General and specific functions of exonic splicing silencers in splicing control

Abstract

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5' and 3' splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5' splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.

Figure 1. ESSs generally influence splice site choice

(A) ESS density (FAS-hex3 hexamers as a fraction of total hexamers at each position, smoothed over a window of 10 bases) as a function of distance from the 5′ss and 3′ss of constitutive exons, plotted separately for exons which have (red) or do not have (blue) an adjacent intronic decoy 5′ss or 3′ss (see Supplemental Data). Dataset sizes were, for 3′ss: 11,944 with decoy, 47,048 without decoy; for 5′ss: 7563 with decoy, 59,048 without decoy. Splice sites are shown as left-facing (5′ss) or right-facing (3′ss) brackets, with solid and dotted lines for authentic and decoy splice sites, respectively. (B) Competing 5′ss and 3′ss reporters. Maximum entropy splice site scores (Yeo and Burge, 2004) are indicated above the corresponding splice site brackets (in bits). Sequences between competing splice sites are given (exons in caps, introns lower-case), with splice junctions indicated by ‘|’ and the inserted sequence marked in red. (C) Body-labeled RT-PCR of total RNA from cells transfected with competing 5′ss reporters with primers targeted to flanking exons. All transfections were repeated at least twice. Results shown for ESS sequences representing all 7 FAS-ESS groups (groups A-G + ‘u’ = unclustered; Table S3) and controls (Table S4). Relative usage of the proximal 5′ss is shown below (determined as ratio of intensity of upper band to sum of upper and lower bands). Mean and range of all control sequences is shown at left (seeFig. S1A for all 6 controls); error bars in remaining lanes indicate range of duplicated experiments. All ESS samples except no. 6 (an unclustered decamer) significantly inhibited use of the intron-proximal site (P < 0.02, rank-sum test, indicated by asterisks). Lane 6 appears to be somewhat under-loaded relative to the other lanes based on duplicate transfection data (not shown). (D) RT-PCR results for competing 3′ss reporters (as in panel C); 18 out of 21 ESS inserts significantly inhibited the intron-proximal 3′ss (P < 0.02, indicated by asterisks). For several ESSs that resemble 5′ss, an additional splice form was observed (indicated by arrow head) resulting from use of the inserted sequence as a 5′ss.

Figure 2. Role of ESSs in control of alternative splice site choice

(A) ESS density (as in Fig. 1A) as a function of distance from the intron-distal splice site for human A5Es (left panel) and A3Es (right panel), with the number of exons in each set listed above. (B) Conserved Occurrence Rate (COR, defined in Experimental Procedures) of ESS hexamers in A5E and A3E extension regions (red dot). Histograms show COR values for 5,000 control sets of hexamers of similar abundance in human/mouse extension regions (Supplemental Data). P-values were calculated based on a normal approximation to the distribution of the controls (purple curve). (C) Splicing of A5E minigene based on human AGER gene exon 9 and flanking introns. Partial sequence between alternative 5′ss is shown, with FAS-hex3 hexamers in red, and introduced mutations in blue. Both wild type and mutant constructs were transfected in duplicate (both shown). Splice site usage assayed by body-labeled RT-PCR using primers targeted to flanking exons. (D) Splicing of A3E minigene based on human H2RSP gene exon 3 and flanking introns (same format as panel C).

Figure 3. Effects of tethering splicing factors between competing splice sites

(A) Results of cotransfection of MS2-splicing factor fusion protein expression constructs with competing 5′ss reporter (as in Fig. 1B, 1C), using MS2 hairpin inserted in place of splicing regulatory element (- indicates mock-transfection). Sequences are shown for MS, an RNA hairpin bound with high affinity by MS2 coat protein; and MSΔ, a hairpin with 1 nt deletion which abolishes coat protein binding. Fusion proteins shown as gray oval (splicing factor) fused to green crescent (MS2 coat protein). Products of body-labeled RT-PCR of total RNA extracted from co-transfected cells using primers in flanking exons. (B) Competing 3′ss reporter with MS2 hairpin insert. Co-transfection and RT-PCR analysis as in panel A. (C) Results of transfecting increasing quantities of hnRNP A1-MS2 coat fusion protein expression construct (0.01 ug to 0.125 ug, proportional to height of grey bars above each lane) co-transfected with 0.5 ug of competing 5′ss reporter containing MS or MSΔ hairpin. RT-PCR as in panel A. The amount of fusion protein was detected by western blot using anti-Flag antibody (second panel from bottom). Bottom panel, loading controls of western blot using anti-actin antibody.

Figure 4. Role of ESS in regulation of intron retention

(A) Multifunctional intron reporter minigene and exon structures of four expected splicing products, ordered by decreasing size. Green boxes represent constitutive GFP exons; purple and yellow boxes represent variable exons; dashed box is retained intron; red box is inserted sequence. (B) Body-labeled RT-PCR of total RNA from cells transfected with intron retention minigene reporters inserted with control and ESS sequences (controls 1 and 2 listed in Table S5; other sequences in Table S3). Transfections were carried out as in Fig. 1C, in duplicate. Bottom panel shows the fraction of spliced product which represents the retained intron isoform in each sample (see Methods for details). Mean and range of control sequences is shown at left; error bars in remaining lanes indicate range of duplicated experiments. (C) Scatter plot showing fraction of fully spliced form and dual-skipped isoforms for ESSs (red dots) and control sequences (blue squares) in the experiment shown in panel B, showing range of dulicate values in each dimension. Red dotted lines illustrate division of ESSs into two activity classes: Class 1 ESSs increased level of dual-skipped isoform above level of horizontal red line; Class 2 ESSs increased level of fully spliced form above level of vertical red line; three ESSs clustered with control sequences (below both red lines). Sequences of tested ESS hexamers and decamers in each class are shown at right, grouped by sequence similarity, with similar segments indicated by shading. Consensus sequences and group names from (Wang et al., 2004) are listed below each cluster.

Figure 5. Distribution of ESS hexamers in retained introns

(A) Clusters of ESS hexamers with similar consensus sequences as Class 1 and Class 2 ESSs tested in Fig. 4. FAS-hex3 hexamers were classified based on these clusters; hexamers whose classification was unclear were omitted. (B) Distributions of Class 1 and Class 2 ESS hexamers in 5′ and 3′ ends of retained introns (5′ end includes 5′ half of intron, up to 200 bases; 3′ end includes 3′ half of intron, up to 200 bases). Histograms show ESS density in 2,000 sets of control constitutive introns with similar length and expected counts of ESS hexamers as retained introns. P-values were calculated based on a normal approximation to the distribution of the controls (blue curves).

Figure 6. General roles of ESSs in alternative splicing

Model of general effects of ESSs on four common types of alternative splicing. Trans-factor represents a factor which binds to the ESS and mediates – directly or indirectly – the indicated effect on splicing. Summary of ESS effects: (A) inhibition of exon inclusion (based on previous bioinformatic and experimental studies – see text); (B) inhibition of intron-proximal 5′ss usage (supported by Figs. 1–3); (C) inhibition of intron-proximal 3′ss usage (supported by Figs. 1–3); (D) inhibition of intron-retention (supported by Figs. 4–5).