Luc7 depletion leads to the loss of Prp40 and Snu71 in U1 snRNP, and extensive splicing defects. (A) Luc7 was depleted after 6 h of β-estradiol and IAA induction, as shown in western blot of whole-cell lysate using antibodies against the HA tag on Luc7 and CBP tag on U1A. R1–3 represent three biological replicates. (B) The growth of the Luc7D strain was retarded upon induction of Luc7 depletion, relative to the uninduced culture or the WT. (C) U1A (probed by an anti-CBP antibody) and U1 snRNA (probed by solution hybridization) levels in whole-cell lysate and after IgG pull down using the TAP tag on U1A are similar between the WT and Luc7D strains. Other snRNAs serve as internal controls. (D) Coomassie-stained SDS-PAGE gel showed that purified U1 snRNPLuc7D after 6 h induction was depleted of not only Luc7, but also Snu71 and Prp40 (indicated by the purple font), compared to the WT U1 snRNP. (E) Negative stain images showed more smaller (likely broken down) particles in purified U1 snRNPLuc7D as compared to the WT. Red and blue circles highlight representative intact and small particles, respectively. (F) The top 10 class averages (sorted by number of particles in each class) from 2D classification (Punjani et al. 2017) of a small cryo-EM data set showed fewer classes and lower number of intact U1 snRNP particles in purified U1 snRNPLuc7D as compared to the WT. (G) Luc7 depletion after induction led to massive splicing defects (manifested as increased %IR). Of 223 introns with sufficient read coverage to quantify, 197 displayed significantly increased IR (FDR < 0.05, ΔIR ≥ 0.05). Arrows indicate the two genes selected for RT-PCR validation in H. (H) Examples of two genes whose splicing was significantly impaired upon Luc7 depletion as shown by RT-PCR analyses.
