The hinge domain of the cleavage stimulation factor protein CstF-64 is essential for CstF-77 interaction, nuclear localization, and polyadenylation.

Because polyadenylation is essential for cell growth, in vivo examination of polyadenylation protein function has been difficult. Here we describe a new in vivo assay that allows structure-function assays on CstF-64, a protein that binds to pre-mRNAs downstream of the cleavage site for accurate and efficient polyadenylation. In this assay (the stem-loop luciferase assay for polyadenylation, SLAP), expression of a luciferase pre-mRNA with a modified downstream sequence element was made dependent upon co-expression of an MS2-CstF-64 fusion protein. We show here that SLAP accurately reflects CstF-64-dependent polyadenylation, confirming the validity of this assay. Using SLAP, we determined that CstF-64 domains involved in RNA binding, interaction with CstF-77 (the "Hinge" domain), and coupling to transcription are critical for polyadenylation. Further, we showed that the Hinge domain is necessary for CstF-64 interaction with CstF-77 and consequent nuclear localization, suggesting that nuclear import of a preformed CstF complex is an essential step in polyadenylation.