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Profiling of RNA-binding protein binding sites by in-situ reverse transcription-based sequencing

ABSTRACT: RNA-binding proteins (RBPs) regulate diverse cellular processes by dynamically interacting with RNA targets. However, effective methods to capture both stable and transient interactions between RBPs and their RNA substrates are still lacking, especially when the interaction is dynamic or materials are limited. Here we present an assay of reverse transcription-based RBP binding sites sequencing (ARTR-seq), which relies on in-situ reverse transcription of RBP-bound RNAs guided by antibodies to identify RBP binding sites. ARTR-seq avoids ultraviolet cross-linking and immunoprecipitation, allowing for efficient and specific identification of RBP binding sites from as few as 20 cells or a tissue section. Taking advantage of rapid formaldehyde fixation, ARTR-seq enables capturing the dynamic binding of RBPs over a short period of time, as demonstrated by the discovery of dynamic RNA binding of G3BP1 during stress granule assembly on a timescale as short as 10 min.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE226161 | GEO | 2023/11/05

REPOSITORIES: GEO

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RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

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2013-11-06 | E-GEOD-49309 | biostudies-arrayexpress

Global Profiling of RNA-Binding Protein Target Sites by LACE-seq

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RNase-mediated protein footprint sequencing reveals protein-binding sites throughout the human transcriptome

2013-11-06 | GSE49309 | GEO

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Global signatures of protein binding on structural RNAs in Saccharomyces cerevisiae

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2013-07-15 | E-GEOD-48888 | biostudies-arrayexpress

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Project description:Two features of eukaryotic RNA molecules that regulate their post-transcriptional fates are RNA secondary structure and RNA-binding protein (RBP) interaction sites. However, a comprehensive global overview of the dynamic nature of these sequence features during erythropoiesis has never been obtained. Here, we use our ribonuclease-mediated structure and RBP-binding site mapping approach to reveal the global landscape of RNA secondary structure and RBP-RNA interaction sites and the dynamics of these features during this important developmental process. We identify dynamic patterns of RNA secondary structure and RBP binding that are consistently anti-correlated throughout mRNAs during erythropoiesis. Additionally, we determine a set of protein-bound sequence motifs and their dynamic structural and RBP binding contexts. Finally, using these dynamically bound sequences, we identify a number of RBPs that have known and putative key functions in post-transcriptional regulation during mammalian erythropoiesis. In total, this global analysis reveals new post-transcriptional regulators of mammalian blood cell development.

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