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Introduction

Accurate pre-mRNA splicing is essential for proper gene expression. Introns must be spliced out of pre-mRNA and exons ligated in order to make mature mRNA. Disease-causing mutations that affect the splicing process are common, and testify to the importance of splicing for normal cellular function. The splicing process is made more complex by the fact that many pre-mRNAs can be spliced in more than one way to give mature transcripts coding for proteins with distinct functions. Such alternative splicing greatly expands the coding capacity of the human genome and contributes to the overall complexity of gene expression [1]. Alternative splicing is often regulated in a tissue- and developmentally-specific manner, and is also affected by signaling pathways.

Intron removal is carried out by the spliceosome, a large complex comprised of protein and RNA components. Among these components are the U1, U2, U4, U5 and U6 small ribonucleoprotein particles (snRNPs) which consist of a specific small nuclear RNA (snRNA) and associated proteins. Assembly of the spliceosome onto pre-mRNA is a dynamic process that involves recognition of splice-site sequences at the ends of introns (reviewed in [2]). For most introns, the conserved splice-site sequences include a 5′ splice-site element surrounding a GT dinucleotide, and a 3′ splice-site AG dinucleotide preceded by a polypyrimidine tract and an upstream branchpoint sequence. These splice-site elements are initially recognized by U1 snRNP, which binds at the 5′ splice site, and by SF1/mBBP, U2AF⁶⁵ and U2AF³⁵, which bind cooperatively to the branchpoint sequence [3], [4], polypyrimidine tract [5], [6] and 3′ splice-site AG dinucleotide [5], [7], [8], [9], respectively.

A conserved family of splicing factors called SR proteins also facilitates the earliest recognition of the 5′ and 3′ splice sites (for review [2]). These first interactions between the spliceosome and the pre-mRNA are important in identifying splice sites and committing an intron to splicing. Once an intron has been initially identified, U2 snRNP becomes stably associated with the pre-mRNA. Recruitment of the U4/U6.U5 tri-snRNP to the transcript initiates the formation of a mature spliceosome that is poised to catalyze intron excision (for review, see [10]).

As a general rule, strong matches to the splice-site consensus sequences are good predictors of efficient splicing. However, there are many cases of introns with weak splice sites that are constitutively spliced, and examples of alternative splicing in which apparently weak splice sites are utlilzed more efficiently than splice sites with stronger matches to the consensus sequence [11], [12]. Our current understanding of the determinants of exon identity and intron splicing is limited. The well-defined consensus sequences at the 5′ and 3′ splice sites do not contain sufficient information to accurately identify bona fide splice sites [13], suggesting that additional sequence features are involved in the recruitment of the spliceosome to the correct location. Some of these features include exonic and intronic enhancer and silencer elements, which are recognized by distinct classes of RNA-binding proteins [14].

Based on biochemical purification of the spliceosome, nearly 200 proteins are estimated to be involved in splicing (for review, see [15]). However, only a fraction of these proteins currently have established roles in splicing. Identification of factors that are important for the recognition of weak splicing signals with poor matches to the splicing consensus sequences is an important goal towards understanding the mechanisms involved in splice-site selection and splicing regulation and fidelity. Such factors may play fundamental roles in tissue-specific or developmentally regulated splicing events, which often result from the use of apparently weak splice sites. In addition, studying the splicing of introns with weak splice sites may facilitate the identification of splicing factors that are important for splicing in vivo, but may be dispensable for detection of basal splicing of introns with strong consensus splice sites, which have traditionally been used in most mechanistic studies of splicing in vitro.

We were interested in identifying splicing factors required for the splicing of weak splice sites. We developed an in vitro splicing complementation assay in which splicing of a substrate with a weakened 3′ splice site is restored upon addition of a fraction of HeLa cell nuclear extract. We identified PUF60 as a protein that stimulated splicing in this assay. PUF60 was previously implicated in splicing [15], [16], though direct evidence for its role in the reaction was lacking. We now provide direct evidence that PUF60 is a splicing factor involved in 3′ splice-site recognition. We find that for some substrates, PUF60 can activate splicing in the absence of the related splicing factor U2AF⁶⁵ and thus may function in a similar capacity. We further demonstrate that PUF60 and U2AF⁶⁵ can function cooperatively in splicing, and that modulating their levels in cells affects specific alternative splicing events. Our results suggest the existence of a splicing regulatory pathway controlled by a class of general splicing factors that is involved in the recognition of the 3′ splice-site region.

Results

Identification of PUF60 as a Splicing Factor

Pre-mRNA splicing in vitro can occur accurately in HeLa cell nuclear extract. The cytoplasmic S100 fraction obtained during the preparation of nuclear extract is also competent for splicing of many substrates when complemented with one or more SR proteins [17], [18]. We identified a pre-mRNA transcript that, as a result of mutations in the pyrimidine tract, requires additional nuclear factors to achieve high levels of splicing in S100 extract. This transcript is derived from the human β-globin gene, with modified sequences between the branchpoint sequence and the 3′ splice site (Figure 1A; [19]). In this case, the parental substrate (WT) is spliced efficiently in HeLa nuclear extract, as well as in HeLa S100 extract complemented with SR proteins, such as recombinant SC35 (Figure 1B). When four pyrimidine residues near the branchpoint sequence were substituted with guanines (PyD) (Figure 1A), there was little difference in the splicing of the two substrates in nuclear extract (Figure 1B). However, splicing of the weakened intron substrate (PyD) was severely compromised in S100 extract complemented with SC35 (Figure 1B). Deficient PyD splicing could be rescued by the addition of a 20-40% ammonium sulfate fraction of nuclear extract (Figure 1B). The ammonium sulfate fraction enhanced splicing of the WT substrate as well, though not to the same degree as it stimulated PyD. We refer to this activity that can rescue splicing of the PyD substrate as RESCUE (Required for Efficient Splicing Complementation in Unproductive Extract).

