RNA induces conformational changes in the SF1/U2AF65 splicing factor complex

Spliceosomes assemble on pre-mRNA splice sites through a series of dynamic ribonucleoprotein complexes, yet the nature of the conformational changes remains unclear. Splicing factor 1 (SF1) and U2 auxiliary factor (U2AF⁶⁵) cooperatively recognize the 3′ splice site during the initial stages of pre-mRNA splicing. Here, we used small-angle X-ray scattering to compare the molecular dimensions and ab initio shape restorations of SF1 and U2AF⁶⁵ splicing factors, as well as the SF1/U2AF⁶⁵ complex in the absence and presence of AdML (adenovirus major late) splice site RNAs. The molecular dimensions of the SF1/U2AF⁶⁵/RNA complex substantially contracted by 15 Å in the maximum dimension, relative to the SF1/U2AF⁶⁵ complex in the absence of RNA ligand. In contrast, no detectable changes were observed for the isolated SF1 and U2AF⁶⁵ splicing factors or their individual complexes with RNA, although slight differences in the shapes of their molecular envelopes were apparent. We propose that the conformational changes that are induced by assembly of the SF1/U2AF⁶⁵/RNA complex serve to position the pre-mRNA splice site optimally for subsequent stages of splicing.     

RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF⁶⁵) and small (hU2AF³⁵) subunits. hU2AF⁶⁵ contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF³⁵ has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF⁶⁵. While the requirement for the three RRMs on hU2AF⁶⁵ is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF³⁸) was complexed with the large-subunit (dU2AF⁵⁰) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF⁵⁰. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF⁶⁵. Surprisingly, the RS domain on dU2AF³⁸ was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF⁵⁰ΔRS) severely impaired its binding activity. However, if the dU2AF³⁸ RS domain was supplied in a complex with dU2AF⁵⁰ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

Solution conformation and thermodynamic characteristics of RNA binding by the splicing factor U2AF65

The U2 auxiliary factor large subunit (U2AF65) is an essential pre-mRNA splicing factor for the initial stages of spliceosome assembly. Tandem RNA recognition motifs (RRM)s of U2AF65 recognize polypyrimidine tract signals adjacent to 3' splice sites. Despite the central importance of U2AF65 for splice site recognition, the relative arrangement of the U2AF65 RRMs and the energetic forces driving polypyrimidine tract recognition remain unknown. Here, the solution conformation of the U2AF65 RNA binding domain determined using small angle x-ray scattering reveals a bilobal shape without apparent interdomain contacts. The proximity of the N and C termini within the inter-RRM configuration is sufficient to explain the action of U2AF65 on spliceosome components located both 5' and 3' to its binding site. Isothermal titration calorimetry further demonstrates that an unusually large enthalpy-entropy compensation underlies U2AF65 recognition of an optimal polyuridine tract. Qualitative similarities were observed between the pairwise distance distribution functions of the U2AF65 RNA binding domain and those either previously observed for N-terminal RRMs of Py tract-binding protein that lack interdomain contacts or calculated from the high resolution coordinates of a U2AF65 deletion variant bound to RNA. To further test this model, the shapes and RNA interactions of the wild-type U2AF65 RNA binding domain were compared with those of U2AF65 variants containing either Py tract-binding protein linker sequences or a deletion within the inter-RRM linker. Results of these studies suggest inter-RRM conformational plasticity as a possible means for U2AF65 to universally identify diverse pre-mRNA splice sites.     

Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65

The essential pre-mRNA splicing factor, U2AF(65), guides the early stages of splice site choice by recognizing a polypyrimidine (Py) tract consensus sequence near the 3' splice site. Since Py tracts are relatively poorly conserved in higher eukaryotes, U2AF(65) is faced with the problem of specifying uridine-rich sequences, yet tolerating a variety of nucleotide substitutions found in natural Py tracts. To better understand these apparently contradictory RNA binding characteristics, the X-ray structure of the U2AF(65) RNA binding domain bound to a Py tract composed of seven uridines has been determined at 2.5 A resolution. Specific hydrogen bonds between U2AF(65) and the uracil bases provide an explanation for polyuridine recognition. Flexible side chains and bound water molecules form the majority of the base contacts and potentially could rearrange when the U2AF(65) structure adapts to different Py tract sequences. The energetic importance of conserved residues for Py tract binding is established by analysis of site-directed mutant U2AF(65) proteins using surface plasmon resonance.     

