Biophysical Characterization and Computational Modeling of Histone mRNA Degradation Intermediates

Abstract

Histone mRNA is the only known metazoan mRNA that is not polyadenylated at its 3′ end; instead, it ends in a highly conserved stem-loop formed by Watson-Crick base pairing. In histone mRNA degradation, the stem-loop is trimmed by 3′ human exonuclease (3′hExo) and uridylated by terminal uridylyl transferase 7. This process creates uridylated intermediates—some of which are more common in the cell than expected. It is unknown how this trimming and uridylation affects the binding interactions between the stem-loop and its protein binding partners, the stem-loop binding protein (SLBP) and 3′hExo. In this work, we characterized the binding interactions of a wild-type stem-loop, a uridylated intermediate, and a truncated stem-loop using a combination of experimental and computational biophysical techniques. The uridylated intermediate represents one of the most prominent degradation intermediates identified in the cell, and the truncated stem-loop represents the stem-loop post-trimming and pre-uridylation. Through native electrophoretic mobility shift assays, we show that SLBP and 3′hExo are able to bind both the uridylated intermediate and the truncated stem-loop despite 3′ end modification. We also performed molecular dynamics simulations (1 µs) to evaluate structural changes induced by stem-loop modification and to understand how these changes impact SLBP and 3′hExo binding. RNA-protein systems were generated by applying AMBER force fields to modified versions of the histone mRNA ternary complex crystal structures (PDB 4QOZ and 4L8R). This work shows that modification of the stem-loop through trimming and uridylation does not interfere with the tertiary structure of the ternary complex.