Figure 1.
Cus2 facilitates refolding of mutually exclusive structures of the U2 snRNA.
(A) Model of Cus2's activity toward the U2 snRNA. Cus2 recognizes and binds the U2-IIc snRNA conformation and facilitates its rearrangement into the U2-IIa conformation. Formation of the U2-IIa conformation activates the snRNP (depicted as a ball) and allows for its stable association with the pre-mRNA substrate to form the pre-spliceosome with the U1 snRNP. The exons of the pre-mRNA are depicted as filled boxes and the intron is depicted as a line. (B) Diagram of the 5′ portion of yeast U2 snRNA mutually exclusive U2-IIc and U2-IIa conformations. The U2 snRNA mutants used in this study promote either U2-IIc or U2-IIa as indicated. The G53 position is shaded red. The sequences that base pair to form the U2-IIc stem are shaded in blue.
Figure 2.
Neither Cus2 nor the U2 snRNA exhibits genetic interactions with p-TEFb-associated cyclin homologs.
(A) Analysis of bur2Δ cus2Δ double mutant. The indicated strains were grown in YPD and four ten-fold serial dilutions were spotted onto YPD plates and grown at 30°C for 3 days. (B) Analysis of ctk2Δ cus2Δ double mutant. Cells were treated as described in (A). (C) Analysis of the U2 snRNA conformational mutants. Strains harboring the wild-type U2 snRNA (URA3 plasmids, pRS316) and the indicated mutant snRNA (LEU2 plasmids, pRS315) were grown in selective SC-ura-leu media and four ten-fold serial dilutions were spotted onto 5-FOA-Leu to select for loss of the wild-type U2 snRNA URA3-marked plasmid. Plates were grown at 30°C for the indicated number of days.
Figure 3.
Phenotypic analysis of bur2Δ double mutant strains.
(A) CUS2 deletion does not confer sensitivity to 6-AU or inositol auxotrophy. Strains used in these studies were transformed with pRS316 (URA3) to allow for growth on media containing 6-azauracil (6-AU). Serial dilutions of the indicated strains were spotted onto solid media medium lacking uracil (Control), in the presence of 6-AU (100 µg/ml), or on media lacking inositol (-INO) and incubated at 30°C for 3–6 days. (B) CUS2 overexpression does not confer 6-AU sensitivity, inositol auxotrophy, or suppress these phenotypes of bur2Δ. The indicated strains carrying URA3-marked plasmids (vector or CUS2 on a 2μ plasmid) were grown in SC-uracil and four ten-fold serial dilutions were spotted onto solid media medium lacking uracil (Control), in the presence of 6-AU (100 µg/ml), or on media lacking inositol (-INO) and incubated at 30°C for 3–6 days. (C) U2 snRNA mutants do not exhibit sensitivity to 6-AU and are not auxotrophic for inositol. Mutant snRNAs were introduced into the indicated strains by plasmid shuffling. Strains were then re-transformed with pRS316 (URA3) and spotted onto the indicated media. Plates were grown at 30°C for the indicated number of 3–6 days.
Table 1.
Summary of phenotypic analysis of cus2Δ and U2 snRNA mutants compared to select transcription elongation factors.
Figure 4.
Neither Cus2 nor the U2 snRNA exhibits an Spt− phenotype.
The strains used in this study all contain the his4-912δ mutation. bur2-1 has been previously characterized as Spt− (A) The indicated strains were grown in YPD and four ten-fold serial dilutions spotted onto media containing (Control) or lacking histidine (-His) and grown at 30°C for 5 days. (B) The indicated strains carrying URA3-marked plasmids (vector or CUS2 on a 2μ plasmid) were grown in SC-uracil and four ten-fold serial dilutions were spotted onto SC-uracil (Control) or onto media lacking uracil and histidine (-His) and incubated at 30°C for 5 days. (C) Mutant snRNAs were introduced into the indicated strains by plasmid shuffling. Strains were grown overnight in SC-leu medium and plated onto SC-leu (Control) or SC-leu-his (-His) media. Plates were grown at 30°C for 5 days.
Figure 5.
Neither Cus2 nor the U2 snRNA co-immunoprecipitate the Bur complex.
Anti-His and anti-TAP immunoprecipitations were performed on whole cell lysates made from formaldehyde cross-linked Bur2-TAP tagged strains carrying a vector or a 6×His tagged GAL1-CUS2 plasmid grown in galactose media. The presence or absence of the epitope tag is denoted by a “+” or “−,” respectively. “IN” indicates the input sample, “IP” indicates the immunoprecipitate. Samples were split and analyzed in parallel for protein interactions by western blotting (WB) and for protein-RNA interactions by primer extension (PE). In the upper panel, the precipitates were blotted with anti-TAP antibody for detection of the Bur2 protein (indicated by the arrow). In the middle panel, precipitates were blotted with anti-His antibody for detection of Cus2 protein. In the bottom panel, total RNA from the precipitates was extracted and probed by primer extension for the presence of U snRNAs using oligos specific to U1, U2, and U4 snRNAs whose products are indicated by arrows. “*” indicates a non-specific product of the primer extension reaction.