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.     

Proteomic analysis identifies a new complex required for nuclear pre-mRNA retention and splicing

Using the proteomic tandem affinity purification (TAP) method, we have purified the Saccharomyces cerevisie U2 snRNP-associated splicing factors SF3a and SF3b. While SF3a purification revealed only the expected subunits Prp9p, Prp11p and Prp21p, yeast SF3b was found to contain only six subunits, including previously known components (Rse1p, Hsh155p, Cus1p, Hsh49p), the recently identified Rds3p factor and a new small essential protein (Ysf3p) encoded by an unpredicted split ORF in the yeast genome. Surprisingly, Snu17p, the proposed yeast orthologue of the seventh human SF3b subunit, p14, was not found in the yeast complex. TAP purification revealed that Snu17p, together with Bud13p and a newly identified factor, Pml1p/Ylr016c, form a novel trimeric complex. Subunits of this complex were not essential for viability. However, they are required for efficient splicing in vitro and in vivo. Furthermore, inactivation of this complex causes pre-mRNA leakage from the nucleus. The corresponding complex was named pre-mRNA REtention and Splicing (RES). The presence of RES subunit homologues in numerous eukaryotes suggests that its function is evolutionarily conserved.     

Functional Cus1p is found with Hsh155p in a multiprotein splicing factor associated with U2 snRNA

To explore the dynamics of snRNP structure and function, we have studied Cus1p, identified as a suppressor of U2 snRNA mutations in budding yeast. Cus1p is homologous to human SAP145, a protein present in the 17S form of the human U2 snRNP. Here, we define the Cus1p amino acids required for function in yeast. The segment of Cus1p required for binding to Hsh49p, a homolog of human SAP49, is contained within an essential region of Cus1p. Antibodies against Cus1p coimmunoprecipitate U2 snRNA, as well as Hsh155p, a protein homologous to human SAP155. Biochemical fractionation of splicing extracts and reconstitution of heat-inactivated splicing extracts from strains carrying a temperature-sensitive allele of CUS1 indicate that Cus1p and Hsh155p reside in a functional, high-salt-stable complex that is salt-dissociable from U2 snRNA. We propose that Cus1p, Hsh49p, and Hsh155p exist in a stable protein complex which can exchange with a core U2 snRNP and which is necessary for U2 snRNP function in prespliceosome assembly. The Cus1p complex shares functional as well as structural similarities with human SF3b.     

Dynamic Contacts of U2, RES,Cwc25, Prp8 and Prp45 Proteins with the Pre-mRNA Branch-Site and 3' Splice Site during Catalytic Activation and Step 1 Catalysis in Yeast Spliceosomes

Little is known about contacts in the spliceosome between proteins and intron nucleotides surrounding the pre-mRNA branch-site and their dynamics during splicing. We investigated protein-pre-mRNA interactions by UV-induced crosslinking of purified yeast B^act spliceosomes formed on site-specifically labeled pre-mRNA, and analyzed their changes after conversion to catalytically-activated B* and step 1 C complexes, using a purified splicing system. Contacts between nucleotides upstream and downstream of the branch-site and the U2 SF3a/b proteins Prp9, Prp11, Hsh49, Cus1 and Hsh155 were detected, demonstrating that these interactions are evolutionarily conserved. The RES proteins Pml1 and Bud13 were shown to contact the intron downstream of the branch-site. A comparison of the B^act crosslinking pattern versus that of B* and C complexes revealed that U2 and RES protein interactions with the intron are dynamic. Upon step 1 catalysis, Cwc25 contacts with the branch-site region, and enhanced crosslinks of Prp8 and Prp45 with nucleotides surrounding the branch-site were observed. Cwc25’s step 1 promoting activity was not dependent on its interaction with pre-mRNA, indicating it acts via protein-protein interactions. These studies provide important insights into the spliceosome''s protein-pre-mRNA network and reveal novel RNP remodeling events during the catalytic activation of the spliceosome and step 1 of splicing.     

A BBP-Mud2p heterodimer mediates branchpoint recognition and influences splicing substrate abundance in budding yeast

The 3' end of mammalian introns is marked by the branchpoint binding protein, SF1, and the U2AF65-U2AF35 heterodimer bound at an adjacent sequence. Baker's yeast has equivalent proteins, branchpoint binding protein (BBP) (SF1) and Mud2p (U2AF65), but lacks an obvious U2AF35 homolog, leaving open the question of whether another protein substitutes during spliceosome assembly. Gel filtration, affinity selection and mass spectrometry were used to show that rather than a U2AF65/U2AF35-like heterodimer, Mud2p forms a complex with BBP without a third (U2AF35-like) factor. Using mutants of MUD2 and BBP, we show that the BBP-Mud2p complex bridges partner-specific Prp39p, Mer1p, Clf1p and Smy2p two-hybrid interactions. In addition to inhibiting Mud2p association, the bbpDelta56 mutation impairs splicing, enhances pre-mRNA release from the nucleus, and similar to a mud2::KAN knockout, suppresses a lethal sub2::KAN mutation. Unexpectedly, rather than exacerbating bbpDelta56, the mud2::KAN mutation partially suppresses a pre-mRNA accumulation defect observed with bbpDelta56. We propose that a BBP-Mud2p heterodimer binds as a unit to the branchpoint in vivo and serves as a target for the Sub2p-DExD/H-box ATPase and for other splicing factors during spliceosome assembly. In addition, our results suggest the possibility that the Mud2p may enhance the turnover of pre-mRNA with impaired BBP-branchpoint association.     

Invariant U2 RNA sequences bordering the branchpoint recognition region are essential for interaction with yeast SF3a and SF3b subunits.

U2 small nuclear RNA (snRNA) contains a sequence (GUAGUA) that pairs with the intron branchpoint during splicing. This sequence is contained within a longer invariant sequence of unknown secondary structure and function that extends between U2 and I and stem IIa. A part of this region has been proposed to pair with U6 in a structure called helix III. We made mutations to test the function of these nucleotides in yeast U2 snRNA. Most single base changes cause no obvious growth defects; however, several single and double mutations are lethal or conditional lethal and cause a block before the first step of splicing. We used U6 compensatory mutations to assess the contribution of helix III and found that if it forms, helix III is dispensable for splicing in Saccharomyces cerevisiae. On the other hand, mutations in known protein components of the splicing apparatus suppress or enhance the phenotypes of mutations within the invariant sequence that connect the branchpoint recognition sequence to stem IIa. Lethal mutations in the region are suppressed by Cus1-54p, a mutant yeast splicing factor homologous to a mammalian SF3b subunit. Synthetic lethal interactions show that this region collaborates with the DEAD-box protein Prp5p and the yeast SF3a subunits Prp9p, Prp11p, and Prp21p. Together, the data show that the highly conserved RNA element downstream of the branchpoint recognition sequence of U2 snRNA in yeast cells functions primarily with the proteins that make up SF3 rather than with U6 snRNA.     

Combined biochemical and electron microscopic analyses reveal the architecture of the mammalian U2 snRNP.

The 17S U2 small nuclear ribonucleoprotein particle (snRNP) represents the active form of U2 snRNP that binds to the pre-mRNA during spliceosome assembly. This particle forms by sequential interactions of splicing factors SF3b and SF3a with the 12S U2 snRNP. We have purified SF3b and the 15S U2 snRNP, an intermediate in the assembly pathway, from HeLa cell nuclear extracts and show that SF3b consists of four subunits of 49, 130, 145, and 155 kD. Biochemical analysis indicates that both SF3b and the 12S U2 snRNP are required for the incorporation of SF3a into the 17S U2 snRNP. Nuclease protection studies demonstrate interactions of SF3b with the 5' half of U2 small nuclear RNA, whereas SF3a associates with the 3' portion of the U2 snRNP and possibly also interacts with SF3b. Electron microscopy of the 15S U2 snRNP shows that it consists of two domains in which the characteristic features of isolated SF3b and the 12S U2 snRNP are conserved. Comparison to the two-domain structure of the 17S U2 snRNP corroborates the biochemical results in that binding of SF3a contributes to an increase in size of the 12S U2 domain and possibly induces a structural change in the SF3b domain.     

Structure-function analysis and genetic interactions of the yeast branchpoint binding protein Msl5

Saccharomyces cerevisiae Msl5 (branchpoint binding protein) orchestrates spliceosome assembly by binding the branchpoint sequence 5'-UACUAAC and establishing cross intron-bridging interactions with other components of the splicing machinery. Reciprocal tandem affinity purifications verify that Msl5 exists in vivo as a heterodimer with Mud2 and that the Msl5-Mud2 complex is associated with the U1 snRNP. By gauging the ability of mutants of Msl5 to complement msl5Δ, we find that the Mud2-binding (amino acids 35-54) and putative Prp40-binding (PPxY(100)) elements of the Msl5 N-terminal domain are inessential, as are the C-terminal proline-rich domain (amino acids 382-476) and two zinc-binding CxxCxxxxHxxxxC motifs (amino acids 273-286 and 299-312). A subset of conserved branchpoint RNA-binding amino acids in the central KH-QUA2 domain (amino acids 146-269) are essential pairwise (Ile198-Arg190; Leu256-Leu259) or in trios (Leu169-Arg172-Leu176), whereas other pairs of RNA-binding residues are dispensable. We used our collection of viable Msl5 mutants to interrogate synthetic genetic interactions, in cis between the inessential structural elements of the Msl5 polypeptide and in trans between Msl5 and yeast splicing factors (Mud2, Nam8 and Tgs1) that are optional for vegetative growth. The results suggest a network of important but functionally buffered protein-protein and protein-RNA interactions between the Mud2-Msl5 complex at the branchpoint and the U1 snRNP at the 5' splice site.     

