Spliceosomal U1A Protein-SL2 RNA Binding Affinity Decreases in Cells

Abstract

While extensive biochemical and biophysical studies have been carried out to elucidate protein-RNA binding mechanisms and dynamics in vitro, most of these studies do not take into consideration the effect of the cellular environment. Here we have experimentally tested the role of the cellular environment on protein transport and binding affinity in one of the most widely studied RNA recognition motifs, the spliceosomal protein U1A, and its binding partner, stem loop 2 (SL2) of the U1 small nuclear RNA. U1A-SL2 localization, stability and binding kinetics were monitored in live U2OS cells by fast relaxation imaging (FreI), which combines a temperature jump with fluorescence microscopy of the FRET (Fluorescence resonance energy transfer)-labeled RNA and protein. U1A protein alone was found to diffuse across the cell, whereas SL2 RNA and the U1A-SL2 complex localized in the nucleus. This demonstrates that SL2 RNA mediates transport of the U1A protein to the nucleus. The binding affinity in live cells was reduced compared to in vitro. The dissociation rate was unchanged in cells; however, the association rate was an order of magnitude lower, resulting in a two order of magnitude decrease in the dissociation constant. Introduction of a macromolecular crowder, Ficoll 70, in vitro further stabilized the complex. This suggests that differences between binding affinities as measured in vitro and in live cells cannot be explained by crowding alone. Instead, high binding affinities as measured in vitro may be necessary for selectivity in vivo, where competition exists between multiple binding partners.