Fig. 1: A −1A bulge induces dynamic RNA binding to U1 snRNP.
From: A sequential binding mechanism for 5′ splice site recognition and modulation for the human U1 snRNP
A Crystal structure of minimal U1 snRNP (PDB: 4PJO). Arrows indicate relative placement of modifications for single molecule measurements. B RNA oligos containing 5′SS. Shaded region indicates predicted base pairing interactions with the U1 snRNA (shown at the top, above a schematic of the exon (box)/intron (line) junction). C SPR sensorgrams showing the association and dissociation of surface-tethered (top) 11bp and (bottom) 11bp-1A RNAs at various U1 snRNP concentrations (0.02 to 100 nM). D Cartoon schematic of the two-color CoSMoS assay for monitoring U1/RNA interactions. E Fields of view under 633 nm (left, immobilized U1 snRNP molecules) and 532 nm (right, interacting RNA oligos) excitation (scale bar = 20 µm). Inset highlights colocalization (scale bar 1 µm). Fluorescent beads were included as fiducial makers (yellow arrow). Images are rendered by averaging three consecutive images and applying uniform brightness and contrast values. F Fluorescence in arbitrary units (au) across time in seconds (s) showing the binding of 9bp (top) and 9bp-1A (bottom) to surface tethered U1 snRNP (0.33 frames/s, Hz). Black lines indicate idealized fit. G Linear regression (solid line) of keq values (circle) on 9bp concentration, given by keq = kon([9 bp]) + koff. The shaded region is the 95% confidence interval of the linear regression. Here, the kon value is fixed from maximum likelihood estimations of unbound dwell times (see F) at kon = 3.9 ×106 M−1s−1, resulting in a koff = 4.6 ± 2.9 ×10−4 s−1. H Cumulative probability distributions of (left) unbound dwell times (0.5 nM, N = 2670; 1 nM, N = 5154; 2 nM, N = 3788; 4 nM, N = 4373) and (right) bound dwell times (0.5 nM, N = 2380; 1 nM, N = 5216; 2 nM, N = 4093; 4 nM, N = 4895) across range of 9bp-1A RNA concentrations. I Cumulative probability distribution of bound dwell times of the 9bp-1A RNA at 1 nM (grey, N = 5216) overlaid with MLE of single (blue dashed) and biexponential (red) distributions. Source data are provided as a Source data file.