Promiscuity in protein-RNA interactions: conformational ensembles facilitate molecular recognition in the spliceosome: conformational diversity in U2AF⁶⁵ facilitates binding to diverse RNA sequences

Here I discuss findings that suggest a universal mechanism for proteins (and RNA) to recognize and interact with various binding partners by selectively binding to different conformations that pre‐exist in the free protein's conformational ensemble. The tandem RNA recognition motif domains of splicing factor U2AF⁶⁵ fluctuate in solution between a predominately closed conformation in which the RNA binding site of one of the domains is blocked, and a lowly populated open conformation in which both RNA binding pockets are accessible. RNA binding to U2AF⁶⁵ may thus occur through the weakly populated open conformation, and the binding interaction stabilizes the open conformation. The conformational diversity observed in U2AF⁶⁵ might also facilitate binding to diverse RNA sequences as found in the polypyrimidine tracts that help define 3′ splice sites. Similar binding pathways in other systems have important consequences in biological regulation, molecular evolution, and information storage.     

Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24

U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24''s four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5′-GAGA-3′), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24''s annealing activity is proposed.     

Prp40 pre-mRNA processing factor 40 homolog B (PRPF40B) associates with SF1 and U2AF65 and modulates alternative pre-mRNA splicing in vivo

The first stable complex formed during the assembly of spliceosomes onto pre-mRNA substrates in mammals includes U1 snRNP, which recognizes the 5′ splice site, and the splicing factors SF1 and U2AF, which bind the branch point sequence, polypyrimidine tract, and 3′ splice site. The 5′ and 3′ splice site complexes are thought to be joined together by protein–protein interactions mediated by factors that ensure the fidelity of the initial splice site recognition. In this study, we identified and characterized PRPF40B, a putative mammalian ortholog of the U1 snRNP-associated yeast splicing factor Prp40. PRPF40B is highly enriched in speckles with a behavior similar to splicing factors. We demonstrated that PRPF40B interacts directly with SF1 and associates with U2AF⁶⁵. Accordingly, PRPF40B colocalizes with these splicing factors in the cell nucleus. Splicing assays with reporter minigenes revealed that PRPF40B modulates alternative splice site selection. In the case of Fas regulation of alternative splicing, weak 5′ and 3′ splice sites and exonic sequences are required for PRPF40B function. Placing our data in a functional context, we also show that PRPF40B depletion increased Fas/CD95 receptor number and cell apoptosis, which suggests the ability of PRPF40B to alter the alternative splicing of key apoptotic genes to regulate cell survival.     

A conditional role of U2AF in splicing of introns with unconventional polypyrimidine tracts

Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3' splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3' splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF59. We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3' splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.     

Pyrvinium pamoate changes alternative splicing of the serotonin receptor 2C by influencing its RNA structure

The serotonin receptor 2C plays a central role in mood and appetite control. It undergoes pre-mRNA editing as well as alternative splicing. The RNA editing suggests that the pre-mRNA forms a stable secondary structure in vivo. To identify substances that promote alternative exons inclusion, we set up a high-throughput screen and identified pyrvinium pamoate as a drug-promoting exon inclusion without editing. Circular dichroism spectroscopy indicates that pyrvinium pamoate binds directly to the pre-mRNA and changes its structure. SHAPE (selective 2′-hydroxyl acylation analysed by primer extension) assays show that part of the regulated 5′-splice site forms intramolecular base pairs that are removed by this structural change, which likely allows splice site recognition and exon inclusion. Genome-wide analyses show that pyrvinium pamoate regulates >300 alternative exons that form secondary structures enriched in A–U base pairs. Our data demonstrate that alternative splicing of structured pre-mRNAs can be regulated by small molecules that directly bind to the RNA, which is reminiscent to an RNA riboswitch.     

