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-RNA binding affinity in the simplest and smallest spliceosomal subunit, the U1 snRNP. We compare binding affinities of the U1 specific proteins U1A and U170K, which have been found to independently bind U1 small nuclear RNA (snRNA). Although the spliceosomal protein U1A and its binding partner, stem loop 2 (SL2) of the U1 snRNA, is one of the most widely studied RNA recognition motifs (RRMs), U170K is relatively less well studied. Intriguingly, U170K has recently been shown to interact with its binding partner, stem loop 1 (SL1) of the U1 snRNA, through an N-terminal helix flanking its RRM, a mechanism unique among RRMs. U1A-SL2 and U170K-SL1 stability and binding kinetics were monitored in live U-2 OS 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. The binding affinity of U1A-SL2 in live cells was reduced compared to in vitro, whereas the binding affinity of U170K-SL1 was maintained in live cells. This suggests that the alternative binding mechanism of U170K may play a functional role inside cells by stabilizing binding through increased interactions between the N-terminal helix of U170K and U1 snRNA. Indeed, removing the N-terminal helix reduces U170K-SL1 binding affinity. Differences between U1A-SL2 binding affinities as measured in vitro and in live cells could not 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.

Fast relaxation imaging (FReI) of live-cell RNA-protein binding affinity and kinetics

256th National Meeting (2018)

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