The removal of non-coding introns and the joining of coding exons via splicing, an essential step in the eukaryotic pre-mRNA processing pathway, requires accurate splice site selection by the spliceosome. Initial recognition of the 5' exon/intron boundary is achieved via base pairing with the U1 snRNA component of the U1 snRNP (see U1A snRNP also known as sans fille), while U2AF (U2 snRNP Auxiliary Factor) is the first factor bound to the 3' splice site (Ruskin, 1988). Biochemical complementation assays have demonstrated that U2AF is required for the subsequent ATP-dependent association of U2 snRNP with pre-mRNA branchpoints (Ruskin, 1988; Valcarcel, 1996). Purified U2AF is a heterodimer composed of large and small subunits in humans (Zamore, 1989), Drosophila melanogaster (Kanaar, 1993; Rudner, 1996), Caenorhabditis elegans (Zorio, 1997; Zorio, 1999a) and Schizosaccharomyces pombe. Functional conservation of this splicing factor is evidenced by (1) the restoration of splicing activity to U2AF-depleted HeLa nuclear splicing extracts via addition of Drosophila U2AF large subunit (dmU2AF^LG; Zamore, 1991) and (2) the ability of human U2AF³⁵ (hsU2AF^SM: see U2 small nuclear riboprotein auxiliary factor 35) to restore growth to a S. pombe strain lacking the small subunit (Webb, 2004a; Webb 2004b and references therein).

The structural domains of U2AF are also conserved except in Saccharomyces cerevisiae, where the large subunit is highly divergent and the small subunit is absent entirely. The small subunit of U2AF consists of two zinc-binding domains (ZBDs) surrounding a central pseudo-RNA recognition motif (˙RRM; Rudner, 1998b), also known as a PUMP (PUF60/U2AF/MUD2 Protein-protein interaction) domain (Page-McCaw, 1999) or a UHM (U2AF Homology Motif; Kielkopf, 2004). These are both highly conserved between S. pombe and humans, followed by a C-terminal domain that consists of RS or RS/glycine repeats in metazoan orthologues that are not present in the fission yeast protein. The three conserved domains including both ZBDs and the ˙RRM of S. pombe U2AF small subunit (spU2AF^SM) contribute to RNA binding and are essential for function in vivo, while the more divergent C-terminal domain is dispensable (Webb, 2004a). A comparable domain ablation analysis in vivo has not been carried out for the U2AF large subunit, which consists of an N-terminal RS domain, a linker region, two classical RRMs (RNA Recognition Motifs) and a ˙RRM (Zamore, 1992). Such an analysis would complement extensive biochemical data demonstrating that this subunit is the major contributor to RNA binding (Rudner, 1998a; Wu, 1999; Kielkopf, 2001) and interacts with multiple protein partners implicated in splicing (Webb 2004b and references therein).

RNA binding assays (Zamore, 1992; Rudner, 1998a) and in vitro selection studies (Singh, 1995; Wu, 1999; Banerjee, 2004) demonstrated that the large subunit of U2AF (U2 small nuclear riboprotein auxiliary factor 50 or U2af50 in Drosophila) interacts with the 3' polypyrimidine tract, while the small subunit (U2 small nuclear riboprotein auxiliary factor 38 or U2af38 in Drosophila) functions in recognition of the 3' AG dinucleotide (Wu, 1999; Zorio, 1999b; Merendino, 1999). The bipartite nature of the RNA target sequences for the two subunits, in combination with biochemical complementation data demonstrating that addition of the human large subunit (hsU2AF^LG) alone to U2AF-depleted HeLa nuclear extracts can rescue splicing of substrates that contain long polypyrimidine tracts (Wu, 1999; Guth, 2001), led to a widely accepted model for 3' splice site recognition by U2AF. The central tenet of this model is that the binding energy contributed by the small subunit/AG interaction is essential only for introns with less extensive polypyrimidine tracts (reviewed in Moore, 2000), consistent with earlier splicing assays of mutant human pre-mRNAs in vitro, which indicated that the requirement for a 3' AG to proceed through the first step of splicing (AG-dependence) could be eliminated by expanding the polypyrimidine tract (Reed, 1989). In S. pombe, mutating the terminal AG dinucleotide prevents the first transesterification reaction for all three introns examined (Romfo, 1997), as well as for a subset of mammalian premRNAs (Reed, 1989; Wu, 1999). However, doubling the length of the polypyrimidine tract in two different fission yeast introns did not render the AG dinucleotide dispensable in vivo (Romfo, 1997), providing the first hint that both subunits of U2AF may be important for initial recognition of a broad spectrum of introns in this organism (Webb 2004b and references therein).

