The SR protein family
© BioMed Central Ltd 2009
Published: 27 October 2009
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© BioMed Central Ltd 2009
Published: 27 October 2009
The processing of pre-mRNAs is a fundamental step required for the expression of most metazoan genes. Members of the family of serine/arginine (SR)-rich proteins are critical components of the machineries carrying out these essential processing events, highlighting their importance in maintaining efficient gene expression. SR proteins are characterized by their ability to interact simultaneously with RNA and other protein components via an RNA recognition motif (RRM) and through a domain rich in arginine and serine residues, the RS domain. Their functional roles in gene expression are surprisingly diverse, ranging from their classical involvement in constitutive and alternative pre-mRNA splicing to various post-splicing activities, including mRNA nuclear export, nonsense-mediated decay, and mRNA translation. These activities point up the importance of SR proteins during the regulation of mRNA metabolism.
Human genes encoding SR proteins
More recent genome-wide studies have identified several other RS-domain-containing proteins, most of wh ich are conserved in higher eukaryotes and function in pre-mRNA splicing or RNA metabolism . Because of differences in domain structure, lack of mAb104 recognition, or lack of a prototypical RRM, these proteins are referred to as SR-like or SR-related proteins. An extensive list of SR-related proteins and their functional roles in RNA metabolism was recently discussed .
All SR proteins share two main structural features: the RS domain and at least one RRM (Figure 1). For the majority of SR proteins with two RNA-binding domains, the second is a poor match to the RRM consensus and is referred to as an RRM homolog (RRMH). The only exception is 9G8, which contains an RRM and a zinc-knuckle domain that is thought to contact the RNA . In the cases where it has been determined, SR proteins have specific, yet degenerate RNA-binding specificities [18, 19]. The RS domains of SR proteins participate in protein interactions with a number of other RS-domain-containing splicing factors [20, 21]. These include other SR proteins, SR-related proteins , and components of the general splicing machinery [20, 21, 23–25]. Furthermore, the RS domain can function as a nuclear localization signal by mediating the interaction with the SR protein nuclear import receptor, transportin-SR [26–28].
In classic cases of alternative splicing, it has been shown that cis-acting RNA sequence elements, known as splicing enhancers, increase exon inclusion by serving as sites for recruitment of the splicing machinery - the spliceosome - which is a complex of ribonucleoprotein splicing factors, such as U1 and U2 small nuclear ribonucleoproteins (snRNPs), and their associated proteins, such as U2 auxiliary factor (U2AF), that splices exons together and releases the intron RNA. Splicing enhancers are usually located within the regulated exon, and are thus known as exonic splicing enhancers (ESEs) [41, 42]. ESEs are usually recognized by at least one member of the SR protein family and recruit the splicing machinery to the adjacent intron [9, 41, 42]. SR proteins act at several steps during the splicing reaction [4, 5, 8, 43–45] and require phosphorylation for efficient splice-site recognition and dephosphorylation for splicing catalysis [46, 47]. A number of SR protein kinases have been identified that specifically phosphorylate serine residues within the RS domain of SR proteins. These include SR protein kinase 1 (SRPK1) , Clk/Sty kinase , cdc2p34 , and topoisomerase . Surprisingly, binding sites for SR proteins are not only limited to alternatively spliced exons, but have also been verified for exons of constitutively spliced pre-mRNAs [52, 53]. It is therefore likely that SR proteins bind to sequences found in most, if not all, exons.
In addition to their exon-dependent functions, SR proteins have activities that do not require interaction with exon sequences . The role of the exon-independent function may be to promote the pairing of 5' and 3' splice sites across the intron or to facilitate the incorporation of the tri-snRNP U4/U6•U5 into the spliceosome  (Figure 5b). U4/U6•U5 is a complex of snRNPs that contains the splicing activity. Although the RRM of the SR protein is essential for its exon-independent activity , it is likely that SR proteins interact with the partially assembled spliceosome or the tri-snRNP through RS domain contacts.
One striking feature of SR proteins is their prevalent location within the pre-mRNA. In nearly all cases SR proteins have been found to interact with exonic sequences of the pre-mRNA. This is a surprising finding considering the fact that their relatively promiscuous binding specificity predicts that introns are littered with potential SR-protein-binding sites. The fact that SR proteins are mainly observed to bind within exonic sequences suggests that additional requirements need to be met for functional SR protein binding to the pre-mRNA. There are, however, some instances of SR proteins binding within the intron, where they function as negative regulators of splicing. The best-characterized example occurs during adenovirus infection . In this case, splicing is repressed by the binding of the SR protein SF2/ASF to an intronic repressor element located upstream of the 3' splice site branchpoint sequence in the adenovirus pre-mRNA. When bound to the repressor element, SF2/ASF prevents the recruitment of the snRNP U2 to the branchpoint sequence, thereby inactivating the 3' splice site (Figure 5c). Other studies have provided further support for the idea that SR proteins bound to introns generally interfere with the productive assembly of spliceosomes . These observations show that exonic splicing enhancers not only function in exon and splice-site recognition, but also act as barriers to prevent exon skipping.
