1. Volume 9, Issue 7, Pages 1040-1050 (July 2016)
The SNW Domain of SKIP Is Required for Its Integration into the Spliceosome and Its Interaction with the Paf1 Complex in Arabidopsis 
Yan Li, Congcong Xia, Jinlin Feng, Dong Yang, Fangming Wu, Ying Cao, Legong Li, Ligeng Ma 
Molecular Plant 
Volume 9, Issue 7, Pages (July 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
Subcellular Localization of the Full-Length and Truncated SKIP Proteins.
(A) Schematic diagrams of the truncated SKIP proteins. The numbers above each protein structure represent the ordinal number of amino acid residues in SKIP.
(B) Subcellular localization of the full-length and truncated SKIP proteins in Nicotiana benthamiana cells. N. benthamiana leaves were transformed with constructs encoding full-length and different deletions of SKIP as indicated in the figure driven by the 35S promoter. The images were recorded using the GFP channel under a confocal microscope. Scale bar, 50 μm.
(C) Subcellular localization of the full-length and truncated SKIP proteins in Arabidopsis roots. Constructs encoding full-length and different deletions of SKIP as indicated in the figure were stably transformed into Arabidopsis driven by its native promoter. Five independent lines for each transgenic event were examined; one representative line for each transgenic event is shown. Roots from transgenic plants at the T3 generation were observed by confocal microscopy. Scale bar, 50 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2 Identification of the NLSs in SKIP.
(A) The predicted (PNLS) and functional (FNLS) NLS1 sequences in SKIP.
(B) Mapping of the NLS1 sequence in SKIP in N. benthamiana cells. Scale bar, 50 μm.
(C) Identification of the FNLS1 sequence in SKIP. Fusion proteins were generated by linking the PNLS1 or FNLS1 to the N-terminus of SKIP, which was localized to the cytosol. Scale bar, 50 μm.
(D) Mapping of the NLS2 sequence located in the C-terminus of SKIP in N. benthamiana cells. Scale bar, 50 μm.
(E) NLS2 is a functional NLS in plant cells. The fusion protein was generated by linking NLS2 to the N-terminus of SKIP, which was localized to the cytosol. Scale bar, 50 μm.
(F) Evaluation of the role of the two NLSs in the nuclear localization of SKIP in N. benthamiana cells. Scale bar, 50 μm.
(G) The two identified NLSs in Arabidopsis SKIP. The peptide sequences for the two identified NLSs are shown below the structure of SKIP.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
The SNW Domain Is Required for the Integration of SKIP into the Spliceosome.
(A) The co-localization of SR45 and transiently expressed full-length or truncated SKIP in N. benthamiana cells. Images from the RFP, GFP, and both (“Merge”) channels are shown. Scale bar, 2 μm.
(B) Assessment of the interaction between SR45 and full-length or truncated SKIP by an acceptor photobleaching FRET assay in N. benthamiana cells. Images from the GFP and RFP channels before and after photobleaching are shown. The difference in fluorescence intensity is represented by different colors as indicated in the barcode. The fluorescence intensities of the donor and acceptor in the pre- and post-bleaching images were determined. The FRET efficiency was calculated according to the formula EFRET = (ID[post] − ID[pre])/ID(post); the data are summarized in Supplemental Table 1.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4
The SNW Domain of Arabidopsis SKIP Is Required for Complementation of the prp45 Phenotypes in Yeast by SKIP.
(A) Cell morphologies of PRP45, prp45(1–169), and prp45(1–169) + truncated SKIP.
(B) The growth of cells transformed with PRP45, prp45(1–169), or prp45(1–169) + truncated SKIP at 30°C or 37°C.
(C) Splicing deficiency detection in cells transformed with PRP45, prp45(1–169), or prp45(1–169) + truncated SKIP by primer extension analysis.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
The SNW Domain Is Required for SKIP to Function as a Splicing Factor in Response to Salt Stress.
(A and B) Salt responses of the wild type, skip-1, and different transgenic lines expressing truncated SKIP in terms of plant survival under salt treatment. Images of the plants are shown in (A); the survival rates are given in (B). Between 8 and 15 independent lines for each transgenic event were examined; two representative independent lines for each transgenic event were selected for analysis. The values in (B) are the mean ± SD from three biological replicates.
(C) Detection of splicing defects in various stress tolerance genes in wild-type, skip-1, and different transgenic lines expressing truncated SKIP by RT–PCR following growth in the presence or absence of salt. Two representative independent lines for each transgenic event were selected for analysis.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
The SNW Domain of SKIP Is Required for Its Interaction with the Paf1c.
(A) The co-localization of ELF7 and full-length or truncated SKIP in transiently transfected N. benthamiana cells. Images from the RFP, GFP, and both (“Merge”) channels are shown. Scale bar, 2 μm.
(B) Assessment of the direct interaction between ELF7 and full-length or truncated SKIP by yeast two-hybrid assays. The interaction between ELF7 and full-length or truncated SKIP was quantitatively evaluated based on the level of β-galactosidase activity. The values are the mean ± SD from three biological replicates.
(C) Assessment of the interaction between ELF7 and full-length or truncated SKIP by acceptor photobleaching FRET assays in N. benthamiana cells. Images from the GFP and RFP channels before and after photobleaching are shown. The difference in fluorescence intensity is represented by different colors as indicated in the barcode. The fluorescence intensities of the donor and acceptor in pre- and post-bleaching images were determined. The FRET efficiency was calculated according to the formula EFRET = (ID[post] − ID[pre])/ID(post); the data are summarized in Supplemental Table 2.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
The SNW Domain of SKIP Is Required for Its Function as a Transcriptional Activator in the Regulation of FLC Transcription and Flowering.
(A) Observation of the flowering time in the wild type, skip-1, and skip-1 plants transformed with full-length or different truncated versions of SKIP. For the transgenic events, 8–15 independent lines were examined; one representative independent line for each is shown.
(B) Measurement of the flowering time in the wild type, skip-1, and skip-1 plants transformed with full-length or different truncated versions of SKIP. For the wild-type and skip-1 plants, the data represent the mean ± SD from three biological replicates (at least 36 individual plants per replicate); for each transgenic SKIP line, the data represent the mean ± SD from the independent lines (8 lines for SKIP/skip-1, 9 lines for SKIPN/skip-1, 15 lines for SKIPSNW/skip-1, 12 lines for SKIPC/skip-1, 11 lines for SKIPNSNW/skip-1, and nine lines for SKIPSNWC/skip-1); and for each independent line, the data represent the mean from three biological replicates (at least 36 individual plants per replicate).
(C) The relative level of stable FLC mRNA expression in 7-day-old wild-type plants, skip-1 plants, and skip-1 plants transformed with full-length or different truncated versions of SKIP by qRT–PCR. Two representative independent lines for each transgenic event are shown. The data represent the mean ± SD from three biological replicates.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions