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Previous Article | Next Article 
Molecular and Cellular Biology, January 2002, p. 221-230, Vol. 22, No. 1
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.22.1.221-230.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390,1 Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 060302
Received 22 June 2001/ Returned for modification 17 July 2001/ Accepted 5 October 2001
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ABSTRACT |
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INTRODUCTION |
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20 nucleotides (nt), and the sequence YAG that defines the end of the 3' splice site. The first step in spliceosome assembly, the formation of the E complex, involves the binding of U1 snRNP to the 5' splice site, SF1/mBBP to the branch point, and U2 snRNP auxiliary factor (U2AF) to the pyrimidine tract and YAG (23, 26). In addition, U2 snRNP is associated with the spliceosome at this point, but not stably bound to the pre-mRNA (6). Subsequently, U2 snRNP replaces SF1 at the branch point to form the A complex (26). Understanding the details of these early events in spliceosome assembly is important because alternative splicing is frequently regulated during these steps (31). U2AF is a heterodimeric complex conserved in all metazoan species. In mammals, this complex consists of 65-kDa (U2AF65) and 35-kDa (U2AF35) subunits (42). U2AF65 contains an N-terminal arginine-serine-rich (RS) domain and three RNA recognition motifs (RRM) (43). U2AF65 is an essential splicing factor in vitro and is required for viability in Drosophila melanogaster (15), Caenorhabditis elegans (20) and Schizosaccharomyces pombe (25). U2AF65 interacts directly with the pyrimidine tract and is involved in stabilizing the interaction of U2 snRNP with the branch point (8, 35). This activity requires the RS domain, which is thought to assist in the formation of the U2 snRNP-pre-mRNA duplex (35), and the third RRM, which interacts with SAP 155, a component of U2 snRNP (8).
The role of U2AF35 in pre-mRNA splicing has been more controversial than that of U2AF65. U2AF35 contains two zinc fingers and a noncanonical RRM (1) in the N terminus of the protein and an RS domain and a glycine tract at the C terminus (44). The precise role of U2AF35 in pre-mRNA splicing has been unclear because some studies have shown that U2AF35 is dispensable for splicing in vitro (14, 41), while other studies indicate that U2AF35 is an essential splicing factor in vitro (12, 49). Nonetheless, the small U2AF subunit is required for viability in Drosophila (15) and C. elegans (48), as is its interaction with U2AF65 (29). One function of U2AF35 in pre-mRNA splicing was recently clarified when three groups demonstrated that U2AF35 associates with the AG at the 3' splice site (21, 40, 47).
In addition to constitutive splicing, U2AF35 appears to play an important role in regulated splicing. The removal of many introns requires cis-acting sequences present in the downstream exon called exonic splicing enhancers (ESEs) (2, 10). Many ESEs are recognized by serine-arginine-rich (SR) proteins—a conserved family of essential splicing factors (10). Several studies have shown that enhancer-bound SR proteins function by recruiting U2AF65 to weak pyrimidine tracts (3, 11, 36, 49). It has been proposed that U2AF recruitment involves protein interactions between enhancer-bound SR proteins and the RS domain of U2AF35 (39, 49). However, other studies indicate that ESEs may function through a mechanism that requires neither U2AF recruitment (14, 19) nor U2AF35 (14). Interestingly, the small U2AF subunits from C. elegans (48) and S. pombe (37) do not contain RS domains. Moreover, transgenic Drosophila expressing a version of dU2AF38 lacking its RS domain as the sole source of dU2AF38 is not only viable but properly regulates the alternative splicing of the enhancer-dependent doublesex pre-mRNA (28). Thus, at least in nonvertebrate systems, the RS domain is not an essential feature of the small U2AF subunit.
Here, we report the identification and characterization of a mammalian gene, U2AF26, capable of encoding a protein product very similar to U2AF35. The N-terminal portion of the U2AF26 polypeptide, which contains two zinc fingers, a noncanonical RRM, and a U2AF65 interaction domain, is 89% identical to U2AF35. However, the C-terminal domain of U2AF26 is quite different from the C-terminal domain of U2AF35—most of the RS dipeptides, as well as the entire glycine tract present in U2AF35, are absent in U2AF26. We find that the relative levels of U2AF35 and U2AF26 mRNAs vary in different mouse tissues. U2AF26 is a nuclear protein that localizes to speckles and can physically associate with U2AF65. Biochemical experiments demonstrate that U2AF26 functions as a pre-mRNA splicing factor and can functionally substitute for U2AF35 in constitutive splicing in vitro. Interestingly, U2AF26 can also replace U2AF35 in enhancer-dependent splicing, demonstrating that although a small U2AF subunit is necessary for efficient enhancer-dependent splicing, its RS domain is not. Finally, we show that U2AF26 enhances the binding of U2AF65 to weak 3' splice sites. These studies identify U2AF26 as a mammalian splicing factor that may participate in the regulation of alternative splicing.