Figure 1. Identification of PUF60 as a Splicing Activator

(A) Schematic of the wild-type (WT) and mutant (PyD) splicing substrates [19]. Boxes represent exons and lines are introns. Mutations are in lower case and the corresponding nucleotides in the wild-type substrate are underlined. Bold A indicates the branchpoint. (B) In vitro splicing assays were carried out in HeLa nuclear extract (NE) or S100 extract complemented with recombinant SC35, with or without a 20–40% ammonium sulfate (AS) fraction from HeLa NE. Quantitation is shown as the percent of the total RNA that is spliced. (C) Scheme for purification of the complementing activity and quantitation of RESCUE activity in HQ column fractions spanning the peak of activity. Splicing activity was normalized to the input material (H*). (see also Figure S2). H* refers to the HQ column input that was denatured with urea and renatured. (D)(top) Silver-stained SDS-PAGE of HQ peak fractions. (bottom) Western blot analysis of heparin (H) and HQ fractions 14-21. PUF* refers to an SDS-resistant dimer of the protein [16]. (E) Western blot analysis of S100 (lanes 1,4; 4 µl), NE (lanes 2,5; 4 µl), and recombinant PUF60 from E. coli (lane 3) using a PUF60 (lane 1–3) or U2AF⁶⁵-specific (lane 4,5) antibody. (F) In vitro splicing assay using the PyD substrate in reactions containing S100 extract with SC35 alone (lane 2; 3 µl) and complemented with PUF60 purified from E. coli (lanes 3–4; 1 and 2 µl).

doi:10.1371/journal.pone.0000538.g001

The splicing factor U2AF⁶⁵ was a possible candidate for RESCUE activity because it recognizes the pyrimidine tract during the splicing reaction [5]. However, western analysis showed a substantial amount of U2AF⁶⁵ in S100 extract (Figure 1E). We also found that recombinant U2AF^65/35 that complements splicing in a U2AF depletion assay (Figure S1A) was not able to complement splicing in our S100 complementation assay (Figure S1B) indicating that U2AF^65/35 are not responsible for RESCUE activity.

We purified RESCUE activity by sequential biochemical fractionation (Figure 1C). Following each step of purification, fractions were assayed for their activity in splicing of PyD pre-mRNA in S100 extract supplemented with SC35. Active fractions were pooled and purified further. As a first step, RESCUE activity in the 20–40% ammonium-sulfate precipitate was subjected to density-gradient centrifugation in cesium chloride (CsCl). The active fractions from the CsCl gradient were loaded on a Poros HE1 heparin column and RESCUE activity eluted at high salt (data not shown). We next disrupted protein-protein interactions in the active fractions by urea denaturation, and separated the pooled fractions on a Poros HQ column in the presence of urea (Figure 1C). Proteins associated with RESCUE activity bound to the column and were eluted at low salt concentrations (Figure S2). The most active fraction (Figure 1C, fraction 18) comprised a limited number of polypeptides, as analyzed by SDS-PAGE (Figure 1D). To identify the proteins, the entire fraction was digested with trypsin and the resulting peptides were identified by liquid chromatography tandem mass spectrometry (LC/MS/MS). Peptides from two proteins were detected: PUF60 (also known as FIR, RoBP1 and siah-bp1) and DDB1. DDB1 is a UV-damaged-DNA binding protein involved in nucleotide excision repair; it is structurally related to the U2-snRNP-associated protein SF3b130, but otherwise has no obvious link to splicing [20].

PUF60 is related to U2AF⁶⁵, which has an important role in 3′ splice site recognition, and is thus a good candidate for RESCUE activity. We confirmed the presence of PUF60 in the HQ fraction with RESCUE activity by western blotting (Figure 1D). PUF60 migrates as a monomer and as an SDS-resistant dimer on SDS-PAGE; the latter form was detected in addition to the monomer in the heparin fraction (Figure 1D).

To confirm that PUF60 is the primary factor responsible for RESCUE activity, we generated recombinant PUF60 in E. coli (Figure 1E). The addition of rPUF60 to S100 extract with SC35 stimulated PyD splicing (Figure 1F) demonstrating that PUF60 activates PyD splicing in the RESCUE splicing assay. Western blot analysis revealed that PUF60 is present predominantly in nuclear extract, with substantially lower levels in S100 extract (Figure 1E), which explains why the addition of PUF60 to the extract stimulates splicing in our complementation assay. In contrast, the level of U2AF⁶⁵ in S100 extract is comparable to the level in nuclear extract (Figure 1E).

PUF60 has been previously identified as a component of purified spliceosomes (reviewed in [15] and is associated with the 17S U2 snRNP [21]. PUF60 was originally found in a highly purified fraction of nuclear extract that contained both PUF60 and the splicing factor SRp54. This fraction, but not purified recombinant PUF60 alone, complemented splicing when combined with U2AF^65/35 in extract depleted of poly(U)-binding proteins [16]. Because this previous report did not demonstrate that recombinant PUF60 could complement splicing, which is the definitive criterion for demonstrating a functional role for a protein, it has been unclear whether PUF60, SRp54 or additional unidentified factors were responsible for the splicing activity of the purified fraction. Thus, our finding that PUF60 can complement splicing in S100 extracts is the first formal demonstration that human PUF60 is a functional splicing factor.

PUF60 Associates with Splicing Factors Involved in Early Spliceosome Assembly

To better understand the role of PUF60 in splicing, we identified PUF60-interacting proteins using a HeLa cell line with stable-integration of PUF60 cDNA fused to tandem N-terminal FLAG and V5 epitope tags (Figure 2A). Nuclear extract was prepared from these cells and PUF60 was immunoprecipitated with anti-FLAG antibody linked to agarose beads (Figure 2B). PUF60 and co-immunoprecipitated proteins were eluted from the beads with excess FLAG peptide, and were then separated by SDS-PAGE (Figure 2B). Prominent polypeptides were excised and identified by mass spectrometry. To thoroughly characterize proteins that specifically associate with PUF60, we performed a parallel experiment in which immunoprecipitates from F-V5-PUF60 HeLa nuclear extract or from control HeLa nuclear extract were digested with trypsin directly on the beads (Figure 2B). Released peptide fragments were subjected to analysis by mass spectrometry. Using these two methods, proteins specifically associated with PUF60 were identified. The results from both experiments were compiled, and only proteins identified in both experiments and absent from control samples were classified as PUF60-associated proteins with a high degree of confidence (Table 1).

