PT  - JOURNAL ARTICLE
AU  - Hanelt, Tiana Nicole
AU  - Treiber, Nora
AU  - Treiber, Thomas
AU  - Lehmann, Gerhard
AU  - Eichner, Norbert
AU  - Rothmeier, Tamara
AU  - Schmid, Georg
AU  - Reichelt, Robert
AU  - Zambelli, Federico
AU  - Pavesi, Giulio
AU  - Grohmann, Dina
AU  - Meister, Gunter
TI  - Endo-bind-n-seq: identifying RNA motifs of RNA binding proteins isolated from endogenous sources
AID  - 10.26508/lsa.202402782
DP  - 2025 Feb 01
TA  - Life Science Alliance
PG  - e202402782
VI  - 8
IP  - 2
4099  - http://www.life-science-alliance.org/content/8/2/e202402782.short
4100  - http://www.life-science-alliance.org/content/8/2/e202402782.full
SO  - Life Sci. Alliance2025 Feb 01; 8
AB  - 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.Sequencing data are available at the SRA database under the accession: PRJNA1097567, Temporary Submission ID: SUB14331669.