In vivo analysis demonstrates that all five domains of spU2AF^LG are essential for viability; a partial deletion of the linker region, which forms the small subunit interface, produces a severe growth defect and an aberrant morphology. A small subunit zinc-binding domain mutant confers a similar phenotype, suggesting that the heterodimer functions as a unit during splicing in S. pombe. Since this is not predicted by the model for metazoan 3' splice site recognition, introns for which the spU2AF^LG and spU2AF^SM make distinct contributions were sought by analyzing diverse splicing events in strains harboring mutations in each partner. Requirements for the two subunits are generally parallel and, moreover, do not correlate with the length or strength of the 3' pyrimidine tract. These and other studies performed in fission yeast support a model for 3' splice site recognition in which the two subunits of U2AF functionally collaborate in vivo (Webb, 2004b).

The dynamics of the splicing process and the complexities of U2AF interactions are reviewed by Kielkopf (2004). To perform its role in RNA splicing, two central canonical RRM domains of U2AF⁶⁵, the large U2AF subunit, recognize the polypyrimidine tract (Py-tract) in the pre-mRNA. Binding of U2AF⁶⁵ to the Py-tract is strengthened by cooperative protein-protein interactions with SF1 at the upstream BPS (Berglund, 1998; Rain, 1998) and with U2AF³⁵ (the small U2AF subunit), which contacts the downstream 3' splice site consensus (Merendino, 1999; Wu, 1999; Zorio, 1999a). The C-terminal U2AF homology motif (UHM) domain of U2AF⁶⁵ (the third RRM motif that is specialized for protein-protein interaction) interacts with the N-terminal domain of SF1 (U2AF⁶⁵-UHM/SF1-ligand; Rain, 1998). At the opposite end of the large U2AF subunit, the N-terminal domain of U2AF⁶⁵ provides a ligand that interacts with the central UHM domain of U2AF³⁵ (U2AF³⁵-UHM/U2AF⁶⁵-ligand; Zhang, 1992; Rudner, 1998b). Subsequently, entry of the U2 snRNP displaces SF1 by interacting with the pre-mRNA branch point sequence (BPS) via the U2 snRNA, and with the U2AF⁶⁵ C-terminal domain via the SF3b subunit, SAP155 (Gozani, 1998; Habara, 1998). Once the U2 snRNP has contacted the pre-mRNA, U2AF is dissociated by conformational rear-rangements of the spliceosome components. In summary, key protein-protein interactions are mediated by the U2AF⁶⁵-UHM, which interacts with SF1 and subsequently SAP155, and by the U2AF³⁵-UHM, which interacts with the U2AF⁶⁵ N terminus (Kielkopf, 2004 and references therein).

To gain an understanding of the mechanisms underlying splice site selection and the control of alternative splicing, genome-wide approaches have been undertaken to pursue a genetic and biochemical investigation of the Drosophila large U2AF subunit (dU2AF⁵⁰). Several groups have reported the identification of temperature-sensitive mutations in the yeast S. pombe large U2AF subunit homolog that reside in conserved amino acids common to all known U2AF large subunits. The S. pombe mutations were transferred to Drosophila and new temperature-sensitive dU2AF⁵⁰ transgenic strains were created. In vitro, these mutant recombinant U2AF heterodimers show a dramatic temperature-dependent reduction in RNA polypyrimidine tract binding, without exhibiting any defect on in vitro splicing of model pre-mRNAs. Genome-wide expression profiles of the mutant flies identify genes that are specifically differentially expressed at the restrictive temperature. These results have allowed the identification of U2AF-sensitive target mRNAs and specific RT-PCR analysis has confirmed that splicing is impaired in the mutant flies. Most interestingly, a high proportion of intronless genes were downregulated in the mutant flies when grown at the restrictive temperature. High-density microarrays and dU2AF⁵⁰ knockdown in cultured cells were used to survey the nucleo-cytoplasmic distribution of all expressed genes. This analysis revealed that more that 28% of mRNAs accumulated in the nucleus upon dU2AF⁵⁰ knockdown, regardless of their intron number. A genome-wide approach analyzing RNAs bound in nuclear RNPs as well as a bioinformatic analysis confirmed that dU2AF⁵⁰ associates with intronless RNAs. These results reveal a previously unknown function for dU2AF⁵⁰ in the nuclear export of intronless mRNAs (Blanchette, 2004).