Figure 2. Identification of PUF60-associated Proteins.

(A) Western blot analysis of nuclear extract (NE) prepared from control untransfected HeLa cells (C, lane 1) or cells expressing the FLAG-V5-tagged PUF60 protein (F-V5-PUF, lane 2). Blots were probed with antibodies specific for PUF60 and U2 snRNP B”. (B) Silver-stained 4–20% gradient SDS-PAGE of FLAG-PUF60 (PUF, lanes 1, 3) or control (C, lanes 2,4) immunoprecipitates, either bound to the α-FLAG beads (lanes 1,2) or eluted from the beads with FLAG peptide (lanes 3,4). The heavy chain (*) and light chain (**) from the FLAG antibody are indicated. (C) Western blot analysis of FLAG-PUF60 immunoprecipitates eluted with FLAG peptide.

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Table 1. Proteins associated with PUF60

doi:10.1371/journal.pone.0000538.t001

Strikingly, most of the proteins that co-immunoprecipitated with PUF60 are known splicing factors. Nine SR proteins, as well as U1 and U2 snRNP-associated proteins make up the majority of the proteins associated with PUF60 (Table 1). These splicing factors function at early steps of the splicing reaction, during the initial recognition and specification of splicing signals. Consistent with the mass-spectrometry data, western analysis confirmed the presence of several proteins identified in the PUF60 immunoprecipitates (Figure 2C).

Functional Redundancy and Synergy between PUF60 and U2AF^65/35

PUF60 has sequence and structural homology to U2AF⁶⁵ [22] suggesting that the two proteins may have related functions in splicing. To investigate this possibility, we compared the ability of PUF60 and U2AF⁶⁵ to complement splicing in extract depleted of both factors. We performed poly(U)-Sepharose chromatography of nuclear extract, which effectively generates extract depleted of these proteins (Figure S3A; [16], [23]. The flow-through (NEΔ) removes more than 98% of poly(U)-binding factors, such as PUF60, U2AF⁶⁵, and U2AF³⁵ (Figure S3B, S3C and S3D) and does not support splicing of PyD or other substrates in vitro (Figure 3). This depletion method does not alter the levels of other nuclear splicing factors tested, such as SF2/ASF (Figure S3B).

Figure 3. Cooperative Activity of PUF60 and U2AF^65/35 in Splicing.

(A) Complementation of PyD splicing in vitro in nuclear extract depleted of PUF60 and U2AF subunits using poly(U)-Sepharose. PyD pre-mRNA spliced in mock-depleted nuclear extract (NE, lane 1), extract depleted of U2AF subunits and PUF60 (ΔNE, lane 2), depleted extract complemented with U2AF^65/35 purified from HEK-293E cells alone (lanes 3–6: 17, 33, 67, and 133 nM final concentration of U2AF⁶⁵, respectively) or with 67 nM of U2AF⁶⁵ plus recombinant PUF60 (lanes 7,8: 1.2 and 2.4 µM PUF60) or with PUF60 alone (lanes 9,10: 1.2 and 2.4 µM). (B) Splicing of β-globin intron 1 in nuclear extract (NE, lanes 1, 8), or in poly(U)-depleted extract alone (ΔNE, lanes 2,9). Depleted extract was complemented with purified U2AF^65/35 (lanes 3–5: 17, 33, and 133 nM final concentration of U2AF⁶⁵), with 670 nM of U2AF⁶⁵ plus recombinant PUF60 (lanes 6,7: 1.2 and 2.4 µM,) or with PUF60 alone (lanes 10,11: 1.2 and 2.4 µM). (C) C12 and (D) δ-crystallin pre-mRNA spliced in nuclear extract (NE, lanes 1), depleted extract (ΔNE, lanes 2) or depleted extract with addition of recombinant PUF60 (lanes 3–5: 1.2, 2.4, and 4.8 µM final concentration; lanes 6–8: 1.2 µM; lanes 12–14, 0.6, 1.2, and 2.4 µM) or purified U2AF^65/35 (lanes 6–8: 33, 67, and 133 nM of U2AF⁶⁵; lanes 9–11: 67, 133, and 200 nM ; lanes 12–14: 670 nM). (E) Quantitation of C12 (left) and δ-crystallin (right) splicing. Splicing was calculated at three concentrations of protein (see C and D). PUF60 corresponds to quantitation of lanes 3–5; PUF60+U2AF⁶⁵ refers to lanes 6–8; U2AF refers to lanes 9–11; and U2AF+PUF60 corresponds to lanes 12–14. The level of splicing expected if the PUF60 and U2AF activity is additive was calculated as the sum of lanes 3+9, 4+10, and 5+11, respectively (Sum).

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To test the ability of U2AF and PUF60 to complement splicing in the depleted extract, we expressed His-tagged PUF60 or His-tagged U2AF³⁵ in HEK-293E cells and purified the proteins by nickel-agarose chromatography (Figure S3C and S3D). His-tagged U2AF³⁵ co-purifies with U2AF⁶⁵ (Figure S3D), reflecting the strong interaction of these two proteins [16]. Purified PUF60 was unable to complement PyD splicing in the depleted extract (Figure 3A) and U2AF^65/35 provided only very marginal complementation. However, when added in combination, PUF60 and U2AF^65/35 stimulated PyD splicing. Our results using this depletion assay confirm our previous results from the S100-extract complementation assay (Figure 1F) that PUF60 has a role in PyD splicing, and suggest a mechanism for its activity that involves U2AF^65/35.

We next tested whether the requirement for PUF60 in splicing and the cooperative action of PUF60 and U2AF^65/35 are specific for PyD, or whether this activity is more general. We tested the natural β-globin intron 1, from which PyD was originally derived, and found that the purified U2AF complex activated β-globin splicing to a higher degree than PyD (Figure 3B). Similar to our results with PyD, the addition of PUF60 and U2AF^65/35 together had a strong cooperative effect on splicing (Figure 3B). This result supports our finding that the PyD substrate is more dependent on PUF60 for splicing (Figure 1). U2AF⁶⁵ is generally considered essential for pre-mRNA splicing [24], [25]. Surprisingly, in the absence of the U2AF heterodimer, PUF60 complemented splicing to some degree (Figure 3B). Overall, our results indicate the PUF60 and U2AF^65/35 function synergistically in splicing, but are also able to functionally replace each other in the splicing of some, but not all substrates.

