Structure and assembly of the SF3a splicing factor complex of U2 snRNP

SF3a is an evolutionarily conserved heterotrimeric complex essential for pre-mRNA splicing. It functions in spliceosome assembly within the mature U2 snRNP (small nuclear ribonucleoprotein particle), and its displacement from the spliceosome initiates the first step of the splicing reaction. We have identified a core domain of the yeast SF3a complex required for complex assembly and determined its crystal structure. The structure shows a bifurcated assembly of three subunits, Prp9, Prp11 and Prp21, with Prp9 interacting with Prp21 via a bidentate-binding mode, and Prp21 wrapping around Prp11. Structure-guided biochemical analysis also shows that Prp9 harbours a major binding site for stem-loop IIa of U2 snRNA. These findings provide mechanistic insights into the assembly of U2 snRNP.     

Mammalian splicing factor SF1 interacts with SURP domains of U2 snRNP-associated proteins

Splicing factor 1 (SF1) recognizes the branch point sequence (BPS) at the 3′ splice site during the formation of early complex E, thereby pre-bulging the BPS adenosine, thought to facilitate subsequent base-pairing of the U2 snRNA with the BPS. The 65-kDa subunit of U2 snRNP auxiliary factor (U2AF65) interacts with SF1 and was shown to recruit the U2 snRNP to the spliceosome. Co-immunoprecipitation experiments of SF1-interacting proteins from HeLa cell extracts shown here are consistent with the presence of SF1 in early splicing complexes. Surprisingly almost all U2 snRNP proteins were found associated with SF1. Yeast two-hybrid screens identified two SURP domain-containing U2 snRNP proteins as partners of SF1. A short, evolutionarily conserved region of SF1 interacts with the SURP domains, stressing their role in protein–protein interactions. A reduction of A complex formation in SF1-depleted extracts could be rescued with recombinant SF1 containing the SURP-interaction domain, but only partial rescue was observed with SF1 lacking this sequence. Thus, SF1 can initially recruit the U2 snRNP to the spliceosome during E complex formation, whereas U2AF65 may stabilize the association of the U2 snRNP with the spliceosome at later times. In addition, these findings may have implications for alternative splicing decisions.     

Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1

SURP domains are exclusively found in splicing‐related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1–α2–3₁₀–α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α‐helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16‐residue α‐helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio‐layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.     

Domains in human splicing factors SF3a60 and SF3a66 required for binding to SF3a120, assembly of the 17S U2 snRNP, and prespliceosome formation

The active 17S U2 small nuclear ribonucleoprotein particle (snRNP), which binds to the intron branch site during the formation of the prespliceosome, is assembled in vitro by sequential interactions of the essential splicing factors SF3b and SF3a with the 12S U2 snRNP. We have analyzed the function of individual subunits of human SF3a (SF3a60, SF3a66, and SF3a120) by testing recombinant proteins, expressed in insect cells, in various in vitro assays. The recombinant subunits readily form the SF3a heterotrimer, where SF3a60 and SF3a66 interact with SF3a120, but not with each other. All SF3a subunits are essential for the formation of the mature 17S U2 snRNP and the prespliceosome. Single subunits engage in interactions with the 15S U2 snRNP (consisting of the 12S U2 snRNP and SF3b), and SF3a60 appears to play a major role in recruiting SF3a120 into the U2 particle. Analysis of functional domains in SF3a60 and SF3a66 identified interaction sites for SF3a120 in their N-terminal portions. C(2)H(2)-type zinc finger domains mediate the integration of SF3a60 and SF3a66 into the U2 snRNP, and we propose a model in which protein-protein interactions between the zinc finger domains and the Sm proteins, common to all spliceosomal snRNPs, contribute to the assembly of the 17S U2 snRNP. Finally, we demonstrate that all domains required for interactions within the SF3a heterotrimer and the formation of the 17S U2 snRNP are also necessary to assemble the prespliceosome.     

