(A) m⁷G-capped P120 substrate RNA was transcribed with T7 RNA polymerase in the presence of [α-³²P]UTP and gel-purified (Tarn and Steitz 1996b; Frilander and Steitz 1999). Stage VI Xenopus laevis oocytes were injected with 9.2 nL containing 8.3 × 10³ cpm (∼2.5 fmol) P120 RNA and 20 mg/mL blue dextran, which serves as a marker for successful nuclear injection, either through the animal pole (GV injection) or the vegetal pole (cytoplasmic injection). Alternatively, mature oocytes were obtained by treatment with progesterone until germinal vesicle breakdown was visualized as a white spot at the animal pole (Smith 1989). All injected oocytes were incubated for 3 h before manually separating the GV from the cytoplasm. RNA was isolated from these fractions or from mature oocytes with TRIZOL reagent (Invitrogen). Eight oocytes were selected and combined for each sample. RNA (cytoplasmic, GV, or total mature) was separated on a denaturing 8% polyacrylamide gel. Analysis with ImageQuant software (Molecular Dynamics) of short (overnight) and long (four days, shown) exposures of the dried gel indicated that the efficiency of splicing was ∼1% and that ∼0.2% splicing could have been detected above background in the cytoplasm. (B) Oocytes were injected in the cytoplasm with water (control) or 92 ng oligodeoxyribonucleotide 5′-GTTGTTATTTTCCTTACTC-3′complementary to U12 snRNA (α-U12 oligo) 15 h prior to injection of the P120 RNA substrate into the nucleus. After 3 h incubation, GV RNA was prepared as in A from 10 oocytes for each sample. RNA was resolved on a denaturing 7% polyacrylamide gel. The positions of the P120 substrate, the spliced product (inhibited by ∼80% by ImageQuant analysis), and the intron-lariat are indicated on the right. (C) The same GV RNA samples were used to verify the knockdown of U12 snRNA by RT-PCR (upper panel) as described previously (Friend et al. 2007), whereas levels of U2 snRNA, U1 snRNA, and U6 snRNA were assessed by Northern blotting (lower panels; probe sequences contained in supplemental information). Lane 1 is the control; lane 2 shows U12 knockdown. (D) Sequencing of the P120 spliced product to verify the accuracy of minor-class splicing in the oocyte system. Both the splicing substrate and spliced product were amplified by nested RT-PCR from GV RNA and sequenced (primer sequences contained in Supplemental Materials). To amplify the spliced product, the pre-mRNA was first digested using RNase H and the DNA oligonucleotide 5′-CTCCTAACTCTTCACTCTGC-3′ complementary to the P120 intron. The arrows mark the 5′ and 3′ splice sites of the substrate and the splice junction of the product. (E) Major-class splicing is inhibited upon GV breakdown. Adenovirus standard splicing substrate (Yu et al. 1998) was microinjected into GVs of immature or the animal pole of mature oocytes (9.0 × 10³ cpm, ∼2.5 fmol) and incubated for 30 min. Total RNA was isolated from nine oocytes per sample and separated on a denaturing 10% polyacrylamide gel. The unspliced precursor and intron-lariat splicing product are schematized. (F) U12 and U6atac snRNAs are highly enriched in the nucleus of Xenopus oocytes. After manual separation of GVs and cytoplasm from five oocytes, RNA was prepared from these samples as well as from five undissected oocytes (total RNA). U12 and U6atac snRNAs were detected by RT-PCR, while U2 snRNA, U6 snRNA, and 5.8S rRNA were visualized by Northern blotting (probe sequences contained in Supplemental Materials).