To further investigate this U2AF-independent splicing phenomenon, as well as the synergistic activity of PUF60 and U2AF^65/35 in splicing, we tested additional substrates, including C12 [26], δ-crystallin [27] and ftz [28]. We found that splicing of these substrates could be complemented by PUF60 in the depleted extract (Figures 3C and 3D and S4). The complementing activities of PUF60 and U2AF^65/35 were comparable for the C12 substrate (Figure 3C). In contrast, U2AF^65/35 was more active than PUF60 in complementing δ-crystallin (Figure 3D) and ftz splicing (Figure S4). The amount of U2AF^65/35 added to the reaction was considerably lower than that of PUF60, because the specific activity of recombinant PUF60 appears to be lower than that of U2AF^65/35. This difference may reflect the intrinsic activities of the proteins in the splicing reaction, or it may be due to differences in the specific activities of the recombinant protein preparations. For example, the PUF60 recombinant protein may lack a cofactor—analogous to the U2AF^65/35 relationship—that could be critical for maximum activity. In any case, our results indicate that U2AF^65/35 is not strictly required for splicing in vitro when PUF60 is present.

A striking synergistic effect of PUF60 and U2AF^65/35 on splicing was observed for all splicing substrates we tested. The addition of increasing amounts of U2AF^65/35 to extract containing a fixed amount of PUF60, or vice-versa (Figures 3C and 3D and S4), resulted in a>5-fold stronger activation of splicing relative to reactions in which comparable amounts of PUF60 or U2AF^65/35 were added to the extract in the absence of the other. The expected level of splicing if the effect of these proteins on splicing is additive was quantitated (Figures 3E and S4B, Sum) and was substantially lower than the observed splicing when PUF60 and U2AF^65/35 were present together in the extract (Figures 3E and S4B). These results demonstrate the cooperative activity of PUF60 and U2AF^65/35 in splicing, and may also suggest differences in substrate-specific requirements for these proteins.

Cooperative Binding of PUF60 and U2AF^65/35 to RNA

To explore the nature of the cooperative activity of PUF60 and U2AF^65/35, we tested whether one protein influences the binding of the other to a 3′ splice site. We performed gel-shift experiments with U2AF^65/35 heterodimer purified from baculovirus-infected SF9 cells and recombinant PUF60 purified from human HEK-293E cells (Figure S5). A 34-nt RNA substrate derived from the 3′ end of adenovirus major late pre-mRNA (Figure 4A) was used as a binding substrate. This RNA has been extensively characterized for binding by U2AF^65/35 and SF1/mBBP [3] and is therefore useful for assessing the general contribution of PUF60 to binding in the 3′ splice-site region. PUF60 and U2AF^65/35 were incubated with the substrate and complexes were separated by native gel electrophoresis and detected by autoradiography and phosphorimage analysis. The proteins in the shifted complexes were identified by transferring the gels to a membrane, followed by western blot analysis using PUF60 and U2AF⁶⁵ antibodies [29](Figure S6).

Figure 4. Cooperative Binding of U2AF^65/35 and PUF60 to the 3′ splice site.

(A) Electrophoretic mobility shift assay using a radiolabeled 34-nt RNA derived from adenovirus major late (AdML) pre-mRNA, and recombinant PUF60 and U2AF^65/35. Complexes are indicated on the left. (B) Quantitation of PUF60 binding to AdML RNA represented as the fraction of total labeled RNA bound by the protein. (C) U2AF⁶⁵ binding represented as the fraction of total labeled RNA bound by the protein.

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When added alone, PUF60 and the U2AF heterodimer each bound the RNA substrate (Figure 4A). When PUF60 and U2AF^65/35 were incubated together with the RNA, PUF60 binding was enhanced as much as four-fold compared to binding in the absence of U2AF^65/35 (Figure 4A, cf. lanes 3–5 with lanes 7–9 and lane 3 with lanes 14–17). Interestingly, increasing amounts of PUF60 binding to the RNA appeared to displace the U2AF complex, as evidenced by the decrease in the U2AF:RNA complex. Thus, U2AF^65/35 strongly facilitates PUF60 binding and in so doing may destabilize its own binding. Alternatively, the presence of PUF60 may alter the binding of U2AF⁶⁵ and result in a less stable interaction with the RNA that does not withstand the separation on the polyacrylamide gel. In any case, the cooperative binding of PUF60 and U2AF^65/35 suggests that the activities of these proteins in splicing may arise from collaboration during their initial binding to the RNA.

In order to help define the interaction between the 3′ splice-site region and PUF60 and the U2AF^65/35 heterodimer and whether these interactions change when the proteins are incubated together, we performed footprinting experiments with the AdML RNA substrate and purified proteins (Figure 5). Previous footprinting analysis of this substrate with purified U2AF⁶⁵ revealed that the protein protects the pyrimidine tract and also the branchpoint sequence to some degree [3]. To test whether both PUF60 and U2AF^65/35 protect the pyrimidine tract, we digested the RNA with RNase 1, which cleaves 3′ of all four bases. RNase1 did not cleave efficiently near the 5′ end of the RNA, even in the absence of protein. Nonetheless, we found that the U2AF^65/35 heterodimer and PUF60 both protected the pyrimidine tract from cleavage (Figure 5A and 5B). We did not observe changes in the protection pattern nor in the level of protection when the two proteins were added in combination (lane 4). However, in order to see efficient protection using this enzyme, a level of PUF60 and U2AF^65/35 was required that was out of the range for cooperative interactions, as judged by the gel-shift experiments. Thus, we were not able to assess the cooperative protection of the RNA by PUF60 and U2AF^65/35 using RNase 1.

