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Evaluation of novel computational methods that identify RNA-binding protein footprints from structural data

ABSTRACT: RNA binding proteins (RBP) play diverse roles in mRNA processing and function. However, from over 1,000 RBPs encoded in the human genome, a detailed molecular understanding of their interactions with RNA is available only for a small fraction. In most cases, our knowledge of the combination of RNA sequence and structure required for specific binding is insufficient for enabling exhaustive prediction of binding sites transcriptome-wide. In that context, the rapidly expanding collection of transcriptomic datasets that map distinct, yet intertwined post-transcriptional marks, such as RNA structure and RBP binding, presents an opportunity to integratively analyze them in order to better characterize binding. A grand challenge faced by our community is that relatively little information on the structural context of RNA-protein interactions has been gleaned from integrating such datasets, partially due to lack of suitable methods. To engage scientists from diverse backgrounds in addressing this gap, the RNA Society organized the RBP Footprint Grand Challenge⸺an international community effort to develop new methods or leverage existing ones for predicting RBP binding sites through analysis of a growing volume of sequence, structure, and binding data and to experimentally validate select predictions. Here, we report the initiative, analyses and methods developed by the participants, validation results, and several new in vivo binding datasets generated for validation. We hope this work will inspire additional innovation in computational methods, further utilization of available data resources, and future endeavors to engage the community in collaborating towards closing other critical data analysis gaps.

ORGANISM(S): Homo sapiens

PROVIDER: GSE262542 | GEO | 2025/05/23

REPOSITORIES: GEO

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Project description:RNA binding proteins (RBPs) play crucial roles in regulating every stage of the mRNA life cycle and mediating non-coding RNA functions. Despite their importance, the specific roles of most RBPs remain unexplored because we do not know their specific RNA binding partners. Current methods, such as crosslinking and immunoprecipitation followed by sequencing (CLIP-seq), have expanded our knowledge of RBP-RNA interactions but are generally limited by their ability to map only one RBP at a time. To address this limitation, we developed SPIDR (Split and Pool Identification of RBP targets), a massively multiplexed method to simultaneously profile global RNA binding sites of dozens to hundreds of RBPs in a single experiment. SPIDR employs antibody-bead labeling coupled with split-pool barcoding to increase the throughput of current CLIP methods by two orders of magnitude. SPIDR identifies precise, single-nucleotide RNA binding sites for diverse classes of RBPs simultaneously. We identified several novel ribosomal RNA binders, including a novel interaction between LARP1 and 18S ribosomal RNA located within the mRNA entry site on the 40S small ribosomal subunit, and resolved this structure at 2.8 A using single-particle cryo-electron microscopy (cryo-EM). This structure provides a potential mechanistic explanation for the role of LARP1 in translational suppression. We next explored SPIDR ability to detect changes in RBP binding upon mTOR inhibition and identified that 4EBP1 acts as a dynamic RBP that selectively binds to 5-untranslated regions of translationally repressed mRNAs only upon mTOR inhibition. These observations provide a potential mechanism to explain the specificity of translational regulation controlled by mTOR signaling. SPIDR enables rapid, de novo discovery of RNA-protein interactions at an unprecedented scale and has the potential to transform our understanding of RNA biology and both transcriptional and post-transcriptional gene regulation.

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Evaluation of novel computational methods that identify RNA-binding protein footprints from structural data

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