SR proteins have been shown to influence translation either indirectly or directly. For example, the splicing activity of SF2/ASF influences alternative splicing of the pre-mRNA for the protein kinase MNK2, a kinase that regulates translation initiation. High levels of SF2/ASF promote the production of an MNK2 mRNA isoform that enhances cap-dependent translation, whereas low levels achieve the opposite . SF2/ASF is also involved in regulating translation directly. It has been shown to associate with polyribosome fractions isolated from cytoplasmic extracts and to enhance the translation efficiency of an ESE-containing luciferase reporter , apparently through mediating the recruitment of components of mTOR (mammalian target of rapamycin) signaling pathway (Figure 6b). As a result of this recruitment, a competitive inhibitor of cap-dependent translation is released .
Importantly, other SR proteins have also been reported to function in translation. SRp20 promotes translation of a viral RNA initiated at an internal ribosome entry site , and 9G8 increases translation efficiency of unspliced mRNA containing a constitutive transport element .
The functional characterization of SR proteins has revealed a wealth of information, placing SR proteins in the context of regulating constitutive and alternative pre-mRNA splicing, mediating efficient transport of mRNAs, and modu lat ing mRNA translation. As such, SR proteins could easily be mistaken for 'Jacks of all trades, masters of none' in mRNA metabolism. However, many studies have demon strated their essential presence in the cell, even with occasional redundancies. Given the enormous functional real estate this family of proteins covers, one is now pressed to find out how it is possible to transition these proteins between their involvements in the various steps of mRNA processing. Clearly, reversible modification, such as serine phosphorylation within the RS domain, is likely to be the ticket for SR protein functional flexibility . The challenge will be to determine the extent and dynamics of such modifications within SR proteins specifically involved in one of these activities and whether changes in modification lend support to the existence of an SR protein-modification code, perhaps similar in principle to the now well-described histone-modification code .
An old foe makes up another challenge: SR protein structure. For more than 15 years attempts have been made to obtain high-resolution structures of SR proteins. So far, these attempts have failed because of problems of low solubility and the likely heterogeneity of RS-domain modifications. As a first step towards gaining ground in this endeavor, clever modification approaches have been used to obtain a high-resolution structure of the SR protein RRM domain. This is a significant first step. However, the much more elusive RS domain is still the big prize, requiring further creative approaches and manipulations to freeze this seemingly unstructured domain in a conformation that permits its structural elucidation.
A different and experimentally challenging puzzle to address is the balance between the relatively low RNA-binding specificity exhibited by SR proteins and their usually specific functional impact. Given that SR proteins generally associate with exon sequences, it is likely that their interaction with the RNA is often aided by other factors. This suggestion is supported by the observation that at least 75% of the nucleotides in a typical human exon are part of sequence motifs that have been found to influence splicing, presumably through the binding of splicing activators, such as SR proteins, or the binding of splicing repressors, such as heterogeneous nuclear RNPs . For example, it is possible that the binding of SR proteins to pre-mRNA is only guaranteed if they are flanked by spliceosomal components such as U2 snRNP auxiliary factor or U1 snRNP, thus establishing a network of protein-protein and protein-RNA interactions. The establishment of such a network would then permit the stable association of SR proteins with many different target sequences, thus enabling SR proteins to recognize the thousands of different exons present in higher eukaryotes . Therefore, the relatively low RNA-binding specificity may have evolved to uphold the suitability of SR proteins to participate effectively in multiple RNA-processing events.
Clearly, SR proteins make up a family of regulators with important functions in RNA metabolism. This realization is exemplified when considering that changes in SR protein function or abundance have frequently been associated with human disease. For example, SF2/ASF has been described as a proto-oncogene  and the misregulation of alternative splicing has been associated with several types of cancer . While the involvement of SR proteins in various aspects of gene expression has been shown to be widespread, it would not be surprising if they emerge as critical players in other important biological processes.
We are grateful to the Hertel laboratory for helpful comments on the manuscript and Lin Li and Rozanne Sandri-Goldin for providing images of SR protein speckles. Our research is supported by NIH grant GM 62287 (KJH).