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. 2020 Sep 8;21(18):6553.
doi: 10.3390/ijms21186553.

Splicing Enhancers at Intron-Exon Borders Participate in Acceptor Splice Sites Recognition

Tatiana Kováčová  1   2 , Přemysl Souček  1   2 , Pavla Hujová  1   2 , Tomáš Freiberger  1   2 , Lucie Grodecká  1
Affiliations

Splicing Enhancers at Intron-Exon Borders Participate in Acceptor Splice Sites Recognition

Tatiana Kováčová et al. Int J Mol Sci. .

Abstract

Acceptor splice site recognition (3' splice site: 3'ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3'ss, of which U2AF35 has a dual function: (i) It binds to the intron-exon border of some 3'ss and (ii) mediates enhancer-binding splicing activators' interactions with the spliceosome. Alternative mechanisms for 3'ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3'ss recognition where the intron-exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3'ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3'ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons' inclusion. Most probably, both factors stochastically bind the 3'ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3'ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants' effects on splicing.

Keywords: SRSF1; U2AF35; acceptor splice site recognition; pre-mRNA splicing; splicing enhancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
GGAGAA motif acts as a splicing enhancer. (A) Schematic representation of no exonic splicing enhancer (ESE) control (NEC) and putative ESE (putESE) sequences inserted into pcDNA-Dup vector middle exon used in an ESE-dependent splicing assay [32,33]. Larger uppercase letters illustrate inserted sequence, and bold letters show the putative ESE motif. (B) Agarose gel image shows the RT-PCR analysis of spliced transcripts expressed from NEC and putESE constructs. In parallel, positive controls (SC35) containing three binding sites for SR protein SC35 and negative control (BRCAi11) containing no known regulatory elements derived from BRCA2 intron 11 were used. (C) Inclusion of the middle exon was quantified by capillary electrophoresis, and it is shown as mean ± SD of three independent experiments. Statistically significant difference is shown (*** p < 0.001). (D) Scores from different prediction tools indicated potential splicing enhancing or silencing properties of analyzed sequences. Enhancer character shown as positive, neutral as zero, and silencer as a negative number; 6-mers/7-mers with the highest score of the overlapping sequence are shown.
Figure 2
Figure 2
Splicing minigene assay to verify a potential ESE element. (A,C) Schematic representation of the wild-type sequence and part of flanking introns of BRCA2 exon 12 (A) and VARS2 exon 17 (C). Uppercase letters show exonic bases, and lowercase letters intronic bases. Bold black letters represent the putative ESE binding motif. The designed potential ESE binding motif variants are indicated below. (B,D) Effects of 3′ splice site (3′ss) mutations on BRCA2 exon 12 splicing (B) and VARS2 exon 17 splicing (D) are shown in graphs. Error bars indicate SD. Statistically significant differences from wild-type inclusion are shown (* p < 0.05, ** p < 0.01, and *** p < 0.001). 3′ss strength (expressed as its max entropy algorithm (MaxEnt) score) and U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF35) score (counted according to Doktor et al. [37]) are shown in the table below the graph. On the right, the correlation of the MaxEnt score of 3′ss and the exon inclusion frequency of both BRCA2 exon 12 (B) and VARS2 exon 17 (D) are shown.
Figure 3
Figure 3
Various variants can have different effects on SRSF1 binding (Z-scores). The maximal correlation coefficient between exon inclusion and potential SRSF1 binding (shown as the Z-score) for BRCA2 (A) when using the SRSF1 binding site starting at the −1 position and VARS2 (B) when starting at the −4 position is shown.
Figure 4
Figure 4
Effect of SRSF1 and U2AF35 overexpression on exon recognition. HeLa cells were co-transfected with SRSF1 [39] or U2AF35 [40] expression vectors and BRCA2 or VARS2 minigenes. An empty vector was used as a control. Graphs show proportions of exon inclusion upon SRSF1 overexpression (A,C) or U2AF35 overexpression (B,D) (light bars) compared to control (dark bars) in BRCA2 exon 12 (blue) (A,B) or VARS2 exon 17 (green) (C,D). Data are shown as mean exon inclusion percentage ± SD obtained from three independent experiments. Statistically significant differences are shown (* p < 0.05, ** p < 0.01, and *** p < 0.001). On the right, SRSF1 or U2AF35 expression levels are shown as fold-change with respect to control transfections. SRSF1 or U2AF35 expression level upon SRSF1 or U2AF35 overexpression relative to SRSF1 or U2AF35 level from control transfection is shown.
Figure 5
Figure 5
Effect of SRSF1 and U2AF35 knockdown. HeLa cells were transfected with either a control (si control) or specific siRNA (si SRSF1 or si U2AF35). Graphs demonstrate the proportions of exon inclusion upon SRSF1 (A,C) or U2AF35 (B,D) knockdown in BRCA2 exon 12 (A,B) or VARS2 exon 17 (C,D). Exon inclusion from different minigenes is compared to control transfection (si control). Error bars represent SDs obtained from three independent experiments. Statistically significant differences are shown (* p < 0.05, ** p < 0.01, and *** p < 0.001). On the right, SRSF1 or U2AF35 expression levels are shown as fold-change compared to control transfections (si control). SRSF1 or U2AF35 expression level upon SRSF1 or U2AF35 knockdown (grey bars) relative to SRSF1 or U2AF35 level upon control knockdown is shown.
Figure 6
Figure 6
Affinity purification of RNA binding proteins (RBPs) of in vitro transcribed RNAs. (A) Table shows SRSF1 binding motif weakening in 1T and 1T2T variants compared to the wild-type construct via their Z-scores. Values correspond to the binding affinity of SRSF1 to each 7-mer [31], and 7-mers with the highest Z-score in analyzed sequences are shown. (B) Western blot analysis after the affinity purification of RNA-binding proteins is shown. Beads alone (Beads) and the HeLa nuclear extract (NE) sample were used as a control. Western blot images and Coomassie gel staining are representative figures in three independent experiments.
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