Figure 5. Footprinting analysis of PUF60 and U2AF^65/35 binding

(A) RNase 1 digestion. Labeled RNA was incubated with purified PUF60, U2AF^65/35, or both PUF60 and the U2AF heterodimer, digested with RNase and separated by denaturing PAGE. The concentration of PUF60 was 0.72 µM (lane 2), or 0.36 µM (lane 4) and that of U2AF⁶⁵ was 1.9 µM (lane 3), or 0.96 µM (lane 4). (B) Graphical representation of footprint data in (A). Relative protection is normalized to digestion in the absence of protein (A, lanes 1, 6). The RNA sequence below the plot indicates the position of cleavage (arrowhead). The pyrimidine tract is underlined. (C) RNase T1 digestion. The concentration of PUF60 was 0.36 µM (lanes 7, 9) and that of U2AF⁶⁵ was 0.48 µM (lanes 8,9). The bands corresponding to the pyrimidine (Py) tract and branchpoint adenosine (A), and full-length RNA (FL) are indicated (see also Berglund et al., 1998). RNase T1 cleavage sites are numbered. A lower exposure of the top of the RNase T1 gel allows visualization of cleavage site 2. (D) Graphical representation of footprinting data in (C). Relative protection is normalized to digestion in the absence of protein (C, lanes 1, 6). The RNA sequence below the plot indicates the position of cleavage (arrowhead). The pyrimidine tract is underlined.

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We also performed footprinting analysis with RNase T1, which cleaves 3′ of guanosines. We observed partial protection of the guanosine at the 3′ splice-site AG dinucleotide by PUF60 and U2AF^65/35 when incubated individually, as evidenced by the decrease in cleavage product 2 (Figure 5C and 5D). When PUF60 and U2AF^65/35 were added together to the reaction, the protection pattern of the RNA was altered. An increase in protection of the AG dinucleotide (product 2) as well as the guanosine in the pyrimidine tract (product 3) suggests that the binding of the proteins changes when both are present. These results suggest that the cooperative binding seen in the gel shift assay may reflect interactions at the pyrimidine tract that in turn stabilize binding to the AG at the 3′ splice site.

We also reproducibly observed U2AF^65/35 protection of the branchpoint sequence region in the absence of PUF60 (Figure 5C, lane 4, products 4,5,6), as previously reported [3]. PUF60 had the opposite effect on accessibility in this region, causing enhanced cleavage in this region (Figure 5C and 5D, product 4, 5, 6). These effects on the branchpoint sequence may reflect non-specific binding of the proteins. Interestingly, these effects were not observed when the proteins were added together, suggesting that the presence of both proteins increases or stabilizes specific binding.

Modulation of Alternative Splicing by PUF60 and U2AF⁶⁵

We have shown that PUF60 and U2AF^65/35 function cooperatively during splicing in vitro, and that the absence of either protein does not eliminate splicing but severely compromises its efficiency. From these results, we reasoned that splicing activity and alternative splicing might be regulated in vivo by variations in the level of these two proteins. We found that the levels of PUF60 and U2AF⁶⁵ do indeed vary between different cell-types. In particular, the levels of PUF60 and U2AF⁶⁵ in HeLa cells are 3-5 fold higher than in the neuronal-like WERI-RB-1 cells (Figure S7). These results indicate that the levels of the proteins are not constant and may be controlled in a tissue-specific manner, which may contribute to differences in alternative splicing between different cells.

We investigated whether alternative splicing can indeed be modulated by changing U2AF⁶⁵ and PUF60 levels in cells. We used an RNAi approach to deplete PUF60 levels in cells. We first created a HeLa cell line (PUFrm) with stable integration of a PUF60 cDNA with silent mutations in the target region for an siRNA, and a control cell line with stable integration of the vector alone. These cell lines were treated with the PUF60 siRNA. As expected, PUF60 levels were reduced in the vector-control cell line (Figure 6A). Because PUF60rm is not targeted by the siRNA, only a slight reduction of PUF60, due to the reduction of endogenous PUF60, was seen in the PUFrm cell line (Figure 6A).

Figure 6. Changes in PUF60 and U2AF^65/35 levels regulate alternative splicing in cells.

(A) Western blot analysis of HeLa cells with stable expression of empty vector (HeLa) or PUF60 cDNA with silent mutations that protect transcripts from siRNA-mediated knockdown (HeLa PUFrm). Cells were treated with PUF60 siRNA (+) or mock-treated (−). Blots were probed with antibodies specific for PUF60, hnRNP A1, and U2 B”. (B) Western blot analysis of HeLa cells treated with siRNA specific for PUF60 (lane 2), a U2AF⁶⁵-specific siRNA (lanes 3 and 4) or mock-treated (lane 1). Antibodies against U2AF⁶⁵, PUF60, or α-tubulin were used. (C) APP and (D) BIN1 alternative splicing analyzed by RT-PCR with [α-³²P]dCTP of endogenous transcripts from stable cell lines mock-treated (lanes 1,3) or treated with PUF60 siRNA (lanes 2,4)(from (A)). (E) APP and (F) BIN1 alternative splicing analyzed by RT-PCR with [α-³²P]-dCTP of endogenous transcripts from HeLa cells mock-treated (−) or treated with PUF60 (P), U2AF⁶⁵ (U) siRNAs, or both (P+U). RT-PCR analysis of APP and BIN1 from untreated Weri-Rb1 (W) cells demonstrates neural splicing patterns.

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PUF60 was previously identified in a yeast three-hybrid assay as a factor that interacts with an intronic splicing enhancer located 36 nucleotides upstream of the 3′ splice-site region of the amyloid precursor protein (APP) transcript; this enhancer promotes inclusion of the alternatively spliced exon 8 of APP transcript [30]. APP alternative splicing is also regulated in a tissue-specific manner: neuronal tissues favor exon 7 and 8 skipping (isoform 69). whereas non-neuronal tissues exhibit nearly complete inclusion of exon 7 and some exon 8 skipping (isoforms 6789 and 679)(reviewed in [31]. To determine whether PUF60 can modulate APP alternative splicing, we performed RT-PCR to analyze alternative splicing of endogenous APP transcripts in the cells depleted of PUF60. We observed a decrease in transcripts that include exon 8, and an increase in transcripts that skip exons 7 and 8, relative to APP transcripts from the control cells (Figure 6C). No change in alternative splicing was observed in the knockdown of PUF60 in the PUFrm cells (Figure 6C), indicating that the change in APP alternative splicing was a direct and specific result of PUF60 depletion. These results are consistent with the aforementioned report that PUF60 interacts with a splicing enhancer that is important for exon 8 splicing [19]. Skipping of APP exons 7 and 8 is also the predominant isoform in WERI cells (Figure 6E), which have lower levels of PUF60 than HeLa cells (Figure S7). These findings may indicate a correlation between skipping of these exons and PUF60 levels in brain.