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MATERIALS AND METHODS |
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Northern blotting. Total RNA was isolated from dissected mouse brain, cerebellum, colon, eyes, heart, kidney, liver, lung, skeletal muscle, small intestine, spleen, testis, uterus, and white adipose tissue using RNA Stat-60 (Tel-Test). Poly(A)+ RNA was isolated by passing the RNA samples twice through oligo(dT) cellulose columns (Gibco). A total of 5 µg of RNA was run on a 1.2% formaldehyde gel, transferred to a Nytran supercharge membrane, and hybridized with 32P-labeled DNA probes. The U2AF26 transcripts were detected with a full-length cDNA probe, while the U2AF35 transcripts were detected with a probe corresponding to the last 476 bp of the mouse cDNA. The blots were also probed with a cyclophilin probe as an internal control for RNA loading. The blots were developed and quantitated with a Fuji BAS1500 phosphorimager. After normalization to the cyclophilin control, the U2AF35 and U2AF26 signals in the lung tissue, which was the tissue expressing the median value of each mRNA, were set to a value of 1.0. The U2AF35 and U2AF26 signals in the remainder of the tissues were normalized to the signal in lung tissue, and these values were used to calculate the ratio of U2AF26 to U2AF35.
Indirect immunofluorescence. HeLa cells were transfected with 1 µg of pcDNA-mU2AF26/V5, which encoded murine U2AF26 (mU2AF26) containing a C-terminal V5 epitope tag, using Fugene6 (Boehringer Mannheim). In addition, cells were transfected with control plasmids expressing either human U2AF35 (hU2AF35) or human SF2/ASF containing C-terminal V5 epitope tags. Twenty-four hours after transfection, the cells were fixed in formaldehyde and probed with anti-V5 antibody (Invitrogen). The cells were then washed with 1x phosphate-buffered saline and probed with an anti-mouse immunoglobulin G-fluorescein isothiocyanate secondary antibody (Jackson ImmunoResearch Laboratories). The cells were washed, stained with DAPI (4',6'-diamidino-2-phenylindole), and visualized with a Zeiss Axioplan 2 microscope, and the images were acquired with a SenSys-cooled charge-coupled device camera using OpenLab software (Improvision).
Expression and purification of recombinant proteins. His6-mU2AF26 and His6-hU2AF35 were cloned into the baculovirus expression vector, pFastBac-1 (Gibco). The N-terminal His6 tag was added by PCR. To construct a virus encoding GST-hU2AF65, hU2AF65 was first cloned into pGEXT-4T (Amersham-Pharmacia), amplified by PCR to isolate the fragment encoding GST-U2AF65, and subsequently cloned into pFastBac-1. Baculoviruses encoding these proteins were produced as described by the manufacturer (Gibco). The U2AF65-U2AF26 and U2AF65-U2AF35 heterodimers were produced by coinfecting the Sf9 cells with the respective viruses, whereas U2AF65 was produced by infecting Sf9 cells with a single virus. At 48 h after infection, the Sf9 cells were centrifuged at 500 x g for 10 min and the pellet was resuspended in lysis buffer (50 mM Tris [pH 8.0], 300 mM NaCl, 0.1% Triton X-100, 5 mM ß-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 10-µg/ml leupeptin). The suspension was sonicated for 1 min and centrifuged at 18,000 rpm in a JA-20 rotor (Beckman) for 25 min. The supernatant was then incubated for 25 min at 4°C with Ni-nitrilotriacetic acid (Ni-NTA) agarose beads (Qiagen) equilibrated with lysis buffer. After incubation, the beads were loaded onto a column, washed with lysis buffer, and eluted with lysis buffer containing 300 mM imidazole. The eluate was then incubated with equilibrated glutathione-Sepharose 4B (Amersham Pharmacia) for 25 min and then loaded onto a column. The beads were washed with lysis buffer and eluted with lysis buffer containing 50 mM glutathione. U2AF65 was purified on glutathione-Sepharose only. The purified proteins were dialyzed overnight in buffer D (100 mM KCl, 20% glycerol, 20 mM HEPES [pH 7.9], 0.2 mM EDTA, 0.1 mM dithiothreitol).
RNAs. Plasmids encoding the ß-globin and dsx-avian sarcoma leukosis virus (ASLV) pre-mRNAs were described previously (27, 32). Templates encoding the RNAs used for the gel shift experiments were generated by PCR from the dsx or ß-globin templates. 32P-labeled RNAs were synthesized with either SP6 or T7 RNA polymerase.