Two Drosophila dU2AF⁵⁰ temperature-sensitive alleles were charcterized in this study; they directly impair U2AF RNA binding to an intron polypyrimidine tract. In addition, the mutations reduced the splicing efficiency of some target genes in vivo. Surprisingly, although U2AF is a known splicing factor, a large number of intronless genes were found to be downregulated in the mutant flies at the restrictive temperature. By looking at the nucleo-cytoplasmic distribution of all expressed genes, it was found that reducing dU2AF⁵⁰ expression had a dramatic and widespread effect on the nucleo-cytoplasmic mRNA localization irrespective of the intron number of the affected genes. Finally, some intronless RNAs were found to be associated with dU2AF⁵⁰ in nuclear RNP complexes. Thus, in addition to the well-known role of U2AF in defining 3′ splice sites in pre-mRNAs, the results reveal an unexpected function for dU2AF⁵⁰ in nuclear export of intronless mRNAs (Blanchette, 2004).

As previously reported, the large U2AF subunit is highly conserved from S. pombe to humans (Kanaar, 1993; Potashkin, 1993; Zamore, 1992; Zorio, 1997) and has been shown to be highly refractory to mutations (Romfo, 1999). Two mutations identified in S. pombe generate new temperature-sensitive dU2AF⁵⁰ alleles in Drosophila. Both mutations, D204N and S284Y, lie on each side of the second RNA binding domain (RRM2) without being part of it. The structures of several RRM-RNA complexes have recently been solved and shown to conform to a canonical ß1alpha1ß2ß3alpha2ß4 fold. In the structure of the human RRM2, asparatic acid 204 is located four amino acids upstream of the first ß sheet (ß1) of RRM2, which is predicted to be part of the RNA interaction platform. Although the D204 residue was not in the structure of RRM2, it is conceivable that asparatic acid 204 is involved in a salt bridge and in absence of this putative salt bridge, the RNA-RRM2 interaction might be less stable leading to the observed temperature-sensitive reduction in RNA binding. The dU2AF⁵⁰ serine 284 residue is conserved in mouse and human U2AF⁶⁵ and is substituted by a cysteine in S. pombe and C. elegans. Interestingly, in the U1A:RNA structure, the C-terminal region next to ß4 contacts the RNA, and, similarly, the same region in U2AF⁶⁵ also appears to contact RNA (Ito, 1999). Thus, changing a serine for a bulky aromatic tyrosine may cause steric effects that might result in reduced RNA affinity (Blanchette, 2004).

Surprisingly, although the temperature-sensitive mutations dramatically affect dU2AF⁵⁰ RNA binding affinity, in vitro splicing of model substrates was not affected. This probably reflects the highly cooperative nature of spliceosome assembly. Reduction in U2AF RNA binding affinity might be compensated for by interaction with other spliceosomal factors. For instance, interaction of the small U2AF subunit with the 3′ splice site together with interaction of the large U2AF subunit with SF1/BBP, which binds to the branchpoint sequence, could be involved in stabilizing U2AF binding to the polypyrimidine tract (Berglund, 1997; Blanchette, 2004).

The most striking observation made in this study is that a very high proportion of intronless genes are downregulated in the dU2AF⁵⁰ mutant flies grown at the restrictive temperature. Although this observation could be interpreted as an indirect effect, the fact that this enrichment for intronless RNAs in the dU2AF⁵⁰ mutants is very different from the average genomic intron distribution suggests a direct role for dU2AF⁵⁰ in the expression of intronless genes. Moreover, the observation that RNAi knockdown of dU2AF⁵⁰ expression results in the nuclear accumulation of a large number of intronless mRNAs, that dU2AF⁵⁰ is found to be associated with intronless mRNAs in purified nuclear RNP complexes, and that the vast majority of intronless genes possess putative U2AF binding sites support a direct role for dU2AF⁵⁰ in the nuclear export of intronless mRNAs. In mammals, U2AF has been shown to directly interact with the protein factor UAP56, a putative DEAD box RNA helicase essential for splicing (Fleckner, 1997), and the essential transport receptor TAP/NXF1 (Zolotukhin, 2002). It has been proposed that UAP56 is recruited to the spliceosome through an interaction with RRM1 of U2AF⁶⁵ (Fleckner, 1997). In Drosophila and yeast, UAP56 is an essential export factor that functions to bridge the mRNA to the export machinery (Gatfield, 2001; Herold, 2003; Jensen, 2001). Interestingly, UAP56 was shown to be required not only for export of spliced mRNAs but also for export of intronless mRNAs (Gatfield, 2001; Jensen, 2001; Strasser, 2001; Strasser, 2002). One attractive possibility is that U2AF, as with intron-containing genes, is involved in the recruitment of UAP56, or other members of the RNA export machinery, for instance, TAP/NXF1, to intronless mRNAs prior to their nuclear export (Blanchette, 2004).

