Determinants of the SMN Tudor Domain Interaction with the RG-tail of SmD3

The Survival of Motor Neurons protein (SMN) forms the core of a large protein complex termed the ‘SMN complex’. The primary characterized function of the SMN complex is to facilitate the specific assembly of seven Sm proteins onto uridine-rich small nuclear RNAs (UsnRNAs) which function in pre-mRNA splicing. Of the three known functional domains of SMN, the Tudor domain forms the centrally-located, structured region and functions in the recognition of a subset of Sm proteins (SmD1, D3, and B) via symmetric dimethyl modification of key arginine residues (sDMA) in the RG-tails. Here, the details of this interaction are explored to understand the nature of sDMA specificity and the role of substrate regions outside of the sDMA modification. Using fluorescence polarization direct binding and competition assays and by selective modification of the SmD3 RG-tail, the factors that contribute to binding are elucidated. We find that the SMN Tudor domain requires methyl modification for interaction and prefers sDMA, but binds with only slightly reduced affinity to MMA and aDMA-modified substrate. Furthermore, μM efficient M binding affinity is achieved only in the context of the full SmD3 tail, where peptide backbone interactions and electrostatics make important contributions to binding. This feature permits the SMN Tudor domain to recognize not only SmD3, but other methylated RG-containing substrates in a similar manner. SMN is also of clinical significance; loss of function via deletion or mutation has been linked to Spinal Muscular Atrophy (SMA). SMA is an autosomal recessive neurodegenerative disease affecting neurons and leading to progressive muscle wasting. To date, 25 mutations of SMN have been identified in patients. Nearly a third (8) of these are found within the Tudor domain and roughly half (12) are located in the C-terminal oligomerization domain. This study also aims to understand the biochemical consequences of these 20 mutations in SMN. Utilizing SmD3 binding assays in conjunction with biophysical experiments to study oligomerization, several SMA patient mutations were determined to be binding deficient, oligomerization deficient, or hyper-oligomerized relative to wild-type SMN. These results provide important new insights into the molecular basis for SMN function and the biochemical basis for SMA