Figure 3: U4atac snRNA secondary structure elements, positional conservation, MOPD1 and Roifman Syndrome causal variants.

Elements of limited or null importance for splicing (dispensable region of 3′ stem–loop) have mutagenesis experiments^20,21 showing no splicing alteration, are enriched in low conservation and do not present any known disease-causing variant. Elements of variable importance for splicing (3′ stem–loop, except dispensable region; single-strand region; 5′ stem–loop, except critical region) have mutagenesis experiments showing modest or no splicing alteration, or have not been probed by mutagenesis, but meet at least one of these criteria: (a) they present at least one MOPD1 causal variant (typically with reduced severity); (b) they are proximal to a MOPD1 causal variant cluster; (c) structural studies^60,61 suggest they may have a functional role; these elements have mixed conservation, and only a few variants at more conserved positions may cause splicing alterations. Elements of major importance for splicing (stem II, critical region of the 5′ stem–loop, stem I, Sm protein-binding site) have mutagenesis experiments producing splicing alterations and/or overlap the MOPD1 variant cluster; in addition, they are expected to have a major functional role based on structural studies; finally, they are enriched in highly conserved positions, the majority of which are expected to cause splicing alterations in presence of variation. Positions are labelled as ‘high conservation’ if placental mammal or 100-vertebrate PhyloP ≥1.75, as ‘diverged’ if placental mammal and 100-vertebrate PhyloP are negative, and ‘moderate or low conservation’ otherwise. Parts of U6atac are displayed only in correspondence of U4atac–U6atac duplex structures. U4atac snRNA coordinate 1 corresponds to hg19 coordinate 122,288,456. Classification of MOPD1 causal variants as ‘full severity’ or ‘reduced severity’ is based on a thorough review of MOPD1 literature^{10,11,15,16,17,18} (for more details, see Supplementary Table 17) and biochemical assays of variant effect²²; the latter is particularly important for less severe MOPD1 forms presenting compound heterozygosity.