Characterization of Plant Polyadenylation Complexes by Using Tandem Affinity Purification
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.
Key wordsPre-mRNA Polyadenylation factor Tandem affinity purification Calmodulin binding protein TEV Protein purification Mass spectrometry
We received funding support from the US National Science Foundation (grant nos. IOS–0817829 and IOS-1353354 to QQL). QQL received funding support from the Fujian Hundred Talent Plan.
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