In vitro-splicing assays. HeLa cell nuclear extract was depleted of U2AF65 and U2AF35 by oligo(dT) chromatography as described previously (11). The depleted extract used in these experiments is the same one used in the experiments reported in reference 11. As shown in reference 11, Western blot analysis suggests that >95% of U2AF65 and U2AF35 was depleted from this extract. Splicing assays were carried out using 30% U2AF-depleted extract as previously described (11).
U2AF binding assays. The recombinant U2AF preparations were incubated with 5 fmol of 32P-labeled RNAs in reaction mixtures containing 800 ng of BSA, 250 ng of tRNA, 1.3% polyvinylalcohol, 1 mM dithiothreitol, and 4 U of RNasin (Promega) in a 10-µl volume for 15 min at 30°C. The reaction mixtures were transferred to ice, heparin was added to 0.05 mg/ml, and the mixtures were resolved by electrophoresis on 4% 80:1 polyacrylamide gels at 13 V/cm for 3 h at 4°C. The gels were quantitated with a Molecular Dynamics Storm PhosphorImager, and the percentage of RNA bound at each protein concentration was calculated. Kd values were calculated for U2AF65 by fitting the data to a single binding isotherm. Kd values for the U2AF heterodimers were calculated by fitting the data to a cooperative binding isotherm where Y = (K1[P] + K1K2KC[P]2)/{1 + (K1 + K2)[P] + K1K2KC[P]2}, where K1 and K2 are the estimated equilibrium association constants for U2AF65 and U2AF35 or U2AF26, Y is the fraction of RNA bound, P is the concentration of protein used, and KC is the cooperativity factor (13, 38).
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RESULTS |
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U2AF35 and U2AF26 share 89% primary amino acid sequence identity within the N-terminal 187 residues of the proteins (Fig. 1A). The N-terminal domain contains two zinc fingers that are identical between the two proteins, as well as a noncanonical RRM (1, 17). The RNP-1 portion of the RRM is identical between U2AF35 and U2AF26 (Fig. 1A). However, the RNP-2 motif of U2AF26 contains two amino acid substitutions with respect to U2AF35—a change from Ala to Val at position 47, and a change from Ile to Leu at position 51. The high degree of conservation of the noncanonical RRM suggests that U2AF26, like U2AF35, has the ability to bind RNA (21, 40, 47). In addition, the majority of the residues in U2AF35 that directly interact with U2AF65 (17, 44) are conserved in U2AF26. For example, the crystal structure of the U2AF35-U2AF65 heterodimer (17) revealed that Glu80, Phe81, Glu84, Glu88, Arg133, and Phe135 of U2AF35 are in intimate contact with U2AF65. Of these residues, only Glu80 is different in U2AF26 (changed to an Asn). Most importantly, Trp134, perhaps the most critical residue involved in the U2AF65-U2AF35 interaction (17), is conserved in U2AF26. This suggests that U2AF26 most likely can physically interact with U2AF65.
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Cloning of the U2AF26 gene. To compare the genomic organization of the U2AF26 gene with that of U2AF35, we cloned and sequenced the gene encoding U2AF26 from a mouse genomic DNA library. The U2AF26 gene is small, spanning only 2,059 bp (Fig. 1B). Experiments with 5' rapid amplification of cDNA ends suggest that transcription initiates approximately 50 bp upstream of the start codon. A poly(A) consensus signal resides 114 bp downstream of the stop codon. The gene contains eight exons that when spliced together generate an mRNA of approximately 900 nt. The exon-intron boundaries of the U2AF26 gene are identical to those in the human U2AF35 gene located on chromosome 21 (18).
U2AF26 is differentially expressed.
To examine the expression of U2AF26 in comparison to U2AF35, we performed Northern blot analyses of poly(A)+ RNA isolated from different mouse tissues (Fig. 2A). Due to the fact that we could not correct for hybridization efficiency of the probes, we have expressed the results as a ratio of U2AF26 to U2AF35 signals in each tissue, which was calculated after normalization to the cyclophilin control (see Materials and Methods). These experiments clearly demonstrate that the relative level of U2AF26 and U2AF35 mRNAs varies in different tissues (Fig. 2B). For example, the ratio of U2AF26 to U2AF35 is highest in brain (3) and lowest in liver (0.5). Thus, U2AF26 is differentially expressed in various mouse tissues.
