FIGURE 4.

Some mutations in hTR204 impair pre-RNP assembly. (A) PAGE analysis of NAF1 IPs conducted on RRL mixtures containing ³⁵S-labeled proteins and ³²P-labeled hTR204 or its derivatives bearing mutations in conserved motifs (see text for details). Input lanes (in) did not contain the ³²P-labeled RNAs. (Bottom) Only the ³²P signal of the protein gel (see legend to Figure 2B for details). (B) NAF1 IPs conducted and analyzed as in (A) with ³²P-labeled hTR204 or its derivatives bearing disease-related mutations C408G, G450A, and Δ378-451. The double mutant C408G-G412C restores base pairing (see text for details). (C) ³²P-labeled hTR204 (arrow) recovered from a NAF1 IP (lane 3) and the IP supernatant (lane 2) was analyzed on an 8% sequencing gel. Untreated ³²P-labeled hTR204 (in) was run in parallel (lane 1). Sizes of ³²P-labeled DNA markers (lane 4) are indicated in nt on the right.