Fig. 2: Prp28 genetically interacts with Brr2 and crosslinks to pre-mRNA 5′SS in the pre-B complex.
From: Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling
a Growth phenotypes of yeast strains harboring various combinations of prp28-K82Δ10 and brr2 alleles spotted in a dilution series to YPD medium at 30 °C. Asterisk represents stop codon. b ChIP analysis revealed that U1 snRNP’s departure is delayed in the prp28-K82Δ10 strain. Short lines in the top panel represent the amplicons used for monitoring U1 snRNP recruitment. In the bottom panel, the amplicon numbers are denoted on X axis. Y axis represents relative enrichment of U1 signal to that of the amplicon 1. Error bars are ± SEM; n = 3 biological repeats; ****P < 0.0001, unpaired two-tailed t-test. c Splicing reactions using radiolabeled ACT1 transcript were done in wild-type extracts at 0.02, 0.05, 0.2, or 2 mM ATP and subjected to 254-nm UV irradiation (even-numbered lanes 6–20). Immunoprecipitations after denaturation were done with anti-Prp28 (lanes 5–12) or with negative-control anti-V5 antibody (lanes 13–20). Relative loadings are 1:1000 for splicing reactions alone (lanes 1–4) vs. immunoprecipitated reactions (lanes 5–20). The experiment was repeated three times with similar results. d Primer extension revealed two strong reverse transcriptase stops. Sequencing ladder (lanes 1–4) allows assignment of the two stops to the high conserved GU dinucleotide (lane 8, triangles) that remain after denaturation (Denat) and immunoprecipitation with anti-Prp28 antibody. Source data are provided as a Source Data file.
