FIGURE 2.

STAU-1 binds double-stranded RNA. A, schematic for the yeast three-hybrid system. Full-length STAU-1 was fused to the Gal4 activation domain. An RNA hybrid was constructed between MS2 RNA and a double-stranded RNA stem-loop (DS1–DS4). STAU-1 binding to the double-stranded RNA stem-loop causes the Gal4 activation domain to come into close proximity to the LexA DNA binding domain, resulting in LacZ reporter expression. LacZ reporter expression indirectly measures the binding affinity of a protein for RNA but has been confirmed to directly correlate with the K_d (45). B, yeast three-hybrid results with full-length STAU-1 and double-stranded RNA stem-loops consisting of the same structure but possessing different RNA sequences (DS1–DS4). STAU-1 and empty vector (which contained no insert) served as a negative control. FBF protein and the FBE RNA served as a positive control. C, electrophoretic mobility shift assays with full-length STAU-1 and DS3 double-stranded RNA (top) or STAU-1 and single-stranded RNA (ssRNA1) consisting of the same sequence as one side of DS3 (middle). Binding curves (bottom) of STAU-1 affinity for double-stranded RNA and ssRNA1 indicate that STAU-1 binds double-stranded RNA with higher affinity (K_d = 16 ± 1.2) than single-stranded RNA (K_d = 160 ± 35). D, supershift assays with DS3 double-stranded RNA and STAU-1 or ΔdsRBD4 protein. The ΔdsRBD4 protein served as a negative control because it lacks the epitope recognized by the α-STAU-1 antibody. Two to three replicates of each experiment are shown. The STAU-1 protein preparations used here exhibited an A₂₆₀/A₂₈₀ ratio of 0.50 (wild-type) and 0.52 (ΔdsRBD4), indicating that they were largely free of nucleic acids. E, analysis of STAU-1 specificity for RNA structure in vitro. The binding of full-length STAU-1 to double-stranded RNAs of variable sequence was determined following five rounds of selection using the SEQRS method (47). In these experiments, the number of reads correlates with binding affinity. Two preparations of STAU-1 (referred to as STAU-1a and -1b) enriched for structured RNAs of increasing stability; GST alone did not. RNA stability was calculated using the Vienna RNA structure algorithm (72). F, STAU-1 does not preferentially enrich for a single-stranded RNA motif. Following five rounds of selection, all possible 8-mer sequences were extracted from the library of random 20-mer sequences and compared across replicates. The 300 most abundant sequences were compared for STAU-1 and C. elegans FBF-2 (which binds single-stranded RNA in a sequence-specific manner, serving as a positive control). Enrichment was highly reproducible for FBF-2 (R² = 0.931) but not for STAU-1 (R² = 0.03). G, mutant versions of STAU-1, in which one double-stranded RNA-binding domain was deleted, were tested in electrophoretic mobility shift assays with DS3 double-stranded RNA. Boundaries of the deletions were designed based on the predicted extents of the dsRBDs as determined from protein alignments with Drosophila Staufen. For dsRBD2 and dsRBD4, the deletions corresponded to C. elegans mutants that delete each domain in the endogenous protein; these deletions remove all of the dsRBD plus small segments to either side. The dsRBD2 deletion mutant (ΔdsRBD2) could not be tested due to protein aggregation. Error bars, S.D.