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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jun 13, 2024 |
| Title |
An in situ method for identification of transcriptome-wide protein-RNA interactions in cells [eCLIP-seq ] |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
RNA-binding proteins (RBPs) play important roles in RNA metabolism including splicing, stability, localization, and translation. RBP-RNA interaction profiles are indicative of many diseases. Existing methods for mapping RBP-RNA interactions transcriptome-wide have notable limitations: immunoprecipitation (IP)-based technologies require large quantities of input materials, and RNA shearing during processing prevents identification of RNA isoforms. Meanwhile, profiling methods using RNA-modifying enzymes require ectopic expression of fusion proteins in cells of interest, potentially distorting interaction profiles. Here we report in situ STAMP, an RBP-RNA profiling method that overcomes the limitations of existing methods. In situ STAMP utilizes a chimeric fusion of the cytosine deaminase APOBEC1 and an IgG-targeting single-domain antibody (nanobody). We demonstrate that this fusion protein can be specifically targeted to proteins of interest including the RBPs RBFOX2 and TDP-43 when combined with primary antibodies targeting these proteins, enabling identification of their binding sites in un-engineered HEK293T cells. The canonical binding motifs of both RBFOX2 (UGCAUG) and TDP43 (UGUGUG) could be identified by de novo motif analysis from in situ STAMP data, demonstrating the method’s high specificity. In situ STAMP preserves intact RNAs and is therefore compatible with direct cDNA PacBio long-read sequencing, enabling the method to distinguish between RNA isoforms. Importantly, in situ STAMP is compatible with multiple fixation methods including methanol and formaldehyde fixation, enabling its application to tissue samples collected in research or clinical settings. Thus, in situ STAMP enables the profiling of authentic RBP-RNA interactions using small quantities of primary cells or tissues, thereby bridging a critical gap in uncovering the roles of RBPs in RNA-related disease mechanisms in authentic biological contexts.
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| Overall design |
eCLIP-seq with RBFOX2 specific antibody in mouse whole brain samples
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| Contributor(s) |
Liang Q, Yu T, Kofman E, Jagannatha P, Rhine K, Yee BA, Song Y, Corbett KD, Yeo GW |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Aug 10, 2023 |
| Last update date |
Jun 14, 2024 |
| Contact name |
Brian Yee |
| E-mail(s) |
brian.alan.yee@gmail.com
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| Organization name |
UCSD
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| Department |
Health
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| Lab |
Yeo
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| Street address |
9500 Gilman Dr
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platforms (1) |
| GPL21103 |
Illumina HiSeq 4000 (Mus musculus) |
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| Samples (2) |
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| This SubSeries is part of SuperSeries: |
| GSE240326 |
An in situ method for identification of transcriptome-wide protein-RNA interactions in cells |
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| Relations |
| BioProject |
PRJNA1004167 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE240521_RAW.tar |
34.2 Mb |
(http)(custom) |
TAR (of BED, BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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