Computational and Biophysical Analysis of RNA/Protein Complexes in Histone mRNA Degradation

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. This stem‐loop and its protein binding partner, the stem‐loop binding protein (SLBP), control every aspect of histone mRNA metabolism. As the cell prepares to degrade histone mRNA in the cytoplasm towards the end of S‐phase, both of these elements are modified; the mRNA is trimmed by 3’ human exonuclease (3’hExo) and uridylated by a terminal uridylyl transferase, while SLBP is dephosphorylated. The trimming and uridylation process creates uridylated intermediates that differ in length as the histone mRNA begins to undergo degradation, while the dephosphorylation of SLBP destabilizes the protein. Both of these events are associated with histone mRNA degradation, their exact roles in histone mRNA metabolism are not fully understood. In this study, we seek to characterize how these changes to SLBP and histone mRNA affect the stability of the ternary SLBP/3’hEXO/mRNA complex using both computational and experimental methods. To computationally characterize the effects of uridylation on stability of the ternary complex, we performed molecular dynamics simulations (1 µs) using AMBER force fields and Nanoscale Molecular Dynamics (NAMD). To experimentally characterize these observations, we used a fluorescence‐based EMSA competition assay to measure the proteins’ difference in affinity for the wild‐type stem‐loop and uridylated intermediates. Through these experiments, we have gained valuable insight into the role of both the uridylation of histone mRNA and the dephosphorylation of SLBP at the beginning of histone mRNA degradation.