Extended Data Fig. 2: Exon length and exonic cis-acting elements contribute to SRRM4 mediated splicing regulation.
(a) Scheme of the strategy used to increase the length of the microexons. To increase their length, we designed sixteen 36-nt long sequences devoid of potential splicing regulatory motifs such as Exonic Splicing Enhancers (ESE) or Silencers (ESS) according to either the Human Splicing Finder tool61 or data from 44 (“designed sequences”; Methods). Then, for each microexon, we increased the length of the microexon through the stepwise addition of 3 nts (color coded) to its center for each of the designed sequences, up to a maximal exon length of 42 nts (Supplementary Table 4). (b) ESRseq scores provided for each hexamer with a 1-nt sliding window44 for each of the 15 sequences used in Fig. 2. Scores range from -1 to 1 depending on whether they are likely to harbor negative or positive regulatory motifs, respectively. Scores close to 0 are less likely to have splicing regulatory elements. (c–f) ESRseq scores obtained upon length variation of LS (c), HS (d), CS (e) and CR (f) events represented in Fig. 2a–c, Extended Data Fig. 2j, k. (g) Pearson correlation matrices represent the impact of each designed sequence when added into LS and HS microexons under two experimental conditions (GFP in the lower triangle, HIGH in the upper triangle), quantified as ΔPSI (VAR-WT). For both LS and HS, ΔPSI values are averaged by sequence and length variation, and correlations are calculated across all sequence and length combinations. (h) PSI across four experimental conditions (GFP vs LOW, MID, HIGH expression of SRRM4) for each CS WT (42 nts) and their most SRRM4-responding shortened variants (in which the indicated number of nucleotides N have been removed, remN). (i) Two examples of CS events whose shortening results in SRRM4-responding variants (EPS15L1-HsaEX6093676 and POSTN-HsaEX6040016). The arrow indicates the maximal value of ΔPSI (HIGH-GFP). (j) RT-PCR assays showing the splicing patterns of various SNAP25-HsaEX6077184 splicing reporters (WT or following the removal of either 9, 11, 18 or 33 nucleotides) under control condition (-) or upon expression of SRRM4 (+) in HEK 293 cells. The PCR amplicons corresponding to inclusion / skipping are indicated with the squares. PSI and standard deviations (std) from at least three biological replicates per minigene variant are provided. (k) Change in CR exon inclusion upon shortening or lengthening was quantified as ΔPSI (VAR-WT) in either the GFP condition or under HIGH level of expression of SRRM4. Each data point corresponds to the effect of adding a given number of nucleotides from one of the fifteen designed sequences (positive number on the x-axis), or removal of a given number of nucleotides from the exonic sequence (negative number on the x-axis). The length of the WT is indicated by the dashed vertical line. The numbers at the right side of each subpanel indicate the Spearman correlation (ρ) and corresponding p-value (P) of the effects in ΔPSI according to length variation. (l) Impact of length variation on the pattern of splicing and regulation under different levels of expression of SRRM4 with respect to GFP condition for each sequence, expressed as ΔPSI (HIGH/MID/LOW-GFP) for CR events.
