(A) Secondary structure of human U4 and U6 snRNAs based on the model of Wersig and Bindereif (8). (B) Secondary structure model of human U4atac and U6atac snRNAs. Shown is the structure proposed by Padgett and Shukla (5). The regions of U4atac snRNA that are examined in this study are bracketed.

(A) Diagram of the in vivo genetic suppression assay. The sequential base pairing interactions between U11 snRNA and the pre-mRNA 5′ splice site and between U6atac snRNA and the 5′ splice site are shown. The boxed residues are those mutated in the suppression assay as shown at the top of the figure. Both the U6atac GG14/15CC and the U11 GG6/7CC mutations are required to fully suppress the effect of the 5′ splice site CC5/6GG mutation. Also shown are the nine mutations introduced by the AthSL mutant into the stem II region of U6atac snRNA and the compensatory mutations introduced into the U4atac snRNA (outlined residues). This suppressor U4atac snRNA serves as the starting construct for the mutants described in this study. (B) Products of RT–PCR amplification of RNA extracted from CHO cells transfected with the listed constructs. The RT–PCR primers are in P120 exons 6 and 7 and amplify the region of intron F in the P120 minigene. The positions of bands corresponding to unspliced RNA, RNA spliced at the normal U12-dependent splice sites (spliced) and RNA spliced at the cryptic U2-dependent splice sites (cryptic) are indicated. Lane 1 is from mock transfected cells, lane 2 is from cells transfected with the empty expression vector, lane 3 is from cells transfected with the control wild type P120 minigene construct. Lanes 4–6 are from cells transfected with the P120 CC5/6GG 5′ splice site mutant (lane 4), both the U11 and the U6atac suppressor mutants (lane 5), and the combination of the U11, U6atac and U4atac suppressor mutants (lane 6). Lane M is a 50 bp DNA size ladder.

(Opposite and above) Splicing phenotypes of U4atac snRNA mutants. (A) Locations of the mutations in the loop region of the 5′ stem–loop element. The first two are three residue substitution mutants and the last mutant is a five residue deletion. (B) RT–PCR products from cells transfected with the indicated U4atac snRNA mutants. Lane 1 in this and subsequent gels is a positive suppression control using the constructs shown in lane 6 of Figure 2B. The experimental lanes in this and subsequent gels are from cells transfected with the P120 CC5/6GG mutant, the GG6/7CC mutant of U11 snRNA, the GG14/15CC + AthSL stem II mutant of U6atac snRNA and the indicated U4atac snRNA mutants. Lanes labeled M are 50 bp DNA size markers. (C) Locations of the mutations in the stem I pairing region of U4atac snRNA. The G60C, C61G mutant would be predicted to disrupt the stem I pairing with U6atac snRNA in the model that we recently proposed (5). The C67G, C68G mutant would be predicted to disrupt the stem I pairing with U6atac snRNA in the model originally proposed by Tarn and Steitz (19). (D) RT–PCR products from cells transfected with the indicated U4atac snRNA mutants. (E) Locations of mutations in the single stranded region of U4atac snRNA. This region is shown base paired to U6atac snRNA according to the model proposed by Jakab et al. (22). (F) RT–PCR products from cells transfected with the indicated U4atac snRNA mutants. (G) Locations of mutations in the 3′ stem–loop element and Sm binding site of U4atac snRNA. Two deletions of the 3′ stem–loop are shown. The first removes the distal portion of the element by deleting residues 92–105 and joining residue 91 to 106 to create a 5 nt terminal loop. The second removes the entire element by deleting residues 84–114 and joining residue 83 to 115. Also shown is the 3 nt mutation of the Sm protein binding site (Sm^–). (H) RT–PCR products from cells transfected with the indicated U4atac snRNA mutants.