1. A Conserved Interaction between SKIP and SMP1/2 Aids in Recruiting the Second-Step Splicing Factors to the Spliceosome in Arabidopsis 
Lei Liu, Fangming Wu, Wei Zhang 
Molecular Plant 
Volume 9, Issue 12, Pages (December 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
SKIP Physically Interacts with SMP1/2 to Recruit Second-Step Splicing Factors to Integrate into Spliceosome.
(A) Physical associations between SKIP and SMP1 and SMP2 detected by yeast two-hybrid assay. The plasmids were co-transformed as indicated combinations. Positive control, pGADT7-T + pGBKT7-53; Negative control, pGADT7-T + pGBKT7-Lam. Scale bar, 5 mm.
(B) Interactions between SKIP and SMP1 and SMP2 tested by FRET assay in tobacco. Nucleuses were imaged using the GFP and RFP channels of a confocal microscope. GFP, GFP channel image; RFP, RFP channel image; Merge, merged images of GFP and RFP; FRET Efficiency, FRET efficiency was measured by sensitized emission. Scale bar, 2 μm.
(C and D) CoIP assays to detect the interactions between SKIP and SMP1 and SMP2. Total proteins extracted from wild-type (WT) and complementation lines were precipitated with anti-Myc and anti-GFP antibodies, and the precipitated proteins were subjected to western blot by anti-SKIP antibody.
(E) Physical interaction between Slu7 and Prp45 in fission yeast, and hSLU7 and hSKIP in human detected by yeast two-hybrid assay. The plasmids were co-transformed as indicated combinations.
(F) Detailed schematics of homologous recombination structures of Prp45-Myc and Slu7-TAP fusion constructs along with hygromycin and G418 antibiotic genes, respectively. Scale bar, 300 bp.
(G) CoIP assay to detect the interaction between Prp45 and Slu7 in fission yeast. Anti-TAP was used to pull down the complex and anti-Myc was used to detect Prp45–Myc fusion protein.
(H) CoIP assay to detect the interaction between hSKIP and hSLU7 in HeLa cells. Normal immunoglobulin G (IgG) was used as a negative control. Total proteins extracted from HeLa cells were precipitated with anti-hSKIP antibody, and the precipitated proteins were subjected to western blot by anti-hSLU7 antibody.
(I) Assessment of the strength of the interaction combinations in fission yeast, Arabidopsis, and human. The values are the mean ± SD of three independent biological replicates.
(J) Physical interactions between SMP1/2 and PRP18, and SMP1/2 and PRP22 were tested by yeast two-hybrid assay.
(K and L) CoIP analysis of the interactions between SMP1/2 and PRP22. Total proteins extracted from WT and complementation lines were precipitated with anti-Myc and anti-GFP antibodies, and the precipitated proteins were subjected to western blot by anti-PRP22 antibody. Anti-PRP22 antibody was raised against peptide PLYDRYHEPNSWRLSKRRA of PRP22 in rabbit.
(M) Enrichment amounts of second-step splicing factors SMP and PRP22 detection in WT, skip-1, and skip-2 transgenic lines with MAC3B-Myc. WT was used as the negative control. The precipitated proteins by anti-Myc were subjected to western blot detected by anti-SMP and anti-PRP22 antibodies to evaluate the amount of SMP and PRP22 in different genetic backgrounds. Anti-SMP antibody was raised against the N-terminal residues 8–165 in rabbit.
(N and O) Enrichment amounts of CDC5 detection in WT, skip-1, and skip-2 transgenic lines transformed with SMP1-Myc (N) and PRP22-Flag (O), respectively. The proteins precipitated by anti-Myc and anti-FLAG were subjected to western blot detected by anti-CDC5 antibody. Anti-CDC5 antibody was raised against the N-terminal 144-amino-acid fragment of CDC5 in rabbit.
(P) Alignment of sequences of WT and mutant versions identified from cloned PCR by sequencing.
(Q) Detection of splicing defects by RT–PCR. The different bands in the left panel confirmed by sequencing are indicated by the schematic diagrams of gene structures in the right panel. ACTIN was used as an internal control.
(R) Measurement of the abundance of splicing defective isoforms by qRT–PCR. The values are the mean ± SD of three independent biological replicates. The primers' locations are indicated by the arrows in the right panel of (Q).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions