s s Brenton R Graveley, University of Connecticut Health Center, Farmington, Connecticut, USA Klemens J Hertel, University of California Irvine, Irvine, California, USA Members of the serine/arginine-rich () protein family have several important functions in mrna biogenesis. proteins are essential splicing factors that also participate in the regulation of alternative splicing and the export of mrnas from the nucleus to the cytoplasm. Advanced article The Family Article contents Roles in Pre-mRNA Processing Splicing Repression Role of Phosphorylation in Regulating Activities Localization and Roles in mrna Export doi: 10.1038/npg.els.0005039 The Family Members of the serine/arginine-rich () protein family function in both constitutive and alternative splicing, and have a role in export of messenger ribonucleic acid (mrna). In humans, the protein family is encoded by nine splicing factor, arginine/ serine-rich genes, designated SF1, SF2, SF3, SF4, SF5, SF6, SF7, SF9 and SF11 (Table 1 and Figure 1). All nine members of the protein family, SF2/ASF, SC35, p20, p40, p55, p75, p30c, 9G8 and p54, have a common structural organization (Figure 2). Each protein contains either one or two amino (N)-terminal ribonucleic acid (RNA)-binding domains and a variable-length arginine/serine-rich () domain at their carboxy (C)-terminus that functions as a protein interaction domain. In addition to their structural similarities, all members of the protein family share several biochemical properties. All proteins contain a phosphoepitope within the domain that is recognized by the monoclonal antibody mab104; individual proteins can complement HeLa cell S100 (the supernatant of a 100,000g spin of a cytoplasmic extract) extracts deficient in proteins; and all proteins can be precipitated in 20 mm MgCl 2. (See Alternative Processing: Neuronal Nitric Oxide Synthase; RNA Processing; Spliceosome; Splicing of pre-mrna; Trans Splicing.) Table 1 Human genes encoding proteins Gene protein Chromosomal location SF1 SF2/ASF/p30a 17q21.3 q22 SF2 SC35/p30b 17q25.1 SF3 p20 6p21.31 SF4 p75 1p35.3 SF5 p40 14q24.2 SF6 p55 20q13.11 SF7 9G8 2p22.1 SF9 p30c 12q24.23 SF11 p54 1p31.1 proteins are highly conserved and exist in all metazoan species (Zahler et al., 1992) as well as some lower eukaryotes such as Schizosaccharomyces pombe. But proteins are not present in all eukaryotes; they are apparently missing from Saccharomyces cerevisiae. As a general rule, the species-specific presence of proteins correlates with the presence of domains within other components of the general splicing machinery. (See Alternative Splicing: Evolution.) All proteins contain one RNA-binding domain of the RNA recognition motif () type. For most proteins with two RNA-binding domains, the second is a poor match to the consensus and is therefore referred to as an homolog (H). The only exception is 9G8, which, in addition to one, contains a zinc-knuckle that is thought to contact RNA. In the cases where it has been determined, proteins have specific but rather degenerate RNA-binding specificities (Liu et al., 1998); however, tight RNA binding does not necessarily correlate with maximal activity. (See RNAbinding s: Regulation of mrna Splicing, Export and Decay.) The domains of proteins participate in protein interactions with several other -domaincontaining splicing factors (Wu and Maniatis, 1993; Kohtz et al., 1994). These include other proteins, a second class of proteins known as the -related proteins (rps) and, most importantly, constituents of the general splicing machinery such as the - (relative molecular mass, M r, 70 000) component of the small nuclear ribonuclear protein (snrnp); U2AF 35 ; and possibly the -domain-containing 100K component of U5 snrnp and the 27K, 65K and 110K components of the U4/U6 U5 tri-snrnp. Although domains have been shown to be dispensable for the ability of proteins to promote splice-site switching in certain pre-mrnas and for the splicing of some precursor mrnas (pre-mrnas) that contain strong splice sites, domains are required for most activities of proteins. ENCYCLOPEDIA OF LIFE SCIENCES & 2005, John Wiley & Sons, Ltd. www.els.net 1