Recently, a subset of the SR family of splicing factors has been shown to be involved in the export of a class of intronless mRNAs (Huang, 2003; Huang, 2001). The SR proteins 9G8, SRp20, and SF2/ASF are proteins that shuttle between the nucleus and cytoplasm and serve as adaptors between the intronless histone mRNA and the export factor TAP. Interestingly, mammalian U2AF has been shown to continuously shuttle between the nucleus and cytoplasm (Gama-Carvalho, 2001; Zolotukhin, 2002), and although there is no known function for U2AF in the cytoplasm, these results are suggestive of a general and direct role of U2AF in the export of intronless, as well as intron-containing mRNAs (Blanchette, 2004).

The genes that are upregulated upon growth of the mutant dU2AF⁵⁰ strain at the restrictive temperature generally contain multiple introns. In addition, their pre-mRNAs are generally longer than the average genomic pre-mRNA length. Intriguingly, no splicing defect was found for any of several individual upregulated genes tested. One possible explanation for this observation could reside in the recent report that the splicing machinery can stimulate the transcription apparatus (Fong, 2001). One might envision that mRNA maturation in the mutant flies might be slower on long pre-mRNAs or on pre-mRNAs containing multiple introns because of reduced dU2AF⁵⁰ RNA binding affinity. This could increase the time during which partially spliced mRNA-containing snRNPs or partially assembled spliceosomes would colocalize on nascent transcripts with the transcriptional machinery. Those spliceosomal components could feed back on the transcription machinery, releasing potentially paused RNA polymerase II complexes. This would lead to an overall increase in transcriptional rate on some genes that are more prone to RNA polymerase II pausing. Although it is not known, it is speculated that genes upregulated in the mutant flies might be part of such a class (Blanchette, 2004).

Over the past few years it has become evident that what were originally thought to be distinct steps in the gene expression pathway are tightly coupled through an extensive network of interactions between the transcriptional RNA processing and RNA export machineries. However, most of this knowledge comes from intron-containing genes, and this, in part, accounts for the relatively poor understanding of the mechanisms controlling expression of intronless genes. The observation that the splicing factor dU2AF⁵⁰ can influence nuclear export of intronless genes suggests that common mechanisms and RNA-protein interactions are probably shared between these two classes of genes (Blanchette, 2004 and references therein).

GENE STRUCTURE

cDNA clone length -

1458

Bases in 5' UTR - 127

Exons - 5

Bases in 3' UTR - 80

PROTEIN STRUCTURE

Amino Acids - 416

Structural Domains

The large subunit of the human pre-messenger RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor (hU2AF65) is required for spliceosome assembly in vitro. A complementary DNA clone encoding the large subunit of Drosophila U2AF (dU2AF50) has been isolated. The dU2AF50 protein is closely related to its mammalian counterpart and contains three carboxyl-terminal ribonucleoprotein consensus sequence RNA binding domains and an amino-terminal arginine- and serine-rich (R/S) domain (Kanaar, 1993).

The large U2AF subunit is a modular protein with an N-terminal RS domain rich in arginine and serine (RS dipeptides). Adjacent to the RS domain on the large U2AF subunit, a proline-rich segment mediates protein-protein interactions with the pseudo-RNA binding domain (RRM for RNA recognition motif) of the small U2AF subunit (Kielkopf, 2001 and Rudner, 1998c). The C terminus of the large U2AF subunit contains three RRMs. Although all three RRMs were initially described as required for the U2AF-RNA interaction (Zamore, 1992), RRM1 and RRM2 were subsequently shown to be sufficient for specific RNA binding (Ito, 1999), while RRM3 is responsible for protein-protein interactions with the branchpoint binding protein SF1/BBP (Berglund, 1998; Rain, 1998 and Selenko, 2003). In addition to interacting with SF1/BBP, U2AF interacts with members of the SR family of splicing factors (McKinney, 1997; Page-McCaw, 1999; Potashkin, 1993; Romfo, 1999; Tronchere, 1997 and Wu and Maniatis 1993), with the U2 snRNP-associated protein, SAP155 (Gozani, 1998), UAP56, a putative RNA helicase required for splicing (Fleckner, 1997) and nuclear export of mRNA, (Gatfield, 2001 and Herold, 2003), and the transport factor TAP/NXF1 (Zolotukhin, 2002; Blanchette, 2004 and references therein).

date revised: 22 January 2005

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