Figure 6.
Both CAB and ACA boxes are required for the association of human U89 C/D-H/ACA scaRNA with WDR79 protein. (A) Secondary structure schematic of tagged human U89 (tU89) scaRNA. Putative CAB boxes in loops 1 (L1) and 2 (L2) as well as H, ACA, C, C′, D and D′ motifs, are indicated. Loop and box ACA substitutions in the mutant tU89 constructs are shown. A 6 nt-long tag generated by changing nucleotides 237–242 into complementary residues is represented by a bar. (B) Northern blot analysis of tU89 scaRNA and its mutants co-immunoprecipitated with Myc-hWDR79 protein. Either wild-type (WT), CAB-mutated (L1+2mt), box ACA-mutated (ACAmt), or combined CAB- and ACA-mutated (L1+2&ACAmt) tU89 scaRNA was co-expressed with Myc-hWDR79 in HEK293 cells. Anti-Myc precipitated RNAs were resolved by 7% denaturing PAGE and analyzed by Northern blot hybridization using oligonucleotide probes (see Supplemental Data). The IP efficiency of either wild-type or mutated tU89 scaRNA represents an average of two experiments with standard error shown (for details see Experimental Procedures).