Interaction domains and nuclear targeting signals in subunits of the U2 small nuclear ribonucleoprotein particle-associated splicing factor SF3a

Human splicing factor SF3a is a component of the mature U2 small nuclear ribonucleoprotein particle (snRNP) and its three subunits of 60, 66, and 120 kDa are essential for splicing in vitro and in vivo. The SF3a heterotrimer forms in the cytoplasm and enters the nucleus independently of the U2 snRNP. Here, we have analyzed domains required for in vitro interactions between the SF3a subunits. Our results indicate that the SF3a66-SF3a120 interaction is mediated by a 27-amino acid region in SF3a120 C-terminal to the second suppressor-of-white-apricot and prp21/spp91 domain and amino acids 108-210 of SF3a66. Neither of these sequences contains known structural motifs, suggesting that the interaction domains are novel. Moreover, an ～100-amino acid region, including the SURP2 domain of SF3a120 but extending into neighboring regions, is sufficient for binding to SF3a60. Analysis of determinants for nuclear import of SF3a demonstrates that SF3a120 provides the major nuclear localization signal and SF3a60 contributes to nuclear import.     

A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.     

Characterization of novel SF3b and 17S U2 snRNP proteins,including a human Prp5p homologue and an SF3b DEAD-box protein

Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5 (a homologue of Saccharomyces cerevisiae Prp5p) were characterized further. Immunodepletion experiments with HeLa nuclear extract indicated that hPrp5p plays an important role in pre-mRNA splicing, acting during or prior to prespliceosome assembly. The SF3b-associated protein SF3b125 dissociates at the time of 17S U2 formation, raising the interesting possibility that it might facilitate the assembly of the 17S U2 snRNP. Finally, immunofluorescence/FISH studies revealed a differential subnuclear distribution of U2 snRNA, hPrp5p and SF3b125, which were enriched in Cajal bodies, versus SF3b155 and SF3a120, which were not; a model for 17S U2 snRNP assembly based on these findings is presented. Taken together, these studies provide new insight into the composition of the 17S U2 snRNP and the potential function of several of its proteins.     

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.     

Functional domains of the human splicing factor ASF/SF2.

The human splicing factor ASF/SF2 displays two predominant activities in in vitro splicing assays: (i) it is an essential factor apparently required for all splices and (ii) it is able to switch utilization of alternative 5' splice sites in a concentration-dependent manner. ASF/SF2 is the prototype of a family of proteins typified by the presence of one or two RNP-type RNA binding domains (RBDs) and a region highly enriched in repeating arginine-serine dipeptides (RS regions). Here we describe a functional analysis of ASF/SF2, which defines several regions essential for one, or both, of its two principal activities, and provides insights into how this type of protein functions in splicing. Two isoforms of the protein, which arise from alternative splicing, are by themselves inactive, but each can block the activity of ASF/SF2, thereby functioning as splicing repressors. Some, but not all, mutations in the RS region prevent ASF/SF2 from functioning as an essential splicing factor. However, the entire RS region can be deleted without reducing splice site switching activity, indicating that it is not absolutely required for interaction with other splicing factors. Experiments with deletion and substitution mutants reveal that the protein contains two related, but highly diverged, RBDs, and that both are essential for activity. Each RBD by itself retains the ability to bind RNA, although optimal binding requires both domains.     

Mer1p is a modular splicing factor whose function depends on the conserved U2 snRNP protein Snu17p

Mer1p activates the splicing of at least three pre-mRNAs (AMA1, MER2, MER3) during meiosis in the yeast Saccharomyces cerevisiae. We demonstrate that enhancer recognition by Mer1p is separable from Mer1p splicing activation. The C-terminal KH-type RNA-binding domain of Mer1p recognizes introns that contain the Mer1p splicing enhancer, while the N-terminal domain interacts with the spliceosome and activates splicing. Prior studies have implicated the U1 snRNP and recognition of the 5′ splice site as key elements in Mer1p-activated splicing. We provide new evidence that Mer1p may also function at later steps of spliceosome assembly. First, Mer1p can activate splicing of introns that have mutated branch point sequences. Secondly, Mer1p fails to activate splicing in the absence of the non-essential U2 snRNP protein Snu17p. Thirdly, Mer1p interacts with the branch point binding proteins Mud2p and Bbp1p and the U2 snRNP protein Prp11p by two-hybrid assays. We conclude that Mer1p is a modular splicing regulator that can activate splicing at several early steps of spliceosome assembly and depends on the activities of both U1 and U2 snRNP proteins to activate splicing.     

Mammalian splicing factor SF3a120 represents a new member of the SURP family of proteins and is homologous to the essential splicing factor PRP21p of Saccharomyces cerevisiae.

