Construction of an O-GlcNAc responsive GFP biosensor.

a. Schematic of the GFP reporter (left, drawn to scale) and predicted changes in reporter splicing and expression upon varying cellular O-GlcNAc conditions (right). ISS – Intronic splicing silencer; LHA – Left homology arm; RHA – Right homology arm; HBB – Hemoglobin subunit β; PGK – Phosphoglycerokinase; CMV – Cytomegalovirus; bGH – Bovine growth hormone; SV40 – Simian virus 40.

b. Semi-quantitative RT-PCR of RNA isolated from the reporter line under different treatment conditions using DNA primers (NC3378 and NC2094) that hybridize within the GFP ORF and just upstream of the polyadenylation signal sequence as shown below. The PCR conditions make it unlikely to detect the full-length detained intron isoform, so only the mRNA is observed.

c. Northern blot analysis of total RNA isolated from the reporter line after treatment with either DMSO, 1 μM TG or 10 μM OSMI-1 for 6 hours. The blot was probed for GFP. The retained intron band is heterogeneous and difficult to discern clearly due to its co-migration with the large ribosomal RNA. Methylene blue stain of the blot (right) is shown as a loading control.

d. GFP fluorescence levels of the reporter line as measured by flow cytometry after treatment with DMSO, TG or OSMI-1 for 24 hours.

e. Validation of GFP reporter protein levels by western blot analysis after treatment of the reporter line with various modulators of cellular O-GlcNAc levels (left). Steps in the hexosamine biosynthesis pathway targeted by the modulators are shown on the right. Treatment with modulators indicated in red are expected to lead to reduced cellular O-GlcNAc levels, while treatment with those indicated in green are expected to lead to increased cellular O-GlcNAc levels. A broad specificity O-GlcNAc antibody (RL2) and β-actin are used as controls.

f. Northern blot analysis of RNA isolated from either 30 nM non-target (siNT) or OGT specific (siOGT) siRNA-treated reporter line. Cells were treated for 6 hr with DMSO, TG or OSMI-1 3 days after siRNA treatment. The blot was probed for GFP as above.

SFSWAP is a negative regulator of OGT intron 4 splicing.

a. Top, timeline of CRISPR screen. Bottom, MAGeCK analysis of CRISPR screen results from TG-treated gain of GFP screen in three biological replicates. Top hits are color coded based on predicted function of the protein. Target genes are arranged alphabetically on the x-axis.

b. GFP fluorescence of TG-treated reporter cells 4 days post treatment with siRNA corresponding to non-target (siNT), OGT (siOGT) or SFSWAP (siSFSWAP). Bottom panels show corresponding brightfield images.

c. Northern blot analysis of RNA isolated from either the TG-treated reporter line (left, probed for GFP) or TG-treated 293A-TOA cells (right, probed for OGT) 4 days after treatment with siRNA corresponding to either non-target (siNT) or SFSWAP (siSFSWAP). Cells were treated with TG for 6 hours just before RNA isolation.

d. RT-qPCR analysis of the splice junctions of interest after treatment of cells with either DMSO, TG or OSMI-1 in the presence or absence of SFSWAP knockdown (n=3). Primers used correspond to either the OGT intron 4 spliced junction (e4-e5) or retained intron junction (RI-e5). p-values are derived from unpaired t-tests against the corresponding non-target control.

SFSWAP is a global regulator of retained intron splicing and exon skipping.

a. Alternate splicing analysis in untreated SFSWAP knockdown (siSFSWAP) cells compared to non-target (siNT)-treated cells using rMATS (n=3). The number of events of each type are plotted as proportion of total events detected. A3SS – alternate 3′ splice site; A5SS – alternate 5′ splice site; MXE – mutually exclusive exon; RI – retained intron; SE – skipped exon. Assignment of sample labels for events was done based on the value of IncLevelDifference (events with positive IncLevelDifference were designated as siNT and negative IncLevelDifference were designated as siSFSWAP).

b. Scatter plots of retained intron (RI) and skipped exon (SE) events plotted using the JC model of rMATS (top). The difference in inclusion values between siNT and siSFSWAP-treated cells is plotted on the x-axis. Only statistically significant events (FDR <= 0.05) are shown. Violin plots of inclusion levels corresponding to the individual samples are shown below. Median inclusion value for the sample is indicated by the black dot. Only significant events with 20% or greater change in inclusion levels are plotted for the violin plots.

c. IGV screenshot of read coverages of a few significant retained intron events. Three biological replicates are shown and the intron of interest is marked by the red rectangle.

d. GC content of relevant regions of significant RI and SE events (FDR <= 0.05, >= 10% change in inclusion levels). Red bars indicate the mode of GC content in each region.

SFSWAP regulates OGT decoy exon inclusion.

a. RT-qPCR analysis of OGT intron 4 splicing in UPF1 knockdown background. SFSWAP knockdown was performed for 5 days and cells were then treated with TG for 6 hours before RNA isolation and reverse transcription using a mixture of dT20 and a DNA oligomer complementary to exon 5 of OGT. qPCR was performed for either the spliced junction (e4-e5), retained intron junction (RI-e5) or decoy-e5 junction as shown. DE – decoy exon. p-values are derived from unpaired t-tests against the corresponding non-target controls.

b. IGV screenshot of aligned reads after nanopore sequencing of semi-quantitative RT-PCR amplicons generated from the above samples using DNA oligomers complementary to exon 3 and exon 8 of OGT. A zoomed version is shown below to better show changes in the decoy exon region.

c. Quantification of OGT decoy exon inclusion from RNA-seq data in the presence or absence of UPF1 knockdown and/or TG treatment. Inclusion levels and p-values are calculated from the JCEC model of rMATS performed after alignment against a custom reference annotation of the human genome containing decoy exon annotations.

SFSWAP is a global regulator of decoy exon splicing.

a. Scatter plot of global decoy exon inclusion level changes upon SFSWAP knockdown in a TG-treated UPF1 knockdown background. Statistically significant events are colored. Not all analyzed cassettes may function as splicing decoys.

b. Exon types of the events shown in (a) classified based on the predicted translation outcome. Events shown in blue introduce an in-frame stop codon in the CDS, thus functioning as poison cassettes.

c. Inclusion level changes in decoy-containing retained introns upon SFSWAP knockdown in a TG-treated UPF1 knockdown background. Significant events are colored.

d. Length distribution of the decoy-containing retained introns compared to non-decoy containing retained introns.

Model for the mechanism of action of SFSWAP on intron retention and exon skipping.

See text for details. In the case of retained introns without decoy exons or cassette exons (top and middle), we suggest that SFSWAP (green oval) restricts splicing subsequent to definition of the exons by U1 and U2. For retained introns with decoys, this inhibition of decoy exon inclusion supports the decoy exon’s function to promote intron retention (see Discussion).

Key resources.

List of primers.