We next tested for changes in additional alternative splicing events following depletion of PUF60 from cells. Alternative splicing of the tumor suppressor BIN1 is similar to that of APP in that there are multiple alternatively spliced exons and a distinct splicing pattern is observed in neuronally-derived samples [32]. Changes in the level of BIN1 isoforms have been linked to tumor progression, which can be induced by modulation of the expression levels of splicing factors [32], [33]. We found that PUF60 knockdown resulted in a reduction of BIN1 isoforms that include exon 12A (Figure 6D). This splicing pattern is similar to that observed in WERI cells (Figure 6F). Thus, knockdown of PUF60 in HeLa cells results in a shift toward a neuronal-type splicing pattern, similar to the shift in splicing observed in APP transcripts following PUF60 depletion.

If different introns have different requirements for PUF60 and U2AF⁶⁵, then depleting U2AF⁶⁵ in cells might be expected to have different effects on alternative splicing than PUF60 depletion. To test this idea, a U2AF⁶⁵-specific siRNA was used to deplete the protein from HeLa S3 cells (Figure 6B). Consistent with above results, PUF60 depletion caused an increase in exon 7 and exon 8 skipping (Figure 6E). In contrast, U2AF⁶⁵ depletion increased exon 8 skipping, as evidenced by an increase in isoform 679 (Figure 6E). U2AF⁶⁵ knockdown also altered BIN1 splicing: unlike PUF60 depletion, which favored exon 12A skipping (Figure 6F), U2AF⁶⁵ depletion promoted exon 12A inclusion (Figure 6F). These results suggest that the splicing of different introns is differentially affected by changes in the levels of U2AF⁶⁵ and PUF60. Knock-down of PUF60 and U2AF⁶⁵ together resulted in a change in APP and BIN1 splicing similar to the pattern seen with U2AF⁶⁵ knockdown alone (Figure 6E and 6F), suggesting that the U2AF⁶⁵ effect may be dominant over the PUF60 effect on splicing. We conclude from these results that modulation of the activity or levels of PUF60 and U2AF⁶⁵ may contibute to the regulation of alternative splicing.

We tested the effect of PUF60 and U2AF⁶⁵ depletion on the splicing of several additional alternatively spliced exons, and observed a spectrum of different responses. A single nucleotide polymorphism in the pyrimidine tract of UBQLN1 intron 7 is associated with partial skipping of exon 8 and has recently been linked to an increased risk of Alzheimer's disease [34]. We found that exon 8 splicing was insensitive to changes in PUF60 levels in HeLa cells (Figure 7A). However, depletion of U2AF⁶⁵ led to a striking increase in exon 8 skipping. We also tested splicing of SMN2 exon 7, which is a well-studied splicing event that is influenced by a number of splicing factors (reviewed in [35]). We found that knockdown of either PUF60 or U2AF⁶⁵ led to a decrease in exon 7 skipping (Figure 7B). Finally, we tested splicing of MAPT exon 10. Human mutations that alter MAPT exon 10 splicing are linked to frontotemporal dementia with Parkinsonism linked to Chromosome 17 (FTDP-17), and many splicing factors have been documented to be involved in the regulation of this splicing event (reviewed in [36]). We found that alternative splicing of exon 10 was not significantly affected by knockdown of PUF60 or U2AF⁶⁵ in HeLa cells (Figure 7). Together, our results indicate that changes in the levels of PUF60 and U2AF⁶⁵ in cells do not alter splicing of all alternative exons. Instead, changes in the quantity of these proteins appear to selectively modulate alternative splicing of a subset of exons.

Figure 7. Complex modulation of alternative splicing by PUF60 and U2AF⁶⁵.

(A) UBQLN1 exon 8 splicing, (B) SMN2 exon 7 splicing, and (C) MAPT exon 10 splicing analyzed by RT-PCR in the presence of [α-³²P]-dCTP of endogenous transcripts from untreated (−) HeLa cells, HeLa cells treated with a PUF60-specific siRNA (P), a U2AF⁶⁵ siRNA (U), or both siRNAs (P+U). Transcripts including the alternative spliced exon (+) or skipping the exon (Δ) are labeled.

doi:10.1371/journal.pone.0000538.g007

Discussion

Introns with splice-site sequences with poor matches to the consensus motifs are common in pre-mRNAs. Despite having weak splicing signals, such introns can be excised efficiently in vivo. The mechanisms responsible for the recognition and selection of authentic splice sites, rather than cryptic sites or alternative splicing pathways, are not clear. In particular, the highly specific recognition of 3′ splice sites is puzzling. The consensus sequence of the 3′ splice site is relatively simple, apparently requiring little more than an AG dinucleotide preceded by a region moderately enriched in pyrimidines and a degenerate branchpoint sequence. Clearly, much more is involved in defining a 3′ splice site, as this combination of sequence elements is plentiful in genomic sequences and yet 3′ splice-site selection is highly specific and subject to inactivation by single point mutations. It is apparent that our current understanding of the 3′ splice site is limited, and has likely overlooked components that may be critical for the efficient and specific recognition of sites that have poor matches to the degenerate 3′ splice-site consensus.

To gain insight into the mechanism of 3′ splice-site recognition, we investigated the factor requirements for splicing of an intron with a weak 3′ splice site/pyrimidine tract, and identified PUF60 as a critical protein for splicing (Figure 1). We showed that PUF60 stimulates splicing, and is in a complex with splicing factors involved in the early steps of spliceosome assembly (Figure 2). Mechanistically, PUF60 collaborated in a cooperative manner with U2AF^65/35 in both RNA binding (Figures 4 and 5) and splicing activation (Figure 3). Surprisingly, however, neither protein was essential for splicing, provided that the other one was present indicating some level of functional redundancy between these structurally related proteins. In addition, modulation of the levels of U2AF⁶⁵ and PUF60 in cells changed alternative splicing patterns (Figures 6 and 7), demonstrating that PUF60 and U2AF⁶⁵ can modulate the efficiency of 3′ splice-site selection in a splice-site-dependent manner, and thereby regulate alternative splicing.