Mammalian splicing factor SF3a consists of three subunits of 60, 66, and 120 kDa and functions early during pre-mRNA splicing by converting the U2 snRNP into its active form. A cDNA encoding the 120-kDa subunit of SF3a has been cloned. The SF3a120 gene was localized to human chromosome 22, and three mRNAs of 3.2, 3.8, and 5.7 kb are ubiquitously expressed. The N-terminal half of the deduced SF3a120 amino acid sequence contains a tandemly repeated motif (the SURP module) that has recently been identified in the essential splicing factor PRP21p of Saccharomyces cerevisiae, the Drosophila alternative splicing regulator suppressor-of-white-apricot, and four proteins from nematodes and mammals; the C-terminal half is organized into a proline-rich region and a ubiquitin-like domain. The spacing between the SURP modules and the protein's essential function in constitutive splicing identify SF3a120 as the mammalian homologue of yeast PRP21p. Binding studies with truncated derivatives of SF3a120 revealed that the SURP domains function in binding to SF3a60, whereas a region of 130 amino acids C-terminal to these domains is essential for contacts with SF3a66.     

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.     

The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.

We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins. In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition. In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p. Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA. Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors. However, it is not required for splicing in vitro. Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the [U4/U6.U5] tri-snRNP or spliceosomal complexes. Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling. Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used. This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing. Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP. Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.     

Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution.

U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site. Purified U2AF comprises two polypeptides of 65 and 35 kd. We have performed biochemical complementation and immunological assays to characterize U2AF in greater detail. First, we use an extract lacking only U2AF activity to show that U2AF is an essential splicing factor. Second, we show that all U2AF activity in vitro resides in the 65 kd U2AF polypeptide. Third, based upon both immunological and functional criteria, we show that U2AF is evolutionarily conserved. Most significantly, a Drosophila melanogaster nuclear extract contains proteins that are antigenically related to both human U2AF polypeptides and can substitute for human U2AF in vitro. Finally, we show that U2AF has an unexpected intranuclear distribution. Although diffusely present throughout the nucleoplasm, U2AF is also concentrated in a small number (between one and five) of nuclear 'centers.' This localization differs strikingly from that reported for snRNP antigens and splicing factors. Our data, in conjunction with those in the accompanying paper [Carmo-Fonseca et al. (1991) EMBO J., 10, 195-206.], suggest that these centers represent novel aspects of nuclear organization.     

Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus

Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro. Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor. A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex. The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure. U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected. Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking. Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export.     

Interactions of the yeast SF3b splicing factor

The U2 snRNP promotes prespliceosome assembly through interactions that minimally involve the branchpoint binding protein, Mud2p, and the pre-mRNA. We previously showed that seven proteins copurify with the yeast (Saccharomyces cerevisiae) SF3b U2 subcomplex that associates with the pre-mRNA branchpoint region: Rse1p, Hsh155p, Hsh49p, Cus1p, and Rds3p and unidentified subunits p10 and p17. Here proteomic and genetic studies identify Rcp10p as p10 and show that it contributes to SF3b stability and is necessary for normal cellular Cus1p accumulation and for U2 snRNP recruitment in splicing. Remarkably, only the final 53 amino acids of Rcp10p are essential. p17 is shown to be composed of two accessory splicing factors, Bud31p and Ist3p, the latter of which independently associates with the RES complex implicated in the nuclear pre-mRNA retention. A directed two-hybrid screen reveals a network of prospective interactions that includes previously unreported intra-SF3b contacts and SF3b interactions with the RES subunit Bud13p, the Prp5p DExD/H-box protein, Mud2p, and the late-acting nineteen complex. These data establish the concordance of yeast and mammalian SF3b complexes, implicate accessory splicing factors in U2 snRNP function, and support SF3b contribution from early pre-mRNP recognition to late steps in splicing.     

Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes.

U6 snRNA sequences required for assembly of U4/U6 snRNP and splicing complexes were determined by in vitro reconstitution of snRNPs. Both mutagenesis and chemical modification/interference assays identify a U6 snRNA domain required for U4/U6 snRNP formation. The results support the existence of a U4/U6 snRNA interaction domain previously proposed on the basis of phylogenetic evidence. In addition, two short U6 snRNA regions flanking the U4/U6 interaction domain are essential to assemble the U4/U6 snRNP into splicing complexes. These two regions may represent binding sites for splicing factors or may facilitate the formation of an alternative U6 snRNA secondary structure during spliceosome assembly.     

Human splicing factor SF3a, but not SF1, is essential for pre-mRNA splicing in vivo

The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B' are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis.