Hanelt, Tiana Nicole and Treiber, Nora and Treiber, Thomas and Lehmann, Gerhard and Eichner, Norbert and Rothmeier, Tamara and Schmid, Georg and Reichelt, Robert and Zambelli, Federico and Pavesi, Giulio and 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. ISSN , 2575-1077
Full text not available from this repository. (Request a copy)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 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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | MESSENGER-RNA; NHL DOMAIN; RECOGNITION; SPECIFICITY; COMPENDIUM; COMPLEX; SITES; |
| Subjects: | 500 Science > 570 Life sciences |
| Divisions: | 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 |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 24 Jul 2025 08:38 |
| Last Modified: | 24 Jul 2025 08:38 |
| URI: | https://pred.uni-regensburg.de/id/eprint/63621 |
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