Sex lethal and U2 small nuclear ribonucleoprotein auxiliary factor (U2AF65) recognize polypyrimidine tracts using multiple modes of binding

The molecular basis for specific recognition of simple homopolymeric sequences like the polypyrimidine tract (Py tract) by multiple RNA recognition motifs (RRMs) is not well understood. The Drosophila splicing repressor Sex lethal (SXL), which has two RRMs, can directly compete with the essential splicing factor U2AF(65), which has three RRMs, for binding to specific Py tracts. We have combined site-specific photocross-linking and chemical cleavage of the proteins to biochemically map cross-linking of each of the uracils within the Py tract to specific RRMs. For both proteins, RRM1 and RRM2 together constitute the minimal Py-tract recognition domain. The RRM3 of U2AF(65) shows no cross-linking to the Py tract. Both RRM1 and RRM2 of U2AF(65) and SXL can be cross-linked to certain residues, with RRM2 showing a surprisingly high number of residues cross-linked. The cross-linking data eliminate the possibility that shorter Py tracts are bound by fewer RRMs. We present a model to explain how the binding affinity can nonetheless change as a function of the length of the Py tract. The results indicate that multiple modes of binding result in an ensemble of RNA-protein complexes, which could allow tuning of the binding affinity without changing sequence specificity.     

A Broad Range of Conformations Contribute to the Solution Ensemble of the Essential Splicing Factor U2AF(65)

U2AF(65) is essential for pre-mRNA splicing in most eukaryotes. Two consecutive RNA recognition motifs (RRM) of U2AF(65) recognize a polypyrimidine tract at the 3' splice site. Here, we use small-angle X-ray scattering to demonstrate that the tandem U2AF(65) RRMs exhibit a broad range of conformations in the solution ensemble. The majority of U2AF(65) conformations exhibit few contacts between the RRMs, such as observed in the crystal structure. A subpopulation adopts tight inter-RRM contacts, such as independently reported based on paramagnetic relaxation enhancements. These complementary structural methods demonstrate that diverse splice sites have the opportunity to select compact or extended inter-RRM proximities from the U2AF(65) conformational pool.     

Structure,phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3′-splice site recognition

Recognition of the 3′-splice site is a key step in pre-mRNA splicing and accomplished by a dynamic complex comprising splicing factor 1 (SF1) and the U2 snRNP auxiliary factor 65-kDa subunit (U2AF65). Both proteins mediate protein–protein and protein–RNA interactions for cooperative RNA-binding during spliceosome assembly. Here, we report the solution structure of a novel helix-hairpin domain in the N-terminal region of SF1 (SF1^NTD). The nuclear magnetic resonance- and small-angle X-ray scattering-derived structure of a complex of the SF1^NTD with the C-terminal U2AF homology motif domain of U2AF65 (U2AF65^UHM) reveals that, in addition to the known U2AF65^UHM–SF1 interaction, the helix-hairpin domain forms a secondary, hydrophobic interface with U2AF65^UHM, which locks the orientation of the two subunits. Mutational analysis shows that the helix hairpin is essential for cooperative formation of the ternary SF1–U2AF65–RNA complex. We further show that tandem serine phosphorylation of a conserved Ser80-Pro81-Ser82-Pro83 motif rigidifies a long unstructured linker in the SF1 helix hairpin. Phosphorylation does not significantly alter the overall conformations of SF1, SF1–U2AF65 or the SF1–U2AF65–RNA complexes, but slightly enhances RNA binding. Our results indicate that the helix-hairpin domain of SF1 is required for cooperative 3′-splice site recognition presumably by stabilizing a unique quaternary arrangement of the SF1–U2AF65–RNA complex.     

