Direkt zum Inhalt

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 und Meister, Gunter (2024) Endo-bind-n-seq: identifying RNA motifs of RNA binding proteins isolated from endogenous sources. Life Science Alliance 8 (2), e202402782.

Veröffentlichungsdatum dieses Volltextes: 05 Dez 2024 06:55
Artikel
DOI zum Zitieren dieses Dokuments: 10.5283/epub.59752


Zusammenfassung

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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Details

DokumentenartArtikel
Titel eines Journals oder einer ZeitschriftLife Science Alliance
Verlag:EMBO Press
Band:8
Nummer des Zeitschriftenheftes oder des Kapitels:2
Seitenbereich:e202402782
Datum2 Dezember 2024
InstitutionenBiologie und Vorklinische Medizin > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Biochemie I > Prof. Dr. Gunter Meister
Biologie und Vorklinische Medizin > Institut für Biochemie, Genetik und Mikrobiologie > Lehrstuhl für Mikrobiologie (Archaeenzentrum) > Prof. Dr. Dina Grohmann
Identifikationsnummer
WertTyp
10.26508/lsa.202402782DOI
Dewey-Dezimal-Klassifikation500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
500 Naturwissenschaften und Mathematik > 540 Chemie
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
StatusVeröffentlicht
BegutachtetJa, diese Version wurde begutachtet
An der Universität Regensburg entstandenZum Teil
URN der UB Regensburgurn:nbn:de:bvb:355-epub-597520
Dokumenten-ID59752

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