Characterization of plant polyadenylation complexes by using tandem affinity purification

Abstract

Messenger RNA in eukaryotic cells is initially produced as a nascent transcript (pre-mRNA) without a polyadenine [poly(A)] tail to the 3′ end. The precise cleavage of the pre-mRNA and addition of a poly(A) track need the communication between cis-elements in the pre-mRNA sequences and transacting protein factors recognizing them. Based on homology analyses, Arabidopsis cleavage and polyadenylation specificity factor (AtCPSF) complex should play a critical role in pre-mRNA 3′ end processing. Here we describe the isolation of AtCPSF complex by using a tandem affinity purification (TAP) method. We demonstrate that TAP is a potent protein complex isolating approach that can fulfill a downstream protein identification purpose based on mass spectrometry techniques.