The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2

Amrein, Hubert; Hedley, Mary Lynne; Maniatis, Tom

doi:10.1016/0092-8674(94)90512-6

Cited by 175 publications

(182 citation statements)

References 52 publications

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“…Al though additional data are necessary to answer this ques tion, we favor the view that the RS domains interact directly. All known SR-interacting proteins themselves contain an RS domain, and where studied this region has been shown to be essential for interaction (Wu and Ma nia tis 1993;Amrein et al 1994;Kohtz et al 1994;Colwill et al 1996;Zhang and Wu 1996). The presence of an RS like domain is the only thing in common between these proteins, supporting the idea that binding involves direct contacts between RS domains.…”

Section: Discussionmentioning

confidence: 65%

Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.

Xiao¹,

Manley²

1997

Genes Dev.

357

355

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ASF/SF2 is a member of a conserved family of splicing factors known as SR proteins. These proteins, which are necessary for splicing in vitro, contain one or two amino-terminal RNP-type RNA-binding domains and an extensively phosphorylated carboxy-terminal region enriched in repeating Arg-Ser dipeptides (RS domains). Previous studies have suggested that RS domains participate in protein-protein interactions with other RS domain-containing proteins. Here we provide evidence that the RS domain of unphosphorylated recombinant ASF/SF2 is necessary, but not sufficient, for binding to the Ul snRNP-specific 70-kD protein (70K) in vitro. An apparent interaction of the isolated RS domain with 70K was observed if contaminating RNA was not removed, suggesting a nonspecific bridging between the basic RS domain, RNA, and 70K. In vitro phosphorylation of recombinant ASF/SF2 both significantly enhanced binding to 70K and also eliminated the RS domain-RNA interaction. Providing evidence that these interactions are relevant to splicing, ASF/SF2 can bind selectively to Ul snRNP in an RS domain-dependent, phosphorylation-enhanced manner. We also describe conditions that reveal for the first time a phosphorylation requirement for ASF/SF2 splicing activity in vitro.

show abstract

Section: Discussionmentioning

confidence: 65%

Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.

Xiao¹,

Manley²

1997

Genes Dev.

357

355

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show abstract

“…Furthermore, like other SR proteins, SF2/ASF influences splice-site selection in part by binding to enhancer sequences that are present frequently in intronic or exonic regions. Upon binding to these elements, the protein activates splicing by recruiting the splicing machinery to an adjacent splice site through protein-protein interactions (Wu and Maniatis 1993;Amrein et al 1994;Zuo and Manley 1994). Arabidopsis atSRp30 was a good candidate for being a splicing modulator, given its similarity to human SF2/ASF and its peculiar expression pattern.…”

Section: Atsrp30 Overexpression Influences Splice-site Choice In Sevementioning

confidence: 99%

“…Furthermore, an important activity of SF2/ASF and other SR proteins is the interaction with exonic enhancer sequences, which stimulate usage of an adjacent weak splice site (Sun et al 1993;Tian and Maniatis 1993;Staknis and Reed 1994;Ramchatesingh et al 1995;Tacke and Manley 1995;Lou et al 1996). The RNA-binding specificity of an SR protein is conferred by the RRM region and adjacent sequences (Cá ceres and Krainer 1993;Zuo and Manley 1993;Tacke and Manley 1995;Allain and Howe 1997;Tacke et al 1997); the various RS domains are responsible primarily for proteinprotein interactions, which are modulated by the phosphorylation status of these regions (Wu and Maniatis 1993;Amrein et al 1994;Kohtz et al 1994;Zuo and Maniatis 1996;Xiao and Manley 1997). In addition, it has been demonstrated that RS domains modulate the RNAbinding activity and subnuclear localization of SR proteins (Li and Bingham 1991;Hedley et al 1995;Cá ceres et al 1997).…”

mentioning

confidence: 99%

atSRp30, one of two SF2/ASF-like proteins from Arabidopsis thaliana, regulates splicing of specific plant genes

