Broad Specificity of SR (Serine⧸Arginine) Proteins in the Regulation of Alternative Splicing of Pre-Messenger RNA

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Abstract

Alternative splicing of pre-messenger RNA (pre-mRNA) is a highly regulated process that allows expansion of the potential of expression of the genome in higher eukaryotes and involves many factors. Among them, the family of the serine- and arginine-rich proteins (SR proteins) plays a pivotal role: it has essential functions during spliceosome assembly and also interacts with RNA regulatory sequences on the pre-mRNA as well as with multiple cofactors. Collectively, SR proteins, because of their capacity to recognize multiple RNA sequences with a broad specificity, are at the heart of the regulation pathways that lead to the choice of alternative splice sites. Moreover, a growing body of evidence shows that the mechanisms of splicing regulation are not limited to the basic involvement of cis- and trans-acting factors at the pre-mRNA level, but result from intricate pathways, initiated sometimes by stimuli that are external to the cell and integrate SR proteins (and other factors) within an extremely sophisticated network of molecular machines associated with one another. This review focuses on the molecular aspects of the functions of SR proteins. In particular, we discuss the different ways in which SR proteins manage to achieve a high level of specificity in splicing regulation, even though they are also involved in the constitutive reaction.

Introduction

More than 25 years after the discovery of introns in higher eukaryotes, the splicing machinery still appears as extraordinary as ever. The pairs of splice sites are recognized and selected with a high fidelity within a multitude of potential, but incorrect, sites distributed along the RNA chains. This outstanding degree of precision is achieved through the involvement of at least 100 factors, associated or not with U small nuclear RNA (snRNA) in the form of small nuclear ribonucleoprotein particles (U snRNPs) and that cooperate in the nucleus at the transcription sites to direct the assembly of functional splicing complexes.

Classically, spliceosome assembly is described in vitro as an ordered process starting with the formation of an early complex (complex E) in which the 5′ splice site is recognized by the U1 snRNP through RNA–RNA interactions and by a set of cofactors. The 3′ splice site and the branch site are recognized cooperatively by the U2 snRNP auxiliary factor (U2AF) and the branch-point-binding protein, establishing a crosstalk between the 5′ and the 3′ splice sites through the intron and⧸or the exon at an early stage (1). In a second step, the U2 snRNP binds to the branch site in an ATP-dependent manner, through RNA–RNA interactions, to form complex A. The subsequent binding of the U4⧸U6.U5 tri-snRNP leads to the formation of complex B and is accompanied by a strong remodeling of RNA–protein, RNA–RNA, and protein–protein interactions to give rise to catalytically active spliceosomes.

Among all the proteins required throughout spliceosome assembly 1, 2, one homogeneous family of polypeptides, called SR proteins (for serine⧸arginine) plays a crucial role in the early steps of splice site selection and also later in the process 3, 4. SR proteins have a modular structure that consists of an RNA-binding region, of the RRM-type, in their amino-terminal (N-terminal) part and an RS domain (rich in dipeptides arginine⧸serine) in their carboxyl-terminal (C-terminal) part. They bind to the pre-mRNA very early, owing to their general RNA-binding properties, and they are required for the formation of complex E. In this aspect of their functions, SR proteins are thought to be largely redundant.

Another level of complexity in splicing is the occurrence of alternative splicing, which could affect 30 to 50% of the genes in higher eukaryotes 5, 6. Alternative splicing is regulated, either in a tissue-specific manner, during embryonic development or cellular differentiation, or under certain physiological conditions (7). This makes the splicing machinery even more fascinating. This regulation requires specific cis-elements present within the regions of the pre-mRNA subjected to alternative splicing. Cis-elements are targeted by trans-acting factors that activate or repress the use of splice signals located in the vicinity. Several recent reviews provide detailed information about the biochemical mechanisms of alternative splicing, the analysis of cis-acting elements, and the consequences of alternative splicing on proteomic diversity 8, 9, 10, 11.

Because of their implication in the early recognition of splice sites, SR proteins represent ideal factors to regulate alternative splicing 3, 4, 12. However, in this process, the RNA-binding specificity of each SR protein becomes essential for its activity. Thus, an extensive analysis of the RNA-binding properties of SR proteins is required to understand the molecular basis of the alternative splicing models in which SR proteins are specifically involved.

Although the specificity of RNA recognition appears to be one of the major determinants underlying the involvement of SR proteins in splicing regulation, only one short but excellent review (12) was specifically dedicated to this aspect of their functions. We therefore update these data in this review and we also summarize the current knowledge about the different pathways by which SR proteins are regulated and their consequences on the control of alternative splicing.

Section snippets

Multiple Ways to Identify SR Proteins

Detailing how the different SR proteins were discovered highlights some of their intrinsic properties. Since 1991, 10 SR proteins, with sizes ranging from 20 to 75 kDa, have been identified in mammals (Fig. 1), and they are relatively well conserved in the animal and plant kingdoms (4). The prototypical SR protein ASF⧸SF2 was discovered independently by two groups using different approaches. SF2 was identified as a factor that restores a full splicing activity in the presence of an incompetent

A General Search for Splicing Enhancer Sequences Specific for Individual SR Proteins

The first evidence for divergent RNA-binding properties came from commitment experiments showing that SC35, ASF⧸SF2, and SRp20 differ in their ability to commit specific pre-mRNA to the splicing pathway (95). This result was in agreement with the observation that individual SR proteins function differently in alternative splicing (96). At the same time came the identification of the first exonic splicing enhancers (ESE), such as purine-rich motifs located in the mouse IgM μ gene (97), in the

Role of SR Cofactors in the Modulation of SR-Protein-Dependent Regulation of Alternative Splicing

Besides the primary function of SR proteins in the regulation of alternative splicing, a large number of studies pointed out the important role that various cofactors play in several regulatory pathways. This section describes how the most characterized of these cofactors influence the activity of SR proteins, for example, by establishing interactions with other components of the splicing machinery or by modulating the specificity of interactions of SR proteins with their target sites on

Regulation of the Activity of SR Proteins

The main question arising from the basic observation that SR proteins regulate alternative splicing is how this regulation is achieved in vivo in the context of a specific cell type or in response to extracellular signals. In the last decade, a large number of reports have shown that SR proteins are targeted at multiple levels to ensure that the appropriate factors are present at the right time and at the right place to accomplish their functions (Fig. 3).

Conclusions and Perspectives

In this review, we summarized how SR proteins are involved in the regulation of alternative splicing events. The identification of the target RNA sequences recognized by specific SR proteins (and other factors), either by in vitro selection or by analysis of natural substrates, has shown that SR proteins have a broad specificity in RNA recognition. We also presented the molecular mechanisms that underlie the activity of these key regulatory factors. This variety of mechanisms contributes to

Acknowledgements

We thank all past and present members of our team, especially Renata Gattoni, Georges Hildwein, and Liliane Kister, as well as all our colleagues at the IGBMC, for their contribution to this work. We are grateful to Renata Gattoni, Nicolas Charlet-Berguerand, and Julian Venables for helpful comments, suggestions, and corrections on the manuscript. We thank Jim Bruzik, Julian Venables, and David Elliott for communicating results prior to publication. Our work was supported by the Centre National

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