The Divergent U12-Type Spliceosome Is Required for Pre-mRNA Splicing and Is Essential for Development in Drosophila  Leo R. Otake, Petra Scamborova, Carl Hashimoto, Joan A. Steitz  Molecular Cell  Volume 9, Issue 2, Pages 439-446 (February 2002) DOI: 10.1016/S1097-2765(02)00441-0

Figure 1 U6atac and U12 Genes (A) Genomic sequences of Drosophila U6atac (DU6atac) (this work), Drosophila U6 (DU6-1,2,3) (Das et al., 1987; Jensen et al., 1998), Arabidopsis U6atac (AU6atac) (Shukla and Padgett, 1999), and human U6atac (HU6atac) (Tarn and Steitz, 1996a) loci are shown. The proximal sequence element A (PSEA) and TATA (Jensen et al., 1998) promoter elements are either bolded or underlined (Shukla and Padgett, 1999). Within the coding sequences, identical residues are indicated by asterisks; dashes indicate gaps. The site of P element insertion in line l(2)k01105 is shown with a black arrowhead after G70. (B) Drosophila U12 snRNA (DU12) coding sequences are aligned with U12 snRNA from human (HU12) (Montzka-Wassarman and Steitz, 1992), mouse (MU12), chicken (CU12) (Tarn et al., 1995), Xenopus (XU12) (Yu et al., 1996), and Arabidopsis (AU12) (Shukla and Padgett, 1999); dashes indicate gaps. Identical residues within nts 3–24 essential for interactions with the 5′ ss and U6atac (see Figure 2B) and in the region of the Sm binding site (nts 104–110) are bolded. Site of P element insertion in EP(3)3147 is shown with a black arrowhead after G121. Molecular Cell 2002 9, 439-446DOI: (10.1016/S1097-2765(02)00441-0)

Figure 2 Predicted Secondary Structures for D. melanogaster U6atac, U4atac, and U12 snRNAs (A) U6atac is shown base paired to the U4atac as recently proposed by Padgett and Shukla (2002). The U4atac snRNA sequence (accession AC007532, positions 155309–155468) exhibits 69% identity to the full-length human query sequence. The U6atac P element insertion site in l(2)k01105 is indicated by the arrowhead. Residues that base pair with the intron 5′ splice site are circled. The putative U4atac Sm binding site is overlined. (B) A hypothetical secondary structure for D. melanogaster U12 snRNA, designed to reflect conserved aspects of U12 structures, is shown. The U12 EP(3)3147 P element insertion site is indicated by an arrow. A second disruption, EP(3)0934, occurring after residue 33, has been lost from the Berkeley Drosophila stock collection. The putative Sm binding site is underlined. Sequences that interact with U6atac are contained in the two boxes adjacent to letter A; sequences that interact with the intron branch point sequence are in the box adjacent to the letter B. (C) Intermolecular base pairing of Drosophila U6atac and U12 snRNAs, as well as interactions with the 5′ splice site and branch point consensus sequences (adapted from Tarn and Steitz, 1996a), form the catalytic core of the U12-type spliceosome. Molecular Cell 2002 9, 439-446DOI: (10.1016/S1097-2765(02)00441-0)

Figure 3 Molecular Evidence for Rescue by the U6atac and U12 Transgenes (A) Sequencing of U6atac RT-PCR amplicons from rescued adults using primers complementary to nts 1–27 and 70–90. The point deletion of thymidylate at position 67 in the U6atac transgene (adjacent to circles in panels 1–3) is indicated by the arrowhead in panel 4 showing the rescued adult. In panel 3, the transgenic sequence is obscured because of its lower abundance compared to endogenous U6atac. Bases near T67 (bold/italic) are identified. (B) PCR performed on total DNA from surviving adults using a primer complementary to nts −16 to −29 upstream of the U12 coding region and either nts 681–711 downstream of the coding region (wt), or within the pCasper-3 vector (to confirm the presence of P{U12} transgene), or within the P element in EP(3)3147 (to confirm the presence of the P element insertion). Individuals heterozygous for disruption of the U12 gene and containing a copy of the U12 transgene are shown in lanes 3–5. U12 homozygous-disrupted individuals rescued by the U12 transgene are shown in lanes 6–8; 30 such rescued flies and their progeny all showed the same PCR profile. To ascertain that DNA was present in lane 6 for the rescued fly, the reaction containing the primers for amplifying wt U12 DNA was allowed to proceed for three cycles; 10% of the PCR reaction was then transferred to a tube containing primers for amplifying the EP(3)3147 amplicon and allowed to proceed for additional 35 cycles. A single PCR product corresponding to the EP(3)3147 amplicon was seen (data not shown). See Supplemental Data online at http://www.molecule.org/cgi/content/full/9/2/439/DC1. Molecular Cell 2002 9, 439-446DOI: (10.1016/S1097-2765(02)00441-0)

Figure 4 Expression of U6atac and Excision of U12-Dependent Introns in U6atac P Element-Disrupted Larvae (A) Northern blot of total RNA from individual early third instar larvae (∼72 hr p.e.l.) (lanes 2–4) or from three larvae isolated at 24–48 hr p.e.l. (lanes 5–7) and 48–52 hr p.e.l. (lanes 8–10) probed for U6atac. Genotypes: +/+, w1118; +/−, l(2)k01105/CyO-GFP; −/−, l(2)k01105/l(2)k01105. The mutant U6atac (U6atac mut) and wild-type U6atac (U6atac wt) snRNAs are indicated. The presence of chimeric P element sequences in the U6atac mut snRNA was confirmed by reprobing the Northern blot with the oligonucleotide PLAC2, complementary to the disrupting P{lacW} element at positions 74–108 (Flybase accession FBtp0000204). (B) Schematic of the alternatively spliced second intron of the D. melanogaster pros transcript. The U2-type intron (black line) is 730 nts long. The U12-type intron contains 59 and 28 nucleotides (diagonal hatched boxes) from the pros-L open reading frame, flanking the U2-type intron. The sequences encoding 5 amino acids of the homeodomain (HD) that are altered by splicing are shown as horizontal-lined and checkered boxes. Positions of primers for RT-PCR analysis are indicated. (C) Regions containing the U12-dependent introns of transcripts CT23545, CT36969, and pros pre-mRNAs were RT-PCR amplified from total RNA of individual third instar larvae (72 hr p.e.l. as in [A]). Amplicons after 25 cycles of PCR corresponding to pre-mRNA (intron still present) are denoted by triangles; mRNA amplicons (intron removed) are indicated by circles or labeled pros-L (U2-type intron removed) or pros-S (U12-type intron removed); a nonspecific amplicon is denoted by x. Molecular Cell 2002 9, 439-446DOI: (10.1016/S1097-2765(02)00441-0)