Domains of CstF-64 and their functions in polyadenylation
Polyadenylation, a critical process for expression of most eukaryotic genes, requires multiple protein factors and pre-mRNA elements. However, the essential nature of polyadenylation proteins precludes in vivo determination of their precise functions. We present a straightforward, sensitive, and adaptable in vivo polyadenylation assay, the stem-loop luciferase assay for polyadenylation (SLAP) as a tool to dissect the functions of critical polyadenylation proteins. Our investigation focused on the CstF-64 subunit of the cleavage stimulation factor (CstF), which binds to the pre-mRNA downstream of the site of cleavage. Using an mRNA with a modified downstream element requiring co-expression of an MS2-CstF-64 fusion protein, we determined that the RNA binding domain (RBD), Hinge, and C-terminal domains (CTD) of CstF-64 were indispensable for polyadenylation in vivo. Furthermore, we showed that the Hinge domain was required for CstF-64 nuclear localization and CstF-77 association, suggesting that CstF complex formation and nuclear import are essential steps in polyadenylation.