Supplementary Figure 9: Cumulative increase of results depending on RNA combinations considered.

Dependencies are shown for each cross-linking experiment – (a) human RNPs, (b) yeast RNPs isolated with TAP purification of Cbp20, (c) yeast RNPs labeled with 4SU and isolated with oligo d(T). The order of cross-linked (oligo)nucleotides from left to right is first depending on increasing length and then on decreasing number of observed cross-links. (a) Around 31% of cross-links (58 of the total 189) were observed to a single U, which corresponds to 82% (49 of the total 60) of the cross-linking sites/regions identified. At least one modification of U, i.e. loss of water, and two dinucleotides (AU and UU) have to be considered to identify 59 of the 60 identified cross-linking sites/regions. (b) [U –H2O] is the most common RNA moiety, found in 25 cross-links (14%) and 21 unique cross-linking sites/regions (33% of the total results). In contrast to human, a much larger number of RNA combinations and modifications has to be taken into account to identify the cross-linking sites/regions reported in this study. (c) All cross-links to 4SU were observed with a net loss of H2S from the RNA. By taking into account a single 4SU as well as all dinucleotides of the cross-linked 4SU and the four native nucleotides, 269 of the 376 cross-links (72%) and 125 of the 133 unique cross-linking sites/regions (94%) could be identified. Overall, considering only a single nucleotide would only permit identification of 14-33% of the reported cross-links and 33-82% of the reported cross-linking sites/regions in the respective experiments, illustrating the benefit of the precursor variant approach over conventional database search treating one (or a few) cross-linked RNA moieties as a post-translational modification.