1. A Catalytically Active Group II Intron Domain 5 Can Function in the U12-Dependent Spliceosome 
Girish C Shukla, Richard A Padgett 
Molecular Cell 
Volume 9, Issue 5, Pages (May 2002)
DOI: /S (02)

2. Figure 1
Comparison of RNA Features in the Spliceosome and Group II Self-Splicing Introns
(A) RNA-RNA interactions in the U12-dependent spliceosome. The specific mutations in the pre-mRNA and the U11 and U6atac snRNAs that are used in the in vivo mutational suppression assay are shown. The pre-mRNA contains a CC-GG mutation in the 5′ splice site sequence that inactivates use of this splice site. This mutation is suppressed by the cotransfection of a U11 snRNA GG6/7CC mutant and a U6atac snRNA GG14/15CC mutant as shown.
(B) Secondary structure of a group II intron showing the six domains. The sequence of the Pl intron domain 5 is shown.
(C) Consensus sequences of the group II intron D5 stem-loop and the U2/U6 and U12/U6atac snRNA stem-loop structures. Each of these structures has been represented in alternative ways by various authors, although none has been solved experimentally. The structures shown here have been selected to emphasize the similarities in the three structures. Circled nucleotides are residues where phosphorothioate substitution at the 5′ side reduces or abolishes function. Outlined residues are contributed by U2 or U12 snRNAs in the spliceosomes. The consensus D5 structure is from Costa et al. (1998). Uppercase letters reflect greater than 90% conservation; lowercase letters, 80%–90% conservation. R (or r) = purine; Y (or y) = pyrimidine; M = A or C; K = G or U. The consensus structure of the major spliceosomal U2/U6 snRNA stem-loop is from Yu et al. (1995). The consensus structure of the minor spliceosomal U12/U6atac snRNA combines the sequences of the human (Tarn and Steitz, 1996a), Arabidopsis (Shukla and Padgett, 1999), and Drosophila (accession # AE003621) snRNAs. Upper case letters are conserved in all three sequences; lower case letters are conserved in two of the three sequences.
Molecular Cell 2002 9, DOI: ( /S (02) )

3. Figure 2 Construction of Chimeric U6atac/D5 snRNAs
(A) Sequence of human U12/U6atac snRNAs.
(B) Sequence of D5 of the Pylaiella littoralis LSU/2 intron (Costa et al., 1997).
(C) Sequence of the chimeric U6atac/D5-Pl snRNA produced by replacing the boxed nucleotides in (A) with the boxed nucleotides in (B). Boxed base pair indicates the nucleotides that were deleted to shorten the upper helix.
(D) Sequence of D5 of the Saccharomyces cerevisiae aI5γ intron.
(E) Sequence of the chimeric U6atac/D5-Sc snRNA produced by replacing the boxed nucleotides in (A) with the boxed nucleotides in (D). Boxed base pair in the upper helix indicates the nucleotides that were deleted to shorten the helix. The boxed base pair in the lower helix and the boxed C residue in the bulge indicate the differences between the two D5 sequences and the changes that were introduced in the intermediate constructs.
Molecular Cell 2002 9, DOI: ( /S (02) )

4. Figure 3
Analysis of In Vivo Splicing Activity of the U6atac/D5 Chimeras
The RT-PCR products from cells transfected with the indicated plasmid DNAs were separated on an agarose gel. The three bands correspond to RNA in which the P120 intron F is retained (Unspliced); RNA in which the P120 intron F has been spliced out at the U12-dependent normal splice sites (Spliced); or RNA in which the P120 intron F has been spliced out using the cryptic U2-dependent splice sites located 13 and 6 nucleotides into the intron for the 5′ and 3′ splice sites, respectively (Cryptic). Lanes 1 and 2, control transfections without DNA or with empty vector plasmid, respectively. Lane 3, transfection with wild-type P120 minigene vector alone. Lanes 4–17, transfection with the P120 5′ splice site mutant CC5/6GG and suppressor U11, U4atac, and U6atac snRNA constructs indicated. The U11 GG6/7CC suppressor snRNA plasmid was included in all lanes except 4 and 6. The U6atac snRNA constructs shown above the lanes all contained the GG14/15CC suppressor mutation and either the wild-type stem-loop (lanes 6 and 7) or D5 chimeric stem-loops as indicated (lanes 8–17). Specific U4atac snRNA suppressor constructs designed for each of the U6atac snRNA chimeric stem-loop constructs were also included where indicated.
Molecular Cell 2002 9, DOI: ( /S (02) )

5. Figure 4 In Vitro Self-Splicing Activity of Group II Intron D5 Mutants
(A) Location of the D5 mutations tested in the Pl.LSU/2 intron construct. The wild-type sequence is shown. All of the mutants contained the A5-U30 substitution. This was combined with either the Δ12,21 deletion in the upper stem, the U32→CC mutation in the lower stem, or all three mutations in a single construct.
(B) Denaturing polyacrylamide gel of representative time points in the reactions of the indicated RNAs. The precursor RNA and the various products of in vitro self-splicing are indicated.
(C) Time course of cis self-splicing of the wild-type and D5 mutant introns. Initial rates were used for the derivation of apparent rate constants. The wild-type and A5-U30 mutant RNAs reacted to over 95%, while endpoints for the reaction of the A5-U30/U32→CC and A5-U30/Δ12,21 mutant RNAs could not be determined. P, product RNA; S, substrate RNA.
Molecular Cell 2002 9, DOI: ( /S (02) )