FIGURE 3.

Transcripts containing multiple introns are present in DHX16-G724N mutant expressing cells. RNA isolated from DHX16-transfected (wt), DHX16-G724N-transfected (GN), or pEGFP-C1-transfected (GFP) cells were analyzed for the presence of transcripts containing multiple introns. Total RNA was used for A and B. Total RNA was incubated with oligo(dT)-cellulose, and the bound and unbound fractions were collected. The oligo(dT)-bound fraction was enriched with poly(A) RNA and was used for D. A, RNA was assayed by RT-PCR using exonic primers flanking two introns and an exon. The two adjacent introns that were assayed in FOS and HSPH1 are marked with black bars in Fig. 1B. PCR products corresponding to RNA containing two introns, one intron, or no intron are depicted on the right. The arrowheads denote primer positions. B, RNA was analyzed by Northern hybridization using oligonucleotides complementary to intron 1, intron 2, or exon 2 of RPL23 (see Fig. 1B). Total RNA (15 μg) was electrophoresed on a 1% agarose formaldehyde gel, transferred, and hybridized with ³²P-labeled oligonucleotides. RNA size markers in kilobases are marked on the left. U, fully unspliced RNA; M, fully spliced RNA; P2, a partially spliced RNA containing intron 2. C, RNA that bound to oligo(dT) (Bound) or remained in the supernatant (Unbound) was assayed by RT-PCR for DNAJB1, GAPDH, and H2AM RNA. D, RNA that bound to the oligo(dT) that was enriched for poly(A) RNA (∼0.8 μg) was probed with oligonucleotides complementary to intron 1, intron 2, or exon 2 of DNAJB1. A diagram of the gene structure is depicted below the Northern blot; the lengths of the introns and exons are numbered in bases. U, fully unspliced RNA; M, fully spliced RNA.