茄子SmU2AF65基因的可变剪接

摘要: U2AF(U2 snRNP auxiliary factor)是参与前体mRNA剪接的重要辅助因子, 在进化上具有较高保守性。文章根据茄子BAC 77N19(GenBank登录号: EF517791)的基因组序列信息和烟草NpU2AF⁶⁵a和NpU2AF⁶⁵b基因的全长cDNA序列, 设计特异引物, 经cDNA末端快速扩增法(RACE)获得了1 986 bp的茄子同源基因(SmU2AF⁶⁵)全长cDNA, GenBank登录号为EU543263。序列分析表明该序列包含1 665 bp的可阅读框, 编码554个氨基酸, 在氨基酸序列的C末端有3个保守的RNA识别结构域RRM。RT-PCR分析表明, SmU2AF⁶⁵基因在不同组织中均有表达,但是该基因通过可变剪接至少能够产生两个转录本, 在根中产生与其他组织中不同的剪切子     

The U1, U2 and U5 snRNAs crosslink to the 5' exon during yeast pre-mRNA splicing

Activation of pre-messenger RNA (pre-mRNA) splicing requires 5′ splice site recognition by U1 small nuclear RNA (snRNA), which is replaced by U5 and U6 snRNA. Here we use crosslinking to investigate snRNA interactions with the 5′ exon adjacent to the 5′ splice site, prior to the first step of splicing. U1 snRNA was found to interact with four different 5′ exon positions using one specific sequence adjacent to U1 snRNA helix 1. This novel interaction of U1 we propose occurs before U1-5′ splice site base pairing. In contrast, U5 snRNA interactions with the 5′ exon of the pre-mRNA progressively shift towards the 5′ end of U5 loop 1 as the crosslinking group is placed further from the 5′ splice site, with only interactions closest to the 5′ splice site persisting to the 5′ exon intermediate and the second step of splicing. A novel yeast U2 snRNA interaction with the 5′ exon was also identified, which is ATP dependent and requires U2-branchpoint interaction. This study provides insight into the nature and timing of snRNA interactions required for 5′ splice site recognition prior to the first step of pre-mRNA splicing.     

Dual function for U2AF(35) in AG-dependent pre-mRNA splicing

The splicing factor U2AF is required for the recruitment of U2 small nuclear RNP to pre-mRNAs in higher eukaryotes. The 65-kDa subunit of U2AF (U2AF(65)) binds to the polypyrimidine (Py) tract preceding the 3' splice site, while the 35-kDa subunit (U2AF(35)) contacts the conserved AG dinucleotide at the 3' end of the intron. It has been shown that the interaction between U2AF(35) and the 3' splice site AG can stabilize U2AF(65) binding to weak Py tracts characteristic of so-called AG-dependent pre-mRNAs. U2AF(35) has also been implicated in arginine-serine (RS) domain-mediated bridging interactions with splicing factors of the SR protein family bound to exonic splicing enhancers (ESE), and these interactions can also stabilize U2AF(65) binding. Complementation of the splicing activity of nuclear extracts depleted of U2AF by chromatography in oligo(dT)-cellulose requires, for some pre-mRNAs, only the presence of U2AF(65). In contrast, splicing of a mouse immunoglobulin M (IgM) M1-M2 pre-mRNA requires both U2AF subunits. In this report we have investigated the sequence elements (e.g., Py tract strength, 3' splice site AG, ESE) responsible for the U2AF(35) dependence of IgM. The results indicate that (i) the IgM substrate is an AG-dependent pre-mRNA, (ii) U2AF(35) dependence correlates with AG dependence, and (iii) the identity of the first nucleotide of exon 2 is important for U2AF(35) function. In contrast, RS domain-mediated interactions with SR proteins bound to the ESE appear to be dispensable, because the purine-rich ESE present in exon M2 is not essential for U2AF(35) activity and because a truncation mutant of U2AF(35) consisting only of the pseudo-RNA recognition motif domain and lacking the RS domain is active in our complementation assays. While some of the effects of U2AF(35) can be explained in terms of enhanced U2AF(65) binding, other activities of U2AF(35) do not correlate with increased cross-linking of U2AF(65) to the Py tract. Collectively, the results argue that interaction of U2AF(35) with a consensus 3' splice site triggers events in spliceosome assembly in addition to stabilizing U2AF(65) binding, thus revealing a dual function for U2AF(35) in pre-mRNA splicing.     

Detection of a Novel ATP-Dependent Cross-Linked Protein at the 5′ Splice Site-U1 Small Nuclear RNA Duplex by Methylene Blue-Mediated Photo-Cross-Linking

Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5′ splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5′ splice site. Moreover, although p65 cross-linking requires only a 5′ splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.     

