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Endo-bind-n-seq: identifying RNA motifs of RNA binding proteins isolated from endogenous sources
Hanelt, Tiana Nicole, Treiber, Nora, Treiber, Thomas, Lehmann, Gerhard, Eichner, Norbert, Rothmeier, Tamara, Schmid, Georg, Reichelt, Robert, Zambelli, Federico, Pavesi, Giulio, Grohmann, Dina
and Meister, Gunter
(2024)
Endo-bind-n-seq: identifying RNA motifs of RNA binding proteins isolated from endogenous sources.
Life Science Alliance 8 (2), e202402782.
Date of publication of this fulltext: 05 Dec 2024 06:55
Article
DOI to cite this document: 10.5283/epub.59752
Abstract
RNA binding proteins (RBPs) are crucial regulators of gene expression and critically depend on the specific recognition of their target RNAs. Accordingly, a selection of methods to analyze RBP specificities has been developed, including protein-RNA crosslinking and sequencing (CLIP) and in vitro selection methods such as SELEX, RNA compete or RNA bind-n-seq. However, limitations like the ...
RNA binding proteins (RBPs) are crucial regulators of gene expression and critically depend on the specific recognition of their target RNAs. Accordingly, a selection of methods to analyze RBP specificities has been developed, including protein-RNA crosslinking and sequencing (CLIP) and in vitro selection methods such as SELEX, RNA compete or RNA bind-n-seq. However, limitations like the availability for purified recombinant proteins and custom microarray platforms (RNAcompete) or extensive sequencing depth and sophisticated bioinformatic data processing (CLIP) may limit a broader implementation of these methods. Here, we present an RNA bind-n-seq method that uses short random RNA pools and enables multiple rounds of selection. This results in strong motif enrichment with low positional variance thus reducing sequencing depth requirements. Furthermore, we have coupled our protocol to immunoprecipitation of tagged or endogenous RBPs from cultured cells or tissue samples, eliminating the need for recombinant proteins. Our method also allows for the identification of indirect RNA motifs of proteins that are integral parts of multiprotein RNPs and result in physically more relevant RNA motifs.
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| Item type | Article | ||||
| Journal or Publication Title | Life Science Alliance | ||||
| Publisher: | EMBO Press | ||||
|---|---|---|---|---|---|
| Volume: | 8 | ||||
| Number of Issue or Book Chapter: | 2 | ||||
| Page Range: | e202402782 | ||||
| Date | 2 December 2024 | ||||
| Institutions | Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie I > Prof. Dr. Gunter Meister Biology, Preclinical Medicine > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Mikrobiologie (Archaeenzentrum) > Prof. Dr. Dina Grohmann | ||||
| Identification Number |
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| Dewey Decimal Classification | 500 Science > 500 Natural sciences & mathematics 500 Science > 540 Chemistry & allied sciences 500 Science > 570 Life sciences | ||||
| Status | Published | ||||
| Refereed | Yes, this version has been refereed | ||||
| Created at the University of Regensburg | Partially | ||||
| URN of the UB Regensburg | urn:nbn:de:bvb:355-epub-597520 | ||||
| Item ID | 59752 |
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