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NE). Previous work had shown that U2AF35 enhances the ability of U2AF65 to restore splicing in NE (11, 12, 49). We first tested whether U2AF26 could function in constitutive splicing. As shown in Fig. 5A, U2AF65 alone weakly restores the splicing of ß-globin pre-mRNA in NE (Fig. 5A, lane 3). However, both U2AF65-U2AF35 and U2AF65-U2AF26 were approximately twofold more effective in restoring splicing of this substrate than U2AF65 alone (lanes 4 and 5). Similar results were observed for the adenovirus major late pre-mRNA (data not shown). These observations indicate that U2AF26 can function as a pre-mRNA splicing factor and that it can functionally substitute for U2AF35 in constitutive splicing.
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NE (Fig. 5B, lane 3). By contrast, both U2AF65-U2AF35 and U2AF65-U2AF26 were five- to sixfold more effective in restoring splicing of the dsx-ASLV pre-mRNA in NE than U2AF65 alone (lanes 4 and 5). Thus, U2AF26 can functionally substitute for U2AF35 in both constitutive splicing and enhancer-dependent splicing. These results demonstrate that an RS domain in the small U2AF subunit is not required for enhancer-dependent splicing. U2AF26 enhances the binding of U2AF65 to weak 3' splice sites. Our data demonstrate that U2AF26 can functionally substitute for U2AF35 in both constitutive and enhancer-dependent splicing. We next wanted to determine the basis for the activity of U2AF26. Given that U2AF26 appeared to be functionally similar to U2AF35, we reasoned that the activity was most likely due to the sequences within the conserved N terminus rather than the divergent C terminus. Given the known function of U2AF35 (21, 40, 47), we thought U2AF26 might act by stabilizing the binding of U2AF65 on weak 3' splice sites and that this activity might involve an interaction between U2AF26 and the AG at the 3' splice site. To test this, we performed binding assays using the purified U2AF preparations and RNAs containing various 3' splice site sequences. As expected, the U2AF65-U2AF35 heterodimer bound much more efficiently to a 100-nt RNA containing the weak dsx 3' splice site (Kd = 65 nM) than did U2AF65 alone (Kd = 2,000 nM) (Fig. 6A). Surprisingly, the affinity of the U2AF65-U2AF26 heterodimer for the dsx 3' splice site (Kd = 60 nM) was nearly identical to that of the U2AF65-U2AF35 heterodimer (Fig. 6A). Thus, both U2AF35 and U2AF26 enhance the affinity of U2AF for the dsx 3' splice site by greater than 30-fold. However, the impact of U2AF35 or U2AF26 on U2AF binding was substantially diminished on a 100-nt RNA containing the pyrimidine tract from the ß-globin pre-mRNA (Fig. 6B) or an RNA containing the consensus U2AF65 binding site (data not shown). In the case of the ß-globin pyrimidine tract, the difference in affinity of the U2AF heterodimers (U2AF65-U2AF35, 120 nM; U2AF6-AF26, 100 nM) compared to U2AF65 alone (270 nM) was only about twofold. This suggests that U2AF26, similar to U2AF35, functions to enhance the binding of U2AF65 to weak 3' splice sites.
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DISCUSSION |
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Orthologues of U2AF26 do not appear to exist in lower eukaryotes, Drosophila, C. elegans, plants, or vertebrates such as Xenopus or zebra fish (data not shown). By contrast, ESTs more similar to U2AF26 than U2AF35 are also found in rat (AW142126, AW142127, and AA848227), pig (BF193007 and BF440828), and cow (BE589680) (GenBank accession numbers are given in parentheses). In addition, humans contain a U2AF26 gene located on chromosome 19 (NT_011296), and several human ESTs in GenBank correspond to this gene. A comparison of the mouse and human U2AF26 gene revealed that the exon-intron boundaries are located in the same positions as in the human U2AF35 gene, although the introns are much smaller in the U2AF26 gene. In addition, the exon sequences of the human and mouse U2AF26 genes are 90% identical at the nucleotide level; the majority of the differences are neutral, third position changes. Thus, the gene encoding U2AF26 appears to have recently arisen in mammals by duplication of the U2AF35 gene. Given the high degree of similarity between the human and mouse U2AF26 genes, and the fact that human and rodents diverged approximately 96 million years ago (24), it is likely that U2AF26 has a unique function.
Although proteins related to U2AF35 have been described previously, U2AF26 is the only one that has been shown to function in a manner similar to U2AF35. U2AF1-RS1 and U2AF1-RS2/Urp are 94% identical to one another and contain stretches that are approximately 50% identical to portions of U2AF35 (33). Like U2AF26, Urp interacts with U2AF65 in a manner likely to be mutually exclusive with U2AF35 (33). However, Urp contains an RS domain, interacts with SR proteins, and is functionally distinct from U2AF35 because U2AF35 cannot complement Urp-depleted extracts (33). In addition to U2AF1-RS1 and U2AF1-RS2-Urp, four proteins related to U2AF35 were recently identified in the draft of the human genome sequence (34). These observations, together with our data, suggest that mammalian cells contain multiple U2AF complexes.
In addition to its role in constitutive splicing, U2AF35 has been shown in some studies to be required for efficient enhancer-dependent splicing (11, 12, 46, 49). One model proposes that SR proteins bound to ESEs act to recruit U2AF to the upstream 3' splice site (3, 11, 36, 46, 49). This activity is thought to require interactions between SR proteins and the RS domain of U2AF35 (40, 49). However, studies with Drosophila have shown that although the small U2AF subunit, dU2AF38, and its interaction with the large U2AF subunit are essential for viability (29, 30), its RS domain is not (28). Moreover, the alternative splicing of the enhancer-dependent dsx pre-mRNA is not affected in flies lacking the dU2AF38 RS domain (28). Additionally, both the C. elegans and S. pombe small U2AF subunits naturally lack RS domains (37, 48). Together these observations suggest that enhancer-dependent splicing does not require interactions between enhancer-bound SR proteins and the RS domain of the small U2AF subunit. Our results support this model because U2AF26, which lacks an RS domain, can functionally substitute for U2AF35 in enhancer-dependent splicing. It remains to be determined whether U2AF26 can directly interact with SR proteins.
Although U2AF26 appears functionally identical to U2AF35 in our assays, it is our anticipation that U2AF26 will have a unique function in vivo that we have yet to uncover. The observation that U2AF35 and U2AF26 are differentially expressed raises the possibility that U2AF26 functions in tissue-specific alternative splicing. Interestingly, with respect to U2AF35, U2AF26 is expressed most strongly in the brain where the occurrence of alternative splicing is unusually high (9). Thus, alternative splicing may be affected by the regulated expression of U2AF26.
What could be the functional differences between these two proteins? One possibility is that the U2AF heterodimers containing either U2AF35 or U2AF26 could recognize distinct, yet overlapping sets of 3' splice sites. In support of this model, it is interesting that in contrast to the RNP-1 sequences, the RNP-2 motif is slightly different between the two proteins. In particular, the first residue of RNP-2 is an Ala in U2AF35 and a Val in U2AF26. Based on modeling with other RRM-RNA crystal structures, it was proposed that Ala47 of U2AF35 may directly contact RNA (17). Thus, although both U2AF26 and U2AF35 appear to bind RNA, and recognize the AG dinucleotide at the 3' splice site, we believe that the binding specificities of the two heterodimers may be slightly different. Thus, it is possible that the two heterodimers recognize both common and distinct sets of 3' splice sites. In this scenario, differences in the expression levels of U2AF35 and U2AF26 could influence alternative splicing because the two U2AF isoforms would recognize and activate different 3' splice sites.
Alternatively, it is possible that the U2AF65-U2AF26 and U2AF65-U2AF35 heterodimers interact differently with splicing regulatory factors. The crystal structure of the core U2AF65-U2AF35 heterodimer revealed that helix A in the U2AF35 RRM is unusually long and amphipathic (17). In particular, glutamate residues 73, 76, 80, 83, 84, 88, 90, and 91; Asp79; and Thr87 (which would be negatively charged if phosphorylated) of U2AF35 all reside on one side of helix A. Kielkopf et al. point out that the highly negatively charged face of this helix may be an interaction surface for other splicing factors—possibly the basic RS domains contained in SR proteins (17). Thus, changes in the residues contained in this alpha helix might alter the interactions between U2AF and other splicing factors. Interestingly, in U2AF26, Glu80 is changed to Asn and Glu90 is changed to Gln. These uncharged amino acids both reside on the negatively charged surface of helix A and will change its electrostatic surface, possibly affecting the types of protein interactions in which U2AF26 can participate. Thus, U2AF may have distinct, yet overlapping, responses to different splicing factors depending on whether U2AF35 or U2AF26 is associated with U2AF65. Given that the levels of U2AF26 and U2AF35 fluctuate with respect to one another in different tissues, we imagine that the relative concentrations of the different U2AF heterodimer isoforms play an important role in determining the splicing patterns of a number of pre-mRNAs.
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ACKNOWLEDGMENTS |
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This work was primarily funded by NIH grant 5 R37 MH59388-03 and an unrestricted endowment fund given to Steve McKnight by an anonymous donor. This work was also supported by NIH grant R01 GM62516-01 (to B.R.G.).
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FOOTNOTES |
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