1. Suppression of Multiple Substrate Mutations by Spliceosomal prp8 Alleles Suggests Functional Correlations with Ribosomal Ambiguity Mutants 
Charles C Query, Maria M Konarska 
Molecular Cell 
Volume 14, Issue 3, Pages (May 2004)
DOI: /S (04)

2. Figure 1 Isolation of Suppressors of Branch Site A-to-G Mutation
(A) Schematic of splicing chemistry for BS-G mutant ACT1-CUP1 reporter in vivo. In WT cells, BS-G lariat intermediates stall prior to the second step of splicing and accumulate (lane 2 of inset); in the isolated suppressor strains, BS-G lariat intermediates proceed through the second step, forming excised lariat and mRNA. Inset: Primer extension analysis of RNAs recovered from WT cells harboring either a WT (BS-A; lane 1) or BS-G (lane 2) reporter.
(B) Strategy for genetic screen for suppressors of BS-G mutation. The 46ΔCUP strain, which is deleted for its chromosomal copy of CUP1, was transformed with the BS-G reporter and UV irradiated. Suppressor strains were selected for growth in the presence of 0.1 mM CuSO4.
(C) Copper growth phenotype of the BS-G mutant in the newly isolated BS suppressors. All suppressor strains containing the BS-G reporter grow on 0.1 mM or higher copper, whereas WT cells do not. Comparable numbers of cells were spotted onto plates containing CuSO4, as indicated, and photographed after 3 days at 30°C.
(D) The isolated suppressors improve splicing of the BS-G mutant in vivo. Primer extension analysis of RNAs recovered from the isolated suppressors harboring the BS-G reporter (lanes 3–12) or from WT cells containing either WT (lane 1) or the BS-G (lane 2) reporter. Primer complementary to the 3′ exon was used to reveal levels of pre-mRNA, mRNA, and lariat intermediate (indicated by icons).
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2
The BS-G Suppressors Also Suppress BS-C, BS-U, and 5′ and 3′SS Mutations
Primer extension of reporter RNAs recovered from the isolated BS suppressors and analyzed as in Figure 1D. Suppressors 1, 2, and 10 (lanes 1–24) as well as 6–11 and M2 (not shown) increase mRNA levels of 5′SS U2A, BS-G, BS-C, BS-U, or 3′SS UuG/ mutant RNAs. For comparison, suppression of 3′SS UuG/ mutation by splice site suppressor alleles of prp8 (8-156 and 8-151) and the cs prp8 allele (8-160) are shown (lanes 25–28).
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3
Previously Isolated Splice Site Suppressor prp8 Alleles Suppress BS as well as 5′ and 3′SS Mutations
(A) Schematic depiction of ACT1-CUP1 reporter pre-mRNA, indicating 5′SS, 3′SS, or BS mutations used in this study. The “/” indicates the position used as the 3′SS when the WT 3′SS is mutated from UAGG to UAcc. Parentheses indicate mutations discussed in the text but not shown in this figure.
(B) Primer extension of RNAs recovered from cells (yJU75) harboring reporters containing various mutations as indicated and either WT PRP8 (odd lanes) or prp8-156 (even lanes). Similar results were obtained with other prp8 alleles (8-122, 8-123, 8-C3, 8-151, 8-152, 8-153, 8-155, 8-157) described in Collins and Guthrie (1999) and Siatecka et al. (1999).
(C) Copper growth of strains harboring either WT-PRP8 or prp8-156 and the same reporters as in (B). Shaded ovals indicate significant suppression.
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4
Effect of prp8-156 and Various prp16 Alleles on Splicing of SS/BS Intron Mutants
(A) Primer extension analysis of RNAs from cells (yCQ06) containing prp8-156 with WT PRP16, or WT PRP8 with WT PRP16, prp16-1, prp16-101, or prp16-302, as indicated. Note that prp8-156 increased mRNA levels for all four mutant reporters shown, whereas prp16 alleles increased lariat intermediate levels.
(B) Quantitation of results presented in (A). For each reaction, the first step efficiency (light bars) was calculated as (M+LI)/(P+M+LI), normalized to the first step efficiency of the WT reporter in the WT strain, set at 100. The second step efficiency (dark bars) was calculated as M/(M+LI), normalized to the WT strain second step efficiency, set at 100. P, pre-mRNA; M, mRNA; LI, lariat intermediate.
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5
prp8 and prp16 Alleles Interact with Each Other Both Functionally and Genetically, Suppressing SS/BS Intron Mutations by Facilitating Different Steps
(A) Suppression effects by prp8-156 and prp16-101, and by prp8-156 and prp16-R686I are additive. Primer extension analysis of RNAs from cells (yCQ06) containing combinations of WT PRP8 or prp8-156, and WT PRP16, prp16-101, or prp16-R686I, as indicated.
(B) Quantitation of results in (A) calculated as in Figure 4B.
(C and D) Copper growth of strains harboring PRP8 and PRP16 alleles and ACT1-CUP1 reporters as indicated (C), or the same PRP8 and PRP16 alleles and the BS-C ACT1-CUP1 reporter as in (A), lanes 17–22 (D). The numbers on the right indicate the highest copper concentration allowing growth. Shaded ovals indicate strong additive effect.
(E) Genetic interaction between PRP8 and PRP16. prp8-156 suppresses the ts growth defect at 37°C of prp16-R686I in the presence of BS-C reporter. A similar, but less pronounced, effect was observed in the absence of reporter.
Molecular Cell  , DOI: ( /S (04) )