PUF60: a Multi-tasking Protein

PUF60 has long been considered a putative splicing factor due to its presence in a number of purified spliceosomes (reviewed in [15]), its similarity to U2AF⁶⁵ [22], as well as its presence in a partially purified fraction of nuclear extract with splicing activity in vitro [16]. In the latter study, PUF60 was shown to bind to poly(U) RNA and was the predominant protein along with SRp54 in a partially purified fraction of nuclear extract that complemented splicing of extract depleted of poly(U) binding factors. However, none of the functional assays done at that time used recombinant PUF60. Thus, despite the suggestive evidence that PUF60 was a splicing factor, rigorous proof of its function in splicing was previously lacking. To demonstrate the activity of PUF60 in splicing, we have used an S100 extract complementation assay, as well as the previous assay involving complementation of poly(U)-depleted extracts. For the latter assay, we used different substrates than Page-McCaw et al. [16], as well as recombinant PUF60 protein purified from mammalian 293 cells for our complementation; thus, it is possible that our PUF60 protein is more active and/or our splicing substrates may be more efficient or responsive to PUF60 activity.

PUF60 has other documented roles in the cell, and appears to be a protein with particularly diverse functions. PUF60 is also known as FBP-interacting repressor (FIR), a regulator of Myc gene expression [37]. In this role, PUF60/FIR represses Myc transcription in a process that involves binding between FIR and FUSE-binding protein (FBP), which binds the Myc promoter region. FIR/PUF60 itself was not found to bind the DNA, but instead enhanced FBP binding. We did not detect an association between FBP and PUF60 by western (data not shown) or by mass-spectrometry of PUF60 immunoprecipitations. However, this result does not preclude a relationship between these two proteins under other conditions. A role for PUF60 in transcription as well as splicing is intriguing, as this implies that PUF60 could contribute to the coupling between these two processes (reviewed in [38]).

Finally, PUF60 is also known as RoBPI (Ro RNA binding protein) and interacts with Ro ribonucleoproteins (RNPs) [39]. Ro RNPs have largely unknown functions, but are currently thought to play a role in quality control of small RNAs (reviewed in [40]).

Cooperation between PUF60 and U2AF^65/35 has a General Role in Splicing

The PyD splicing substrate with a weak pyrimidine tract was used to initially identify PUF60 as a splicing factor. Previous analysis of this substrate revealed that it requires both U2AF⁶⁵ and U2AF³⁵ for splicing, whereas splicing of the wild-type parental substrate is not dependent on U2AF³⁵ ([41], Hastings&Krainer, unpublished results). U2AF³⁵ recognizes the 3′ splice-site AG dinucleotide and stabilizes binding of U2AF⁶⁵ to the pyrimidine tract [5], [7], [8], [9]. This role for U2AF³⁵ may be particularly important in substrates with weak pyrimidine tracts that are not bound efficiently by U2AF⁶⁵. Similarly, PUF60 may be required in addition to U2AF⁶⁵ and U2AF³⁵ to facilitate splice-site recognition. In these instances, in which splicing is inefficient, the synergistic activity of these proteins may be critical for splice-site identification.

We propose that 3′ splice-site selection efficiency is dictated in part by the ability of the site to be recognized by U2AF^65/35 and PUF60. Splicing efficiency, as well as alternative splicing patterns, could thereby be dictated by the availability, modifications, or expression levels of these proteins. One possible function of the proteins may be to displace inhibitory factors from the pyrimidine tract. Indeed it has been reported that modulation of the levels of U2AF⁶⁵ and the inhibitory protein PTB (polypyrimidine-tract-binding protein) can influence alternative 3′ splice-site selection [42]).

Mechanistic Considerations for PUF60 in Splicing

Cooperation between PUF60 and U2AF^65/35 was observed for all the splicing substrates tested, suggesting that this activity is an integral part of the splicing process. Synergy between proteins in splicing may reflect cooperative binding to a functional element(s), or multiple, simultaneous interactions between the activators and other components of the splicing machinery (reviewed in [43]). Indeed, we find that having both PUF60 and U2AF^65/35 present not only stimulates splicing in vitro in a cooperative manner, but also influences their binding to the 3′ splice-site region (Figures 4 and 5). Our gel shift experiments suggest that PUF60 and U2AF^65/35 may bind sequentially, rather than simultaneously to the RNA. One possible mechanism is that U2AF^65/35 binds initially and recruits PUF60, which subsequently or concomitantly displaces U2AF from the RNA. It is also possible that U2AF is not fully displaced, but that its interaction with the 3′ splice-site is weakened in the presence of PUF60. This change in affinity could reflect an important transition in the spliceosomal complex as splicing proceeds. Although our analysis of the PUF60 complex confirmed the presence of U2AF⁶⁵, only two peptides were found by mass spectrometry (Table 1), suggesting that interactions between the proteins may be relatively transient.

Spliceosome assembly in the 3′ splice-site region of the intron is very dynamic. Early in the process, interactions between SF1 and the U2AF heterodimer allow for cooperative RNA binding that is important for initial branchpoint sequence recognition [3]. An interaction between SF3b155 and U2AF⁶⁵ replaces the U2AF⁶⁵-SF1 interaction and is important for stable U2 snRNP binding to the branchpoint sequence [44]. U2AF^65/35 binding to the RNA also becomes destabilized during this process [45]. In our PUF60 complex (Table 1) we identified SF3b155 but not SF1. One possible scenario is that SF1 binds cooperatively with U2AF⁶⁵, which then recruits PUF60. The arrival of PUF60 could recruit SF3b155 and initiate the replacement of U2AF-SF1 with SF3b155, as well as the stable U2 snRNP association, accompanied by destabilization of U2AF^65/35 binding. Many alternatives can also be envisioned, including the possibility that PUF60 functionally overlaps with SF1 in the recruitment of U2AF⁶⁵ to the RNA. Such a mechanism could explain why SF1 does not appear to be essential for splicing in cells [46]. More detailed experiments aimed at understanding the mechanistic interplay of PUF60 and U2AF⁶⁵ are required to better define the interactions of these proteins and the precise role of PUF60 in 3′ splice site selection.

