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Hanelt, Tiana Nicole ; Treiber, Nora ; Treiber, Thomas ; Lehmann, Gerhard ; Eichner, Norbert ; Rothmeier, Tamara ; Schmid, Georg ; Reichelt, Robert ; Zambelli, Federico ; Pavesi, Giulio ; Grohmann, Dina ; Meister, Gunter

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.



Involved Institutions


Details

Item typeArticle
Journal or Publication TitleLife Science Alliance
Publisher:EMBO Press
Volume:8
Number of Issue or Book Chapter:2
Page Range:e202402782
Date2 December 2024
InstitutionsBiology, 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
ValueType
10.26508/lsa.202402782DOI
Dewey Decimal Classification500 Science > 500 Natural sciences & mathematics
500 Science > 540 Chemistry & allied sciences
500 Science > 570 Life sciences
StatusPublished
RefereedYes, this version has been refereed
Created at the University of RegensburgPartially
URN of the UB Regensburgurn:nbn:de:bvb:355-epub-597520
Item ID59752

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