Identification of a stem-loop structure important for polyadenylation at the murine IgM secretory poly(A) site

We have previously shown that a distal GU-rich downstream element of the mouse IgM secretory poly(A) site is important for polyadenylation in vivo and for polyadenylation specific complex formation in vitro. This element can be predicted to form a stem-loop structure with two asymmetric internal loops. As stem-loop structures commonly define protein RNA binding sites, we have probed the biological activity of the secondary structure of this element. We show that mutations affecting the stem of the structure abolish the biological activity of this element in vivo and in vitro at the level of cleavage and polyadenylation specificity factor/cleavage stimulation factor complex formation and that both internal loops contribute to the enhancing effect of the sequence in vivo. Lead (II) cleavage patterns and RNase H probing of the sequence element in vitro are consistent with the predicted secondary structure. Furthermore, mobility on native PAGE suggests a bent structure. We propose that the secondary structure of this downstream element optimizes its interaction with components of the polyadenylation complex.