s SF1 (SF2/ASF) 2412 bp SF2 (SC35) 3193 bp SF3 (p20) 9043 bp SF4 (p75) 33 692 bp SF5 (p40) 4824 bp SF6 (p55) 3406 bp SF7 (9G8) 7739 bp SF9 (p30c) 7994 bp SF11 (p54) 30 389 bp Figure 1 Organization of the human genes that encode proteins. The exon intron organization of each human protein gene is shown. Noncoding portions of each exon are represented by white boxes, coding portions of exons are indicated by black boxes. Alternatively spliced exons are indicated in gray. The splicing of the alternative exons is autoregulated by the protein encoded by the gene. The exons and introns for each gene are drawn to scale and the size of each gene is indicated. p20 SC35 p54 Roles in Pre-mRNA Processing The best-characterized activities of proteins are those that involve their binding to exon sequences and SF2/ASF H p30c H p40 H p55 H p75 H 9G8 Zn Figure 2 The human protein family. The structural organization of the nine human proteins is shown. : RNA recognition motif; H: homology; Zn: zinc-knuckle; : arginine/serine-rich domain. are therefore referred to as exon-dependent. It is now well documented that proteins bind to sequences within regulated exons and thereby activate exon inclusion. These binding sites have been designated exonic splicing enhancers (ESEs). When bound to an ESE, proteins can enhance the recognition of suboptimal upstream 3 0 splice sites, possibly by directly recruiting the essential splicing factor U2AF to the pyrimidine tract (Zuo and Maniatis, 1996; Figure 3a). However, proteins may also recruit components of the splicing machinery through interactions with other essential splicing factors or by relieving the inhibitory effect of splicing repressor elements (Figure 3a). (See Exonic Splicing Enhancers.) In addition, proteins regulate choice of 5 0 splice site when bound to ESEs that are positioned upstream of 5 0 splice sites (Ryner et al., 1996). Analogous to the U2AF recruitment model for activation of 3 0 splice sites, proteins may recruit snrnp to the regulated 5 0 splice site by directly interacting with the - component of snrnp (Figure 3b). In support of this view, the protein SF2/ASF has been shown to recruit snrnp to a 5 0 splice site containing RNA (Kohtz et al., 1994). But further experiments are required to delineate decisively the mechanism of -protein-dependent 5 0 splice-site activation. (See Splice Sites.) 2
s (a) m160 Inhibitor snrnp U2 snrnp U2AF U2AF65 35 Exon 1 A Py AG ESE Exon 2 ESS 27K U4/U6 U5 tri-snrnp 100K (b) ESE Exon 1 snrnp (c) U2AF U2AF65 35 Py AG ESE Exon snrnp Figure 3 Exon-dependent functions of proteins. (a) ESE-bound proteins may function by activating upstream 3 0 splice sites. This can be achieved through recruitment of the general splicing factor U2AF, through interactions with the splicing coactivator m160, or by antagonizing the activity of splicing inhibitors. (b) Alternative 5 0 splice sites may be activated on the recruitment of snrnp by ESE-bound proteins. (c) proteins may function in constitutive splicing by simultaneously interacting with U2AF bound to the upstream 3 0 splice site and snrnp bound to the downstream 5 0 splice site. Py: pyrimidine tract. Surprisingly, proteins promote constitutive splicing in an exon-dependent manner (Mayeda et al., 1999). It is thought that proteins bound to constitutively spliced exons simultaneously interact with U2AF 35 that is bound to the upstream 3 0 splice site and with - of snrnp that is bound to the downstream 5 0 splice site (Wu and Maniatis, 1993; Figure 3c). These cross-exon interactions most probably facilitate exon definition and thus promote incorporation of the exon into the resulting mrna. These observations suggest that proteins are essential not only for the recognition of alternatively spliced exons but also for the definition of constitutively spliced exons. The degenerate RNA-binding specificities of proteins may ensure that at least one member of the protein family can bind to each constitutively spliced exon. In addition to their exon-dependent functions, proteins have activities that do not require interactions with exon sequences. A potentially important exonindependent function is the pairing of 5 0 and 3 0 splice sites (Figure 4). Similar to the exon-bridging model, this activity requires simultaneous interactions with - and U2AF 35 (Wu and Maniatis, 1993). In this case, however, the interactions span across the intron and thus allow juxtaposition of the splice sites that are to be joined together. proteins have also been shown to facilitate the incorporation of the U4/U6 U5 tri-snrnp into the spliceosome (Figure 4). This activity probably involves interactions between snrnp U2 snrnp U2AF 35 U2AF 65 Exon 1 A Py AG Exon 2 Figure 4 Exon-independent functions of proteins. proteins have two exon-independent functions. First, proteins facilitate splice-site pairing by