Fig. 8
From: A cryptic RNA-binding domain mediates Syncrip recognition and exosomal partitioning of miRNA targets
Eliminating the RNA-binding activity of the NURR domain impairs Syncrip interaction with miR-3470 in the cell. a Surface representation of human Syncrip N-terminal domain, residues affected by RNA binding are coloured blue and the two mutated residues coloured orange. b Model of Syncrip NeR1 R60A G97L mutant. The side chains of residues A60 and L97 are coloured red. The central image shows a cartoon representation of the protein, the two side panels show the side chains of residues close to the mutations in space filling representation. The model was built using SwissMode, using the structure of the Drosophila NeR1 two-domain protein as template, as the G97 residue is close to the N-eR1 interface. c The R60A G97L double mutation does not change the structure of the protein but impairs RNA binding. (Left) Superimposition of the 15N-correlation spectra of the wild-type (blue) and mutant (orange) NeR1 protein, showing that only minor changes are observed upon mutation and that the structure of the protein is conserved. d The R60A G97L double mutation eliminates RNA binding by the N-terminal domain. The changes in a representative peak of the wild-type (top) and mutant (bottom) proteins upon addition of a 1:1 and 1:2 ratio of hEXO RNA targets are displayed in this spectral superimposition. Peaks in the RNA-binding surface change upon titration of the RNA into the wild-type, but not the mutant protein. e RIP assays with anti-SYNCRIP antibody and, as control, preimmune IgG, on lysates from hepatocytes knocked-down for endogenous Syncrip by short-hairpin RNA (shRNAs) approach (shSyncrip). A scrambled shRNA was used as control (shCTR) as previously reported10. The cells were transduced with either an empty vector (Vector) or vectors overexpressing human WT Syncrip (full length), human ΔNURR (dNURR) and human mutant Syncrip (R60A-G97L). miRNA 3470a (left) and miRNA 92a-1-5p (right) (hEXO miRNA and non-hEXO miRNA, respectively) levels in immune-precipitated samples were determined by RT-qPCR and reported as IP/IgG. Data are means ± SEM of three independent experiments. Statistical significance was determined by parametric paired t-test analysis (*p < 0.05)
