hnRNP A1 and the SR Proteins ASF/SF2 and SC35 Have Antagonistic Functions in Splicing of β-Tropomyosin Exon 6B

Abstract

Mutually exclusive splicing of exons 6A and 6B from the chicken ␤-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them. Splicing is the process by which introns from premessenger RNAs are removed in eukaryotes. Pre-mRNA splicing takes place within the spliceosome, which is a large, highly dynamic complex composed of four small ribonucleoprotein particles (snRNP 1 U1, U2, U4/U6, and U5) and multiple non-snRNP factors (1, 2). One of the most intriguing questions that remains in RNA splicing is how the 5Ј and 3Ј splice sites are selected and paired together within large RNA sequences (3). This question takes on particular importance in alternative splicing, where the selection of certain splice sites is modulated depending on the developmental stage, on tissue differentiation, or on metabolic changes of the cells (3). Numerous studies have demonstrated that regulatory sequences within the pre-mRNA that lie outside the splicing signals play a crucial role in controlling the choice of splicing sites in a given cellular context (reviewed in Refs. 4 and 5).Among these sequences are the splicing enhancers. These elements are found in a wide variety of metazoan pre-mRNAs, either within exons or introns. Purine-rich splicing enhancers (known as ESE) are a well characterized class of exonic splicing enhancers that mostly interact with specific subsets of SR proteins (reviewed in Refs. 6 and 7). SR proteins belong to a family of essential splicing factors that are highly conserved between Drosophila and mammals and that are involved in both constitutive and regulated splicing events (reviewed in Refs. 8 and 9). It has been proposed that the function of SR proteins is to stimulate the recognition of weak upstream 3Ј splice sites, by recruiting U2AF 65/35 , or to f...

Serine/arginine-rich protein-dependent suppression of exon skipping by exonic splicing enhancers

“…Computational analyses indicate that sequence motifs for ESEs are more frequent in exons than in introns (28). However, there are a number of examples in which SR protein binding sites are located within the intron and can function to promote splicing of upstream 5Ј splice sites (8,(29)(30)(31)(32). At the same time, negative intronic regulatory elements with which SR proteins interact have also been found (20,29,33,34).…”

Dichotomous splicing signals in exon flanks

“…This reiteration is suggestive of cooperative protein binding within a flank. Some of these particular sequences resemble binding sites of known factors, e.g., CTCTCT in clcn7-3 has been seen in core polypyrimidine-binding proteinbinding sites (Southby et al 1999;Carstens et al 2000), AGGG in thbs4-12 is part of several hnRNP A1-binding sites (Burd and Dreyfuss 1994;Boerkoel et al 1995;Abdul-Manan et al 1996;Expert-Bezancon et al 2004), GGGA in thbs4-12 is the core element of the hnRNP H family (Caputi and Zahler 2001) and sequences bearing G-triplets can potentially bind U1 snRNP (McCullough and Berget 2000). We also compared the effect of flank deletions on winner content and phenotype.…”

Hide and Go Seek: Activation of the Secretory-Specific Poly (A) Site of Igh by Transcription Elongation Factors

“…The balance between these has been shown to control many genes, reviewed in (House and Lynch 2008). Changes in the levels of the splicing factors in various B cell types (Bruce, Dingle et al 2003) and during development have been shown to alter splicing patterns (Expert-Bezancon, Sureau et al 2004). For, example, SRp20 plays an important role in alternative splicing of the ED1 exon (de la Mata and Kornblihtt 2006).…”