High-Resolution Mapping of 3' Extremities of RNA Exosome Substrates by 3' RACE-Seq.
Fiche publication
Date publication
janvier 2020
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Mme KOECHLER Sandrine
Tous les auteurs :
Scheer H, De Almeida C, Sikorska N, Koechler S, Gagliardi D, Zuber H
Lien Pubmed
Résumé
The main 3'-5' exoribonucleolytic activity of eukaryotic cells is provided by the RNA exosome. The exosome is constituted by a core complex of nine subunits (Exo9), which coordinates the recruitment and the activities of distinct types of cofactors. The RNA exosome cofactors confer distributive and processive 3'-5' exoribonucleolytic, endoribonucleolytic, and RNA helicase activities. In addition, several RNA binding proteins and terminal nucleotidyltransferases also participate in the recognition of exosome RNA substrates.To fully understand the biological roles of the exosome, the respective functions of its cofactors must be deciphered. This entails the high-resolution analysis of 3' extremities of degradation or processing intermediates in different mutant backgrounds or growth conditions. Here, we describe a detailed 3' RACE-seq procedure for targeted mapping of exosome substrate 3' ends. This procedure combines a 3' RACE protocol with Illumina sequencing to enable the high-resolution mapping of 3' extremities and the identification of untemplated nucleotides for selected RNA targets.
Mots clés
3′ Adapter ligation, 3′ RACE-seq, Exosome, Illumina sequencing, MiSeq, Rapid amplification of cDNA 3′ end, Untemplated nucleotides, rRNA maturation
Référence
Methods Mol. Biol.. 2020 ;2062:147-167