High-Resolution Mapping of 3’ Extremities of RNA Exosome Substrates by 3’ RACE-Seq
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 wordsExosome rRNA maturation Rapid amplification of cDNA 3′ end 3′ RACE-seq 3′ Adapter ligation Illumina sequencing MiSeq Untemplated nucleotides
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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