datasets. Furthermore, by leveraging the pyRBDome results, we have enhanced the sensitivity and specificity of RBS detection through training new ensemble machine learning models. pyRBDome analysis of a human RBDome dataset, compared with known structural data, revealed that while UV cross-linked amino acids were more likely to contain predicted RBSs, they infrequently bind RNA in high-resolution structures. This discrepancy underscores the limitations of structural data as benchmarks, positioning pyRBDome as a valuable alternative for increasing confidence in RBDome datasets.