In Vitro Reconstitution of the Drosophila melanogaster CCR4-NOT Complex to Assay Deadenylation

Filip Pekovic; Elmar Wahle

doi:10.1007/978-1-0716-3481-3_2

In Vitro Reconstitution of the Drosophila melanogaster CCR4-NOT Complex to Assay Deadenylation

Methods Mol Biol. 2024:2723:19-45. doi: 10.1007/978-1-0716-3481-3_2.

Authors

Filip Pekovic^{1

2}, Elmar Wahle³

Affiliations

¹ Institute of Biochemistry and Biotechnology and Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle, Germany. filip.pekovic@nih.gov.
² RNA Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, MD, USA. filip.pekovic@nih.gov.
³ Institute of Biochemistry and Biotechnology and Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, Halle, Germany. ewahle@biochemtech.uni-halle.de.

PMID: 37824062
DOI: 10.1007/978-1-0716-3481-3_2

Abstract

The CCR4-NOT complex is a multi-subunit poly(A)-specific 3' exoribonuclease that catalyzes the deadenylation of mRNA. In this chapter, we describe procedures to express and purify recombinant Drosophila melanogaster CCR4-NOT. Furthermore, we provide protocols for preparing radioactively labeled RNA substrates and conducting in vitro deadenylation assays.

Keywords: CCR4-NOT; Deadenylases; Deadenylation; Poly(A) tail; mRNA decay.

MeSH terms

Animals
Drosophila Proteins* / genetics
Drosophila Proteins* / metabolism
Drosophila melanogaster* / genetics
Drosophila melanogaster* / metabolism
RNA Stability
Ribonucleases / metabolism

Substances

Ribonucleases
CCR4 protein, Drosophila
Drosophila Proteins