Lopato¹,

Kalyna²,

Dorner³

et al. 1999

Genes & Development

148

199

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SR proteins are nuclear phosphoproteins with a characteristic Ser/Arg-rich domain and one or two RNA recognition motifs. They are highly conserved in animals and plants and play important roles in spliceosome assembly and alternative splicing regulation. We have now isolated and partially sequenced a plant protein, which crossreacts with antibodies to human SR proteins. The sequence of the corresponding cDNA and genomic clones from Arabidopsis revealed a protein, atSRp30, with strong similarity to the human SR protein SF2/ASF and to atSRp34/SR1, a previously identified SR protein, indicating that plants possess two SF2/ASF-like proteins. atSRp30 expresses alternatively spliced mRNA isoforms that are expressed differentially in various organs and during development. Overexpression of atSRp30 via a strong constitutive promoter resulted in changes in alternative splicing of several endogenous plant genes, including atSRp30 itself. Interestingly, atSRp30 overexpression resulted in a pronounced down-regulation of endogenous mRNA encoding full-length atSRp34/SR1 protein. Transgenic plants overexpressing atSRp30 showed morphological and developmental changes affecting mostly developmental phase transitions. atSRp30-and atSRp34/ SR1-promoter-GUS constructs exhibited complementary expression patterns during early seedling development and root formation, with overlapping expression in floral tissues. The results of the structural and expression analyses of both genes suggest that atSRp34/SR1 acts as a general splicing factor, whereas atSRp30 functions as a specific splicing modulator.

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“…One potential explanation stems from the domain structure of TRA2, one of the protein components of the enhancer complex (31). TRA2 contains two separate RS domains, one on the N terminus and the other on the C terminus (62). It is conceivable that each of the bipartite RS domains is responsible for the recruitment of specific factors to either the 5Ј-or 3Ј-splice site (Fig.…”

Section: Discussionmentioning

confidence: 99%

Enhancer-dependent 5′-Splice Site Control of fruitless Pre-mRNA Splicing

Lam

Bakshi

Ekinci

et al. 2003

Journal of Biological Chemistry

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The Drosophila fruitless (fru) gene encodes a transcription factor that essentially regulates all aspects of male courtship behavior. The use of alternative 5 -splice sites generates fru isoforms that determine gender-appropriate sexual behaviors. Alternative splicing of fru is regulated by TRA and TRA2 and depends on an exonic splicing enhancer (fruRE) consisting of three 13-nucleotide repeat elements, nearly identical to those that regulate alternative sex-specific 3 -splice site choice in the doublesex (dsx) gene. dsx has provided a useful model system to investigate the mechanisms of enhancer-dependent 3 -splice site choice. However, little is known about enhancer-dependent regulation of alternative 5 -splice sites. The mechanisms of this process were investigated using an in vitro system in which recombinant TRA/TRA2 could activate the female-specific 5 -splice site of fru. Mutational analysis demonstrated that one 13-nucleotide repeat element within the fruRE is required and sufficient to activate the regulated femalespecific splice site. As was established for dsx, the fruRE can be replaced by a short element encompassing tandem 13-nucleotide repeat elements, by heterologous splicing enhancers, and by artificially tethering a splicing activator to the pre-mRNA. Complementation experiments showed that Ser/Arg-rich proteins facilitate enhancer-dependent 5 -splice site activation. We conclude that splicing enhancers function similarly in activating regulated 5 -and 3 -splice sites. These results suggest that exonic splicing enhancers recruit multiple spliceosomal components required for the initial recognition of 5 -and 3 -splice sites.Alternative pre-mRNA splicing is commonly used to regulate the expression of genes and to enrich the proteomic diversity of higher eukaryotic organisms (1-4). Current estimates suggest that ϳ60% of human genes are alternatively spliced (5), sometimes leading to thousands of different mRNA isoforms from a single gene. Numerous examples describe how alternative splicing regulates gene expression in humans, but the mechanisms involved are understood in only a few cases (6 -17). In contrast, the use of genetics in Drosophila has aided in the identification of proteins that regulate alternative splicing. In particular, the genes involved in Drosophila sex determination have become prototypes for the study of positive and negative control of alternative splicing (18). The best characterized gene is the doublesex (dsx) 1 gene, in which the use of two alternative 3Ј-splice sites leads to the expression of male-or female-specific gene products that initiate gender-specific sexual differentiation. Female-specific splicing of dsx requires the production of two proteins, Transformer (TRA) and Transformer2 (TRA2), and an exonic splicing enhancer (ESE) element located within the untranslated region of the fourth exon (19 -21). Both TRA and TRA2 contain Arg/Ser-rich (RS) domains, protein interaction domains characteristic of the Ser/Arg-rich (SR) family of essential splicing factors. SR prot...

show abstract

The role of specific protein-RNA and protein-protein interactions in positive and negative control of pre-mRNA splicing by Transformer 2

Cited by 175 publications

References 52 publications

Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.

Phosphorylation of the ASF/SF2 RS domain affects both protein-protein and protein-RNA interactions and is necessary for splicing.

atSRp30, one of two SF2/ASF-like proteins from Arabidopsis thaliana, regulates splicing of specific plant genes

Enhancer-dependent 5′-Splice Site Control of fruitless Pre-mRNA Splicing

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