Analysis of Human Endogenous mRNA Deadenylation Complexes by High-Resolution Gel Electrophoresis

In eukaryotic cells, poly(A) tails stabilize mRNA molecules and play a pivotal role in enhancing translational efficiency. Consequently, the enzymatic shortening of these poly(A) tails by deadenylase enzymes has a critical role in post-transcriptional gene regulation. However, deadenylases are usually large, multisubunit, and multifunctional complexes, which complicates their biochemical analysis. This chapter presents a methodology for isolating human deadenylation complexes from endogenous sources and conducting an in vitro deadenylation assay to examine their enzymatic activity. The reactions involving fluorescently labeled synthetic polyadenylated RNAs are subsequently analyzed using high-resolution denaturing polyacrylamide gel electrophoresis.