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High-Resolution Mapping of 3’ Extremities of RNA Exosome Substrates by 3’ RACE-Seq

  • Hélène Scheer
  • Caroline De Almeida
  • Natalia Sikorska
  • Sandrine Koechler
  • Dominique GagliardiEmail author
  • Hélène ZuberEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2062)

Abstract

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.

Key words

Exosome rRNA maturation Rapid amplification of cDNA 3′ end 3′ RACE-seq 3′ Adapter ligation Illumina sequencing MiSeq Untemplated nucleotides 

Notes

Acknowledgments

This work was supported by the Centre National de la Recherche Scientifique (CNRS, France) and research grants from the French National Research Agency as part of the “Investments for the Future” program in the frame of LABEX ANR-10-LABX-0036_NETRNA and ANR-15-CE12-0008-01 to D.G, and in the frame of the IdEx Unistra to H.Z.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Hélène Scheer
    • 1
  • Caroline De Almeida
    • 1
  • Natalia Sikorska
    • 1
  • Sandrine Koechler
    • 1
  • Dominique Gagliardi
    • 1
    Email author
  • Hélène Zuber
    • 1
    Email author
  1. 1.Institut de Biologie Moléculaire des Plantes, Centre National de la Recherche Scientifique (CNRS)Université de StrasbourgStrasbourgFrance

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