A Role for SRp54 during Intron Bridging of Small Introns with Pyrimidine Tracts Upstream of the Branch Point

One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 in Saccharomyces cerevisiae) to the 3′ splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3′ splice site. Many small introns, especially those in nonvertebrates, lack a classical 3′ pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5′ splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5′ and 3′ splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3′ pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.Pre-mRNA splicing is a conserved process occurring in a wide variety of eucaryotes with differing exon/intron architectures (reviewed in references 4, 6, 9, 15, 20, and 26). Vertebrates typically have small exons and large introns. Nonmetazoans frequently have the opposite genetic organization, with introns smaller than the minimum permissible for splicing of a vertebrate intron. Drosophila melanogaster possesses a mixture of these two classes of intron sizes (16, 23). In addition, more than half of the small introns in Drosophila are missing a prominent vertebrate splicing signal, the 3′ polypyrimidine tract (23). For these reasons, Drosophila provides a model system in which to study potential mechanistic variations operating during recognition of splicing signals.In the general model of early vertebrate spliceosome complex assembly, U1 snRNP binds to the 5′ splice site and U2 snRNP auxiliary factor (U2AF) binds to the 3′ polypyrimidine tract, thereby facilitating U2 snRNP interaction with the branch point. Various members of the serine/arginine (SR) family of proteins may participate by promoting or stabilizing these interactions (reviewed in references 13, 22, and 31). This family of proteins may also act as exon or intron bridging factors via their SR-mediated interaction with SR domains on the small subunit of U2AF (U2AF³⁵) and the U1 70K protein (32, 33, 38). SF1, originally discovered as an essential splicing factor in reconstitution assays (19), has also been observed to bind to the branch point (7, 8). In yeast, BBP (branch point bridging protein), the ortholog to SF1, functions as an intron bridging factor via interactions with U1 snRNP-associated proteins and the large subunit of U2AF (U2AF⁶⁵) (1, 2). It is assumed that vertebrate SF1 can play a similar role, although the mammalian equivalents to the yeast U1 snRNP proteins that interact with BBP have not yet been identified. Furthermore, the relationship between bridging by SR proteins and that afforded by SF1 is unclear.We have previously examined the cis-acting sequences required for efficient splicing of a constitutively spliced small (59-nucleotide [nt]) intron from the D. melanogaster mle gene that lacks a well-defined pyrimidine tract between the branch point and 3′ splice site (18, 29). Assembly of initial ATP-dependent spliceosomes (complex A) on the mle intron requires both the 5′ and 3′ splice sites, suggesting concerted recognition of the entire intron (29). Instead of a classic pyrimidine tract, the mle intron contains two C-rich tracts located between the 5′ splice site and branch point that are necessary for efficient splicing of this intron (18). In addition to a requirement for maximal splicing efficiency, the pyrimidine stretches are also necessary for binding of U2AF, interaction of factors with the 5′ splice site, and proper assembly of the active spliceosome, suggesting that these sequences affect early assembly events at both ends of this small intron. Interestingly, the upstream C-rich tracts are inhibitory if a classical 3′ pyrimidine tract is introduced between the branch point and 3′ splice site (18). This observation suggests competing pathways of factor binding to this substrate and also raises the possibility of alternative gene-specific modes of association of constitutive factors with introns.Here we demonstrate that both U2AF and an SR protein, SRp54, interact with the C-rich tracts in the mle intron. The central location of the pyrimidine tracts, their importance for maximal splicing, and the ability of human SRp54 to interact with U2AF⁶⁵ instead of U2AF³⁵ (37) suggested that the binding of SRp54 to the tracts could replace SF1 in bridging this intron. Immunoprecipitation studies using an antibody specific for SF1 indicated that SF1 did not contact mle precursor RNA unless a pyrimidine tract was introduced downstream of the branch point. Furthermore, antibodies against either SRp54 or U2AF immunoprecipitated both halves of a precleaved mle splicing substrate, suggesting that these factors either directly or indirectly interact with both the 5′ and 3′ splice sites. We suggest that SRp54 participates in bridging the small mle intron via its ability to bind both the C-rich tracts and the large subunit of U2AF.