7. Figure 6
prp8 and U6-U57C Alleles Interact with Each Other Both Functionally and Genetically, Suppressing SS/BS Intron Mutations by Facilitating Different Steps
(A) Comparison of effects of prp8-156 and U6-U57C alleles on splicing of SS/BS mutants. Primer extension of RNAs isolated from cells (yCQ05) containing WT PRP8 and WT U6, prp8-156 and WT U6, WT PRP8 and U6-U57C, or prp8-156 and U6-U57C, and the mutated reporters as indicated.
(B) Quantitation of results in (A) calculated as in Figure 4B.
(C) Copper growth of strains harboring the PRP8 or and WT U6 or U6-U57C alleles and the BS-C reporter. The numbers on the right indicate the highest copper concentration allowing growth. The shaded oval indicates additive suppression by prp8-156 and U6-U57C.
(D) Genetic interaction between PRP8 and U6-U57. prp8-156 suppresses the ts growth defect at 37°C of U6-U57C and U6-U57G in the presence of BS-C reporter. Similar, but less pronounced, effects were observed in the absence of reporter.
Molecular Cell  , DOI: ( /S (04) )

8. Figure 7
Model of Structural Rearrangements within the Spliceosome at the Time of Transition between the First and Second Step of Splicing
The first and second catalytic steps require different conformations of the spliceosome, the equilibrium between these conformations being modulated by interactions of Prp8, U6 snRNA, and Prp16 (and possibly other factors). By analogy to the ribosomal decoding process, the initial signal for this conformational change is presented by the branch structure formed in the first catalytic step. This induced-fit conformational change is facilitated by SS/BS prp8 and U6-U57A alleles but inhibited by U6-U57C allele. Thus, U6-U57C allele disfavors exit from the first step conformation, improving first step catalysis of mutant substrates and inhibiting the second step; conversely, SS/BS prp8 and U6-U57A alleles promote progression into the second step conformation, improving splicing of mutant intermediates. Prp16 facilitates this transition, and, upon hydrolysis of ATP by Prp16, the substrates are repositioned for the second catalytic step. At this stage, substrates that are improperly positioned are discarded by the WT spliceosome. Thus, slowed action by prp16 alleles improves the first step by inhibiting exit from the first step conformation and by reducing discard of mutant intermediates.
Molecular Cell  , DOI: ( /S (04) )