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. 2004 Jul 15;381(Pt 2):343-50.
doi: 10.1042/BJ20040408.

The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5'-splice site selection

Vita Dauksaite  1 , Göran Akusjärvi
Affiliations

The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5'-splice site selection

Vita Dauksaite et al. Biochem J. .

Abstract

The human splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2) is modular in structure with two RNA-binding domains (RBD1 and RBD2) and a C-terminal domain rich in arginine-serine dipeptide repeats. ASF/SF2 is an essential splicing factor that also functions as an important regulator of alternative splicing. In adenovirus E1A (early region 1A) alternative pre-mRNA splicing, ASF/SF2 functions as a strong inducer of proximal 5'-splice-site selection, both in vitro and in vivo. In the present study, we tested the functional role of individual domains of ASF/SF2 in alternative splicing in vitro. We show that ASF/SF2-RBD2 is the critical domain controlling E1A alternative splicing. In fact, RBD2 alone is sufficient to mimic the activity of the full-length ASF/SF2 protein as an inducer of proximal 5'-splice-site selection in vitro. The RBD2 domain induces a switch to E1A-proximal 5'-splice-site usage by repressing distal 12 S splicing and simultaneously stimulates proximal 13 S splicing. In contrast, the ASF/SF2-RBD1 domain has a more general splicing enhancer phenotype and appears to stimulate preferentially cap-proximal 5'-splice-site selection. Furthermore, the SWQDLKD motif, which is conserved in all SR proteins (serine/arginine-rich proteins) containing two RBDs, and the ribonucleoprotein-1-type RNA recognition motif were both found to be necessary for the alternative splice-site-switching activity of ASF/SF2. The RNP-1 motif was necessary for efficient RNA binding, whereas the SWQDLKD motif most probably contributes by functioning as a surface-mediating critical protein-protein contact during spliceosome assembly.

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Figures

Figure 1
Figure 1. Role of structural domains of ASF/SF2 in the regulation of E1A alternative splicing
(A) Structure of the MS2–ASF/SF2 hybrid proteins used to examine the domain requirements of ASF/SF2 in E1A alternative splicing. Hybrid proteins encode for the following ASF/SF2 sequences: MS2–ASF/SF2, amino acids 1–248; MS2–RBD2+RS, amino acids 92–248; MS2–RBD2, amino acids 92–197; MS2–RBD1, amino acids 1–92; and MS2–RS, amino acids 196–248. (B) Schematic representation of the E1A pre-mRNA showing the position of splice sites and the splicing events that generate the 13 and 12 S mRNAs respectively. (C) Alternative splicing enhancer activity of the MS2–ASF/SF2 hybrid proteins. In vitro splicing reactions were performed under limiting splicing conditions in HeLa-NE using the E1A pre-mRNA and the indicated MS2–ASF/SF2 fusion proteins (30 pmol). The products were resolved by gel electrophoresis and visualized by autoradiography. Positions of the pre-mRNA, free first exons and splicing products are indicated by arrowheads. Lane 1 shows a splicing reaction incubated under optimal splicing conditions.
Figure 2
Figure 2. ASF/SF2-RBD1 and -RBD2 have different effects on E1A alternative 5′-splice-site selection
(A) Schematic representation of the structure of His–RBD1 and His–RBD2 proteins. His–RBD1 contains amino acids 1–92 and His–RBD2 contains amino acids 92–197 from ASF/SF2. (B) Schematic representation of the E1A pre-mRNA showing the position of splice sites and the splicing events that generate the 13 and 12 S mRNAs respectively. (C, D) In vitro splicing reactions were performed under limiting splicing conditions in HeLa-NE using the E1A pre-mRNA and the indicated His–RBD fusion proteins (150 pmol). The products were resolved by gel electrophoresis and visualized by autoradiography. Positions of pre-mRNA, splicing products and free first exons are indicated by arrowheads.
Figure 3
Figure 3. The conserved SWQDLKD motif and the RNP-1-type RNA recognition motif are essential for the proximal 13 S 5′-splice-site enhancer activity of ASF/SF2-RBD2
(A) Schematic representation of the structure of MS2–RBD2+RS mutant proteins. The amino acid sequence of RBD2 is shown on the top and the residues changed in each mutant protein are indicated. The conserved SWQDLKD motif is shown boxed in the sequence. (B) In vitro splicing reactions were performed under limiting splicing conditions in HeLa-NE using the E1A pre-mRNA and the indicated MS2–RBD2+RS hybrid proteins (24.5 pmol). The products were resolved by gel electrophoresis and visualized by autoradiography. Positions of pre-mRNA, splicing products and free first exons are indicated by arrowheads.
Figure 4
Figure 4. The RNP-1-type RNA recognition motif of ASF/SF2-RBD2, but not the SWQDLKD motif, is required for protein binding to the E1A 13 S 5′-splice site
(A) RNA binding was tested by a UV cross-linking assay in which ASF/SF2-RBD2 mutant proteins (schematically depicted in Figure 3A) were incubated and cross-linked to a short 32P-labelled RNA, containing the E1A 13 S wild-type (lanes 1–4) or mutant 5′-splice site (lanes 5–8). The products were resolved on a 12% SDS/polyacrylamide gel and visualized by autoradiography. (B) Coomassie Blue-stained gel of the ASF/SF2-RBD2 mutant proteins used in the experiments shown in Figures 3 and 4.
Figure 5
Figure 5. Effects of His–RBD1 and His–RBD2 on the activity of individual E1A 5′-splice sites
(A) Schematic representation of the three E1A pre-mRNAs used to assay the activity of the ASF/SF2-RBDs on E1A 13 or 12 S 5′-splice-site activity, alone or in competition. (B) In vitro splicing reactions were performed under limiting splicing conditions in HeLa-NE using the indicated E1A pre-mRNAs and supplemented with the His–RBD1 or His–RBD2 proteins (150 pmol), as depicted in Figure 2(A). The products were resolved by gel electrophoresis and visualized by autoradiography. Positions of the pre-mRNA, free first exons and splicing products are indicated.
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