RBPscan: A Quantitative, In Vivo Tool for Profiling RNA-Binding Protein Interactions

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 4, 2025

SUMMARY RNA-binding proteins (RBPs) are essential regulators of gene expression at post-transcriptional level, yet obtaining quantitative insights into RBP-RNA interactions in vivo remains a challenge. Here we developed RBPscan, method that integrates RNA editing with massively parallel reporter assays (MPRAs) to profile RBP binding . RBPscan fuses the catalytic domain ADAR interest, using recorder mRNA as readout events. We demonstrate its utility zebrafish embryos, human cells, and yeast, where it quantifies strength, resolves dissociation constants, identifies high-specificity motifs for variety RBPs, links affinities their impact on stability. also provides positional information conserved novel Pumilio-binding sites lncRNA NORAD With simplicity, scalability, compatibility across systems, offers versatile tool investigating complements established methods studying regulatory networks. GRAPHICAL ABSTARCT

Language: Английский

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Sequencing technologies to measure translation in single cells

Nature Reviews Molecular Cell Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 20, 2025

Language: Английский

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Harnessing RNA base editing for diverse applications in RNA biology and RNA therapeutics

Advanced Biotechnology, Journal Year: 2025, Volume and Issue: 3(2)

Published: April 8, 2025

Abstract Recent advancements in molecular engineering have established RNA-based technologies as powerful tools for both fundamental research and translational applications. Among the various developed, RNA base editing has recently emerged a groundbreaking advancement. It primarily involves conversion of adenosine (A) to inosine (I) cytidine (C) uridine (U), which are mediated by ADAR APOBEC enzymes, respectively. been applied biological therapeutic contexts. enables site-directed within target transcripts, offering reversible, dose-dependent effects, contrast permanent or heritable changes associated with DNA editing. Additionally, editing-based profiling RNA-binding protein (RBP) binding sites facilitates transcriptome-wide mapping RBP-RNA interactions specific tissues at single-cell level. Furthermore, sensors utilized express effector proteins response species. As continue evolve, we anticipate that they will significantly drive therapeutics, synthetic biology, research.

Language: Английский

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Decoding RNA–Protein Interactions: Methodological Advances and Emerging Challenges

Advanced Genetics, Journal Year: 2025, Volume and Issue: unknown

Published: May 12, 2025

Abstract RNA–protein interactions are fundamental to cellular processes such as gene regulation and RNA metabolism. Over the past decade, significant advancements in methodologies have transformed ability study these with unprecedented resolution specificity. This review systematically compares RNA‐ protein‐centric approaches, highlighting their strengths, limitations, optimal applications. RNA‐centric methods, including hybridization‐based pulldowns, proximity labeling, CRISPR‐assisted techniques, enable identification of proteins interacting specific RNAs, even low‐abundance or transient partners. Protein‐centric strategies, immunoprecipitation‐based CLIP‐seq, emerging proximity‐tagging map interactomes RNA‐binding nucleotide precision. evaluates key innovations like LACE‐seq ARTR‐seq, which minimize cell input requirements, HyPro‐MS, bypasses genetic modifications. Guidelines for method selection provided, emphasizing experimental goals, abundance, interaction dynamics, technical constraints. Critical challenges also discussed, capturing low‐affinity interactions, resolving structural complexities, integrating multi‐omics data. underscores importance method‐tailoring biological contexts, offering a roadmap researchers navigate evolving landscape studies. By bridging practical recommendations, this aims accelerate discoveries biology, therapeutic development, precision medicine.

Language: Английский

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Viral RNA Interactome: The Ultimate Researcher’s Guide to RNA–Protein Interactions

Viruses, Journal Year: 2024, Volume and Issue: 16(11), P. 1702 - 1702

Published: Oct. 30, 2024

RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with variety regulatory consequences. Often, interactions these facilitated complex secondary and tertiary structures molecules. Viral RNAs especially known to be heavily structured interact many RBPs, roles including genome packaging, immune evasion, enhancing replication transcription, increasing translation efficiency. As such, RNA-protein interactome represents critical facet viral cycle. Characterization is necessary for development novel therapeutics targeted at disruption essential cycle events. In this review, we aim summarize various shaping interactome, interactions, as well up-to-date methods developed characterization directions novel, RNA-directed therapeutics.

Language: Английский

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Accurate in silico predictions of modified RNA interactions to a prototypical RNA-binding protein with λ-dynamics

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 11, 2024

RNA-binding proteins shape biology through their widespread functions in RNA biochemistry. Their function requires the recognition of specific motifs for targeted binding. These binding elements can be composed both unmodified and chemically modified RNAs, which over 170 chemical modifications have been identified biology. Unmodified sequence preferences widely studied, with numerous methods available to identify preferred motifs. However, only a few techniques detect modifications, no current method comprehensively screen vast array hundreds natural modifications. Prior work demonstrated that λ-dynamics is an accurate silico predict base antibody. This extends effort by using human Pumilio, prototypical protein. A library was screened at eight nucleotide positions along predicted affect Pumilio Computed affinities were compared experimental data reveal high predictive accuracy. In force field accuracies also evaluated between CHARMM Amber fields determine best parameter set use calculations. demonstrates interactions bona fide protein without requirements reagents or new experimentally test bench. Advancing like will unlock frontiers understanding how

Language: Английский

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