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Human splicing factor ASF/SF2 encodes for a repressor domain required for its inhibitory activity on pre-mRNA
Vita Dauksaite1, Göran Akusjärvi
1Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 582, 751 23 Uppsala, Sweden.
Abstract:
The essential splicing factor ASF/SF2 activates or represses splicing depending on where on the pre-mRNA it binds. We have shown previously that ASF/SF2 inhibits adenovirus IIIa pre-mRNA splicing by binding to an intronic repressor element. Here we used MS2-ASF/SF2 fusion proteins to show that the second RNA binding domain (RBD2) is both necessary and sufficient for the splicing repressor function of ASF/SF2. Furthermore, we show that the completely conserved SWQDLKD motif in ASF/SF2-RBD2 is essential for splicing repression. Importantly, this heptapeptide motif is unlikely to be directly involved in RNA binding given its position within the predicted structure of RBD2. The activity of the ASF/SF2-RBD2 domain in splicing was position-dependent. Thus, tethering RBD2 to the IIIa intron resulted in splicing repression, whereas RBD2 binding at the second exon had no effect on IIIa splicing. The splicing repressor activity of RBD2 was not unique to the IIIa pre-mRNA, as binding of RBD2 at an intronic position in the rabbit beta-globin pre-mRNA also resulted in splicing inhibition. Taken together, our results suggest that ASF/SF2 encode distinct domains responsible for its function as a splicing enhancer or splicing repressor protein.
Insights
The splicing factor ASF/SF2
Area of Science:
- Molecular Biology
- RNA Splicing Mechanisms
- Gene Regulation
Background:
- Alternative splicing is crucial for gene expression regulation.
- The splicing factor ASF/SF2 (also known as SF2/ASF) can activate or repress splicing.
- Previous work showed ASF/SF2 inhibits adenovirus IIIa pre-mRNA splicing.
Purpose of the Study:
- To identify the specific domain of ASF/SF2 responsible for splicing repression.
- To investigate the role of the SWQDLKD motif in ASF/SF2-mediated repression.
- To determine the positional requirements for ASF/SF2's repressor activity.
Main Methods:
- Utilized MS2-ASF/SF2 fusion proteins to study splicing activity.
- Employed site-specific tethering assays to analyze domain function.
- Investigated splicing of adenovirus IIIa and rabbit beta-globin pre-mRNAs.
Main Results:
- The second RNA binding domain (RBD2) of ASF/SF2 is necessary and sufficient for splicing repression.
- A conserved SWQDLKD motif within RBD2 is essential for this repressive function.
- Repressor activity of RBD2 is position-dependent, inhibiting splicing when bound to an intron but not an exon.
- RBD2-mediated repression was observed in both adenovirus IIIa and beta-globin pre-mRNA systems.
Conclusions:
- ASF/SF2 possesses distinct domains mediating its enhancer and repressor functions.
- RBD2 and its SWQDLKD motif are key components of ASF/SF2's splicing repressor activity.
- The mechanism of repression involves specific interactions dependent on the binding location of RBD2 on pre-mRNA.