The guide sRNA sequence determines the activity level of box C/D RNPs

Andrea Graziadei, Frank Gabel, John Kirkpatrick, Teresa Carlomagno

eLife 2020;9:e50027    https://doi.org/10.7554/eLife.50027

Here, we provide the first structural evidence that, in the context of the Box C/D RNP, the affinity of the catalytic module fibrillarin for the substrate–guide helix is dependent on the RNA sequence outside the methylation site, thus providing a mechanism by which both the substrate and guide RNA sequences determine the degree of methylation. To reach this result, we develop an iterative structure-calculation protocol that exploits the power of integrative structural biology to characterize conformational ensembles.

Figure-4 paper

Fibrillarin binds the substrate–guide duplex more strongly in the substrate D’-loaded sRNP.

(a) The structural ensemble selected by the pseudo-genetic scoring algorithm (Methods) for the substrate D’-loaded sRNP, containing two [on,off]-state and one [off,off]-state conformers, with fibrillarin shown in shades of blue. The fits to the experimental SAS curves are shown on the right. All SANS curves were measured in 42%:58% D2O:H2O. (b) Structural ensemble selected by the pseudo-genetic scoring algorithm for the substrate D-loaded sRNP, containing three [on,off]-state and eight [off,off]-state conformers. In both a and b, the mean and standard deviation of the percentage of [on,off]-state structures in the three top-scoring ensembles across three independent scoring runs is shown in the title. The structural ensembles yield much better agreement with the SAS curves than do the individual [on,off]- and [off,off]-state structures (Figure 3—figure supplement 5).