Abstract
To better understand splicing regulation, we used a cell-based screen to identify ten diverse motifs that inhibit splicing from introns. Motifs were validated in another human cell type and gene context, and their presence correlated with in vivo splicing changes. All motifs exhibited exonic splicing enhancer or silencer activity, and grouping these motifs according to their distributions yielded clusters with distinct patterns of context-dependent activity. Candidate regulatory factors associated with each motif were identified, to recover 24 known and new splicing regulators. Specific domains in selected factors were sufficient to confer intronic-splicing-silencer activity. Many factors bound multiple distinct motifs with similar affinity, and all motifs were recognized by multiple factors, which revealed a complex overlapping network of protein-RNA interactions. This arrangement enables individual cis elements to function differently in distinct cellular contexts, depending on the spectrum of regulatory factors present.
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Acknowledgements
We thank J. Hui (Shanghai Institute of Biological Science, Shanghai, China) and A. Willis (University of Nottingham, UK) for providing expression constructs of trans factors and B. Graveley (University of Connecticut Health Center, Farmington, Connecticut, USA) for constructs containing RS domains. We thank T. Nilsen and A. Berglund for critical reading of manuscripts and Z. Dominski and B. Marzluff for helping in RNA affinity purifications. This work was supported by an American Heart Association grant (0865329E) and US National Institutes of Health grant (R01CA158283) to Z.W. and (2-R01-GM085319) to C.B.B.
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Y.W., C.B.B. and Z.W. designed the research. Y.W., Z.W., J.Z., K.L. and R.C. performed the experiments. M.M., X.X. and A.R. developed computational methods to analyze the data. Y.W., C.B.B. and Z.W. wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Identification of FAS-ISS decamers.
(a) Flow cytometry profile of 293 FlipIn cells transfected with the pZW11 reporter inserted with random library. After selection for stable integration, all hygromycin resistant cells were pooled and analyzed by flowcytometry. Both red and green fluorescence signals were measured to correct for self-fluorescence background. The GFP-positive cells (R1 region) were sorted using a Cytomation MoFlo high-speed sorter into 96 well plates to recover all of the ISS sequences. (b) Validation of FAS-ISS decamer activity. To validate the silencer activities of the newly identified ISS decamers, 293T cells were transiently transfected with pZW11 containing 16 arbitrarily selected ISS decamers and control sequence. The GFP-positive cells were examined by flow cytometry at 24h after transfection. (c) The frequencies of mononucleotide in the screened ISS decamer set. (d) The frequencies and odds ratios of dinucleotides in the screened ISS decamers set.
Supplementary Figure 2 Diverse functions of ISSs when inserted in the exons or between two 5′ SS.
(a) The ISS exemplars of each group were inserted into the exon of a splicing reporter and transiently transfected into both HeLa and 293T cells. After 48 hours, RNAs were isolated from the transfected cells to determine the functions of ISSs by RT-PCR. (b) The representative ISSs of each group were inserted between two 5′ SS in the exonic extension region of the mini-gene reporter. The resulting reporters were transiently transfected into HeLa and 293T cells. After 48 hours, RNAs were extracted from the transfected cells and the functions of ISSs were examined by RT-PCR and shown in the gel figures.
Supplementary Figure 3 The positional frequency of FAS-ISS k-mers in different premRNA locations.
Distribution of ISS k-mers near the constitutive exon (black) and skipped exon (red). The number of transcripts containing ISS k-mers divided by total number of transcripts at each position is plotted as ISS frequency. The first and last 50 bases of exons and the first and last ~200 bases of introns are shown, excluding the last 20 bases of the upstream intron and the first 10 bases of the downstream intron to avoid overlaps with the splice site motifs.
Supplementary Figure 4 Depletion or overexpression of hnRNP L and YB1 proteins.
(a) 293T cells were transfected with the siRNA of HNRNP L and YB1. After 48 hours of siRNA transfection, we transfected 0.2 μg splicing mini-gene reporters containing ISS group U or group D into the cells respectively, and harvested the cells 24 hours after the second transfection to examine the protein levels of HNRNP L and YB1 through Western blot, and the tubulin level was detected as loading control. GRSF1 siRNA was used as the specificity control. The asterisk indicated a non-specific protein that cross-react with HNRNP L antibody. (b) 293T cells were transiently transfected with expression vectors of HNRNP L and YB1. After 72 hours, proteins were extracted from the transfected cells to determine the expression levels of HNRNP L and YB1 with western blot. The asterisk indicated a non-specific protein that cross-react with HNRNP L antibody. The tubulin level was examined as loading control. (c) 293T cells were transfected with siRNA of scramble control or HNRNP L. After 48 hours of siRNA transfection, the cells were cotransfected with 0.2 μg splicing reporter containing ISS group U or group D and FLAGYB1 expression vector (lane 3 and 6). The cells were harvested after another 24 hours for the protein analysis. The tubulin level was measured as loading control.
Supplementary Figure 5 The SDS-PAGE gel of putative protein factors that bind to different ISS groups.
Biotinylated RNA oligos of each ISS group were incubated with HeLa whole cell extract, bound to streptavidin beads and washed, the RNA-protein complex were eluted and separated on a SDS-PAGE gel. The specific bands (marked with a dot and labeled according to each group) were cut and identified by mass spectrometry. Two batches of the affinity purification experiments were carried out for group D ISS, and we separated two samples in the same gel for protein identification.
Supplementary Figure 6 Purification of recombinant proteins for measurement of direct RNA-protein binding.
The putative trans-factors binding to groups F, H, I were cloned into bacterial expression system (pT7HtB ) and purified with His GraviTrap Kit. The final protein products were assayed with SDS-PAGE to check purity, and were later used in Biacore assay to measure the direct RNA-protein binding. Three fractions eluded from Ni column were shown for hnRNP A0, A2, A1 and D, and the elution fractions were combined for hnRNP DL
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Wang, Y., Xiao, X., Zhang, J. et al. A complex network of factors with overlapping affinities represses splicing through intronic elements. Nat Struct Mol Biol 20, 36–45 (2013). https://doi.org/10.1038/nsmb.2459
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DOI: https://doi.org/10.1038/nsmb.2459
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