The isolated PUF60 complex (Figure 2 and Table 1) offers some clues to the role of PUF60 in splicing. This complex is composed mainly of splicing factors with functions in early spliceosome assembly, including SR proteins and U1 and U2 snRNP components, as well as a putative human homolog of PRP5, an RNA-dependent ATPase. Interestingly, yeast PRP5 forms a bridge between U1 and U2 snRNPs during pre-spliceosome assembly, an association that appears to be important for U2 snRNP interaction with the pre-mRNA [47]. The presence of these particular components in the PUF60 complex further suggests that PUF60 is involved in early spliceosome assembly, perhaps by helping to recruit or stabilize U2 snRNP binding. Collectively, our results suggest that PUF60 associates with a subset of splicing factors that likely reflect its function in splicing during early events of the reaction.

PUF60 and U2AF⁶⁵ as a Functional Class of Splicing Factors

One model for the mechanism of PUF60 in splicing supported by our results is that PUF60 and U2AF⁶⁵ have distinct functions in splicing, but these functions may be partially interchangeable or conditionally dispensable. Although it has been generally accepted that U2AF⁶⁵ is required for pre-mRNA splicing in metazoans ([23] and reviewed in [22]), we demonstrate that splicing in vitro can occur in the absence of U2AF^65/35 (Figure 3). Under these conditions, PUF60 is required in the extract to sustain splicing. At the same time, these two proteins act cooperatively to stimulate splicing at a level more than 5-fold greater than expected if the activities of PUF60 and U2AF⁶⁵ were independent of each other. Thus, although splicing can occur in the absence of either protein, it is much more efficient when both are present.

Splicing was previously shown to occur in the absence of U2AF⁶⁵ under certain experimental conditions. One report provides evidence that when nuclear extract is prepared from cells infected with adenovirus, in vitro splicing of some substrates is dependent on the presence of U2AF⁶⁵ [48]; however, splicing of other substrates can occur in the absence of U2AF⁶⁵. Another study suggesting the dispensability of U2AF⁶⁵ reported that in vitro splicing can be restored in U2AF-depleted extract by the addition of an excess of the SR protein SC35 [49].

Our results raise the possibility that PUF60 and U2AF⁶⁵ may belong to a family of factors that can modulate splicing based on substrate-specific, early recognition of distinct 3′ splice sites. Another protein, HCC1, which is structurally related to PUF60 and U2AF⁶⁵ [50] may be another factor involved in this mode of regulation. HCC1 has been shown to interact with splicing factors such as SRp54 [51] and SRrp53 [52] and is found in the spliceosome (reviewed in [15]). Related to the notion that these proteins may represent a class of regulatory factors, a recent RNAi screen in Drosophila aimed at identifying splicing regulators found that knockout of hfp, the PUF60 ortholog, influences alternative splicing of a partially overlapping set of substrates, compared to knockout of HCC1 and U2AF50, the U2AF⁶⁵ ortholog [53].

Regulation of Alternative Splicing by PUF60 and U2AF⁶⁵

If PUF60 and U2AF⁶⁵ can indeed modulate splicing based on differential splice-site strengths and/or different requirements for their activities in the splicing of particular introns, then regulation of individual pathways via control of PUF60 and U2AF⁶⁵ expression levels, localization, or activities could play an important role in alternative splicing and tissue-specific splicing. Indeed, we have identified several alternative splicing events that are altered by such fluctuations in cells (Figures 6 and 7).

Our observation that PUF60 depletion from HeLa cells shifts APP and BIN1 processing to favor brain-specific splicing (Figure 6) suggests that PUF60 may be one factor that helps determine non-neuronal splicing patterns, and the relatively low levels of PUF60 in neuronal cell lines may be partially responsible for the observed skipping of exon exons 7 and 8 in these cells. More extensive experiments testing the effect of PUF60 over-expression in neuronally-derived cells are required to confirm this activity. In this first documented role of PUF60 in alternative splicing, the protein appears to influence splicing of some regulated exons. Interestingly, U2AF⁶⁵ had different effects on APP and BIN1 splicing compared to PUF60.

The regulation of splicing by PUF60 and U2AF⁶⁵ appears to be complex, and at this point not readily predictable. We have identified splicing events that are only altered by U2AF⁶⁵, others that are altered in a similar fashion by both proteins, and still other transcripts that are apparently unaffected by the depletion of either protein. There are no obvious sequence patterns in the 3′ splice sites of these transcripts that correlate with PUF60 or U2AF⁶⁵ sensitivity. Identifying such features will be an important goal in understanding the mechanism of regulation by these splicing factors.

For some transcripts, such as BIN1 and SMN2, the depletion of U2AF⁶⁵ (BIN1) or both U2AF⁶⁵ and PUF60 (SMN2) results in an increase in exon inclusion. These results argue that as yet unknown features of a splice site dictate its dependence on one or the other protein. For example, in the case of SMN2, the predominant skipping of exon 7 has been attributed to the disruption of a splicing enhancer in exon 7 [54]. This splicing enhancer is intact in the SMN1 gene—a paralog of SMN2—whose transcripts efficiently include exon 7. Exonic splicing enhancers recruit U2AF⁶⁵ to upstream 3′ splice sites [55], [56]. Thus, SMN2 exon 7 skipping may be a direct consequence of inefficient U2AF⁶⁵ binding. It is possible that the depletion of U2AF⁶⁵ weakens the recognition of the exon 8 3′ splice site, but has little effect on exon 7 splicing, which is already compromised in its ability to recruit U2AF⁶⁵. Thus, the strength of the exon 7 and exon 8 3′ splice sites may be equalized by U2AF⁶⁵ or PUF60 depletion, and thus these sites become more competitive for pairing with the 5′ splice site of exon 6. The outcome of this shift in splice-site recognition would predict an increase in exon 7 inclusion, as observed in Figure 7. Indeed, masking the exon 8 3′ splice site with an antisense oligonucleotide results in more efficient exon 7 inclusion [57].