simultaneously interacting with snrnp and U2AF across the intron. Second, proteins in the partially assembled spliceosome are involved in recruiting the U4/U6 U5 tri-snrnp. an protein within the partially assembled spliceosome and an -domain-containing component of the tri-snrnp such as U5-100K or the 27K, 65K or 110K proteins of the tri-snrnp complex. Splicing Repression In certain situations, proteins can also function as negative splicing regulators. The best-characterized example of this occurs during adenovirus infection (Kanopka et al., 1996), where splicing is repressed by the binding of the protein SF2/ASF to an intronic repressor element located upstream of the branch site of a regulated 3 0 splice site in the adenovirus premrna. When bound to the repressor element, SF2/ ASF prevents the interaction of U2 snrnp with the branch site, which inactivates the 3 0 splice site. Because proteins are thought to interact predominantly with exonic sequences, binding to intronic sequences may be a general mechanism of splicing repression. Role of Phosphorylation in Regulating Activities All activities of proteins are modulated by phosphorylation within the domain. Whereas both hypo- and hyperphosphorylated proteins are inactive in splicing assays (Sanford and Bruzik, 1999), moderately phosphorylated proteins can 3
s participate in the splicing reaction. The phosphorylation level of proteins, and thus their activity, is regulated throughout development (Sanford and Bruzik, 1999) and during the course of a single round of intron removal. These observations suggest that protein phosphorylation and dephosphorylation are highly dynamic and essential aspects of their biological activities. Several kinases have been identified that phosphorylate proteins, including protein kinases 1 and 2 (PK1 and PK2), Clk/Sty and DNA topoisomerase I. As the domain provides a rather extensive platform for phosphorylation, the extent and specificity of modifications required for protein activity are only beginning to be understood. Localization and Roles in mrna Export Virtually all proteins involved in pre-mrna splicing, including the proteins, are enriched in numerous nuclear compartments called speckles, which consist of two distinct structures: interchromatin granule clusters (IGCs) of 20 25 nm in diameter, which act as storage or reassembly sites for pre-mrna splicing factors; and perichromatin fibrils (PFs) of *5 nm in diameter, which form the site of actively transcribing genes and cotranscriptional splicing. The proteins are one prominent component of nuclear speckles, and biochemical analyses have indicated that domains are responsible for targeting the proteins to speckles. As the nuclear organization of proteins is dynamic, proteins are recruited from the IGC storage clusters to the site of cotranscriptional splicing (PFs). Notably, both the RNA-binding domains and the domains are required for the recruitment of proteins from IGCs to PFs, as is phosphorylation of the domain. Some proteins SF2/ASF, p20 and 9G8 shuttle continuously between the nucleus and the cytoplasm. The movement of these proteins requires an appropriately phosphorylated domain and the RNA-binding domain. These unique intracellular transport properties suggest that a subset of proteins function not only in pre-mrna processing but also in mrna export. In fact, the proteins 9G8 and p20 promote nuclear export of the intronless histone H2a mrna in mammalian cells and Xenopus oocytes (Huang and Steitz, 2001) by binding to a 22-nucleotide sequence within the H2a mrna. In addition, the S. cerevisiae protein Npl3p a protein that is closely related to proteins assists (a) Intronless mrna Nucleus 5' (b) Pre-mRNA 3' 5' 3' hnrnp particle 5' 3' 5' 3' Export machinery? ALY/REF ALY/REF hgle2 TAP hgle p15 hdpb5 Conserved export machinery Figure 5 Models of mrna export. (a) Intronless mrnas such as H2a contain high-affinity binding sites (black box) for proteins that shuttle continuously between the nucleus and the cytoplasm. Association of shuttling proteins with the intronless mrna leads to nuclear export via an unknown pathway. (b) Before splicing, nascent pre-mrnas are coated by hnrnps (heterogeneous nuclear ribonucleoproteins). During spliceosomal assembly, proteins bind to each exon with sufficient affinity to replace hnrnps effectively. During intron removal, conserved export factors, in particular ALY/REF, are recruited to the exons. Because of their association with exonic sequences throughout the splicing cycle, proteins may assist in linking pre-mrna splicing to mrna export either directly by interacting with the export machinery or indirectly by inhibiting the association of nuclear retention factors such as hnrnps with the mrna. The spliced mrna is then exported from the nucleus via a pathway involving TAP/p15, Gle, Gle2 or Dbp5 (nuclear export factors). 4
