The balance between the poly(A) length and the downstream post-poly(A) length affects deadenylation efficiency. (A) Schematic representation of a series of Mini-let-7 reporter constructs. These reporter RNAs include four let-7 target sites and were capped with [α-32P] GTP. The subscripts show the length of an RNA sequence including the shown base. The asterisks indicate a radiolabel. (B) Deadenylation assay for Mini-let-7 reporter RNAs with S2 cell lysate overexpressed Ago1. (C) The signal intensity of the bands in B was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini-let-7-A20 effectively shifted to A0. While the length of the post-poly(A) sequence negatively correlated with the deadenylation efficiency, elongating the internal poly(A) sequence improved. The graph shows means and standard deviations (n = 3). (D) Deadenylation assay for Mini-let-7-A60C10 and Mini-let-7-N40A20C10 with S2 cell lysate overexpressed Ago1. (E) The signal intensity of the bands in D was quantified, and the ratio of the intensity from the lower band for the total intensity was calculated and plotted. Mini-let-7-A60C10 was deadenylated more effectively than Mini-let-7-N40A20C10. The graph shows means and standard deviations (n = 3).