s in mrna export in yeast. Once again, phosphorylation of specific serine residues within the domain seems to control the functional efficiency of mrna export mediated by Npl3p. It is not known yet whether proteins also promote the export of spliced mrnas; however, given the fact that proteins are essential for splicing, remain associated with the spliced mrna after intron removal, and shuttle between the nucleus and the cytoplasm, it seems highly likely that proteins have an important role in the export of spliced mrnas. In the proposed model (Figure 5), proteins participate in the export of mrnas not only through interactions with specific cis-acting export elements of intronless mrnas, but also by assisting the functions of other export factors through stable associations with spliced mrnas after intron removal. (See mrna Export.) See also Exonic Splicing Enhancers Posttranscriptional Processing Splicing of pre-mrna References Huang Y and Steitz JA (2001) Splicing factors p20 and 9G8 promote the nucleocytoplasmic export of mrna. Molecular Cell 7: 899 905. Kanopka A, Muhlemann O and Akusjarvi G (1996) Inhibition by proteins of splicing of a regulated adenovirus pre-mrna. Nature 381: 535 538. Kohtz JD, Jamison SF, Will CL, et al. (1994) protein interactions and 5 0 splice site recognition in mammalian mrna precursors. Nature 368: 119 124. Liu HX, Zhang M and Krainer AR (1998) Identification of functional exonic splicing enhancer motifs recognized by individual proteins. Genes and Development 12: 1998 2012. Mayeda A, Screaton GR, Chandler SD, Fu XD and Krainer AR (1999) Substrate specificities of proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mrna exonic elements. Molecular and Cellular Biology 19: 1853 1863. Ryner LC, Goodwin SF, Castrillon DH, et al. (1996) Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. Cell 87: 1079 1089. Sanford JR and Bruzik JP (1999) Developmental regulation of protein phosphorylation and activity. Genes and Development 13: 1513 1518. Wu JY and Maniatis T (1993) Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell 75: 1061 1070. Zahler AM, Lane WS, Stolk JA and Roth MB (1992) proteins: a conserved family of pre-mrna splicing factors. Genes and Development 6: 837 847. Zuo P and Maniatis T (1996) The splicing factor U2AF 35 mediates critical protein protein interactions in constitutive and enhancerdependent splicing. Genes and Development 10: 1356 1368. Further Reading Blencowe BJ, Bowman JAL, McCracken S and Ronina E (1999) -related proteins and the processing of messenger RNA precursors. Biochemistry and Cell Biology 77: 277 291. Caceres JF, Screaton GR and Krainer AR (1998) A specific subset of proteins shuttles continuously between the nucleus and the cytoplasm. Genes and Development 12: 55 66. Graveley BR (2000) Sorting out the complexity of protein functions. RNA 6: 1197 1211. Kan JLC and Green MR (1999) Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor. Genes and Development 13: 462 471. Li Y and Blencowe BJ (1999) Distinct factor requirements for exonic splicing enhancer function and binding of U2AF to the polypyrimidine tract. Journal of Biological Chemistry 274: 35074 35079. Prasad J, Colwill K, Pawson T and Manley JL (1999) The protein kinase Clk/Sty directly modulates protein activity: both hyper- and hypophosphorylation inhibit splicing. Molecular and Cellular Biology 19: 6991 7000. Reed R and Magni K (2001) A new view of mrna export: separating the wheat from the chaff. Nature Cell Biology 3: E201 E204. Roscigno RF and Garcia-Blanco MA (1995) proteins escort the U4/U6 U5 tri-snrnp to the spliceosome. RNA 1: 692 706. Schaal TD and Maniatis T (1999) Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mrna. Molecular and Cellular Biology 19: 261 273. Spector DL (1993) Macromolecular domains within the cell nucleus. Annual Reviews in Cell Biology 9: 265 315. Web Links Splicing factor, arginine/serine-rich 1 (SF1); LocusID: 6426. Locus Link: http://www.ncbi.nlm.nih.gov/locuslink/locrpt.cgi?l=6426 Splicing factor, arginine/serine-rich 1 (SF1); MIM number: 600812. OMIM: http://www.ncbi.nlm.nih.gov/htbin-post/omim/ dispmim?600812 5