U2 snRNA-protein contacts in purified human 17S U2 snRNPs and in spliceosomal A and B complexes

The 17S U2 snRNP plays an essential role in branch point selection and catalysis during pre-mRNA splicing. Much remains to be learned about the molecular architecture of the U2 snRNP, including which proteins contact the functionally important 5' end of the U2 snRNA. Here, RNA-protein interactions within immunoaffinity-purified human 17S U2 snRNPs were analyzed by lead(II)-induced RNA cleavage and UV cross-linking. Contacts between the U2 snRNA and SF3a60, SF3b49, SF3b14a/p14 and SmG and SmB were detected. SF3b49 appears to make multiple contacts, interacting with the 5' end of U2 and nucleotides in loops I and IIb. SF3a60 also contacted different regions of the U2 snRNA, including the base of stem-loop I and a bulge in stem-loop III. Consistent with it contacting the pre-mRNA branch point adenosine, SF3b14a/p14 interacted with the U2 snRNA near the region that base pairs with the branch point sequence. A comparison of U2 cross-linking patterns obtained with 17S U2 snRNP versus purified spliceosomal A and B complexes revealed that RNA-protein interactions with stem-loop I and the branch site-interacting region of U2 are dynamic. These studies provide important insights into the molecular architecture of 17S U2 snRNPs and reveal U2 snRNP remodeling events during spliceosome assembly.     

The SF3b155 N-terminal domain is a scaffold important for splicing

Recruitment of U2 snRNP to the branch point sequence of introns is a necessary step in pre-mRNA splicing. In the current model, U2AF65, bound at the polypyrimidine tract of the intron, recruits U2 snRNP to the branch point sequence by interacting with the U2 snRNP protein SF3b155. We demonstrate that the N-terminal domain of SF3b155 contains multiple U2AF65 binding sites that are distinct from the binding site for the U2 snRNP protein p14, mapped to amino acids 396-424 of SF3b155. The N-terminal domain of SF3b155 appears to adopt a primarily unfolded structure but is functional to inhibit splicing in vitro. RNA binding studies show that the N-terminal domain of SF3b155 binds RNA nonspecifically and that the sites for U2AF65 binding and RNA binding are overlapping (or the same) within SF3b155. We propose that the N-terminal domain of SF3b155 adopts a primarily unfolded structure that functions as a scaffold to facilitate SF3b155's multiple protein-protein and protein-RNA interactions. The multiple U2AF65 binding sites on SF3b155 suggest a model in which multiple U2AF65 molecules bound to the intron could enhance U2 snRNP recruitment to the branch point sequence.     

Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing

The p14 subunit of the essential splicing factor 3b (SF3b) can be cross-linked to the branch-point adenosine of pre-mRNA introns within the spliceosome. p14 stably interacts with the SF3b subunit SF3b155, which also binds the 65-kDa subunit of U2 auxiliary splicing factor (U2AF65). We combined biochemical and NMR techniques to study the conformation of p14 either alone or complexed with SF3b155 fragments, as well as an interaction network involving p14, SF3b155, U2AF65, and U2 snRNA/pre-mRNA. p14 comprises a canonical RNA recognition motif (RRM) with an additional C-terminal helix (alphaC) and a beta hairpin insertion. SF3b155 binds to the beta-sheet surface of p14, thereby occupying the canonical RNA-binding site of the p14 RRM. The minimal region of SF3b155 interacting with p14 (i.e., residues 381-424) consists of four alpha-helices, which are partially preformed in isolation. Helices alpha2 and alpha3 (residues 401-415) constitute the core p14-binding epitope. Regions of SF3b155 binding to p14 and U2AF65 are nonoverlapping. This allows for a simultaneous interaction of SF3b155 with both proteins, which may support the stable association of U2 snRNP with the pre-mRNA. p14-RNA interactions are modulated by SF3b155 and the RNA-binding site of the p14-SF3b155 complex involves the noncanonical beta hairpin insertion of the p14 RRM, consistent with the beta-sheet surface being occupied by the helical SF3b155 peptide and p14 helix alphaC. Our data suggest that p14 lacks inherent specificity for recognizing the branch point, but that some specificity may be achieved by scaffolding interactions involving other components of SF3b.     

Protein localisation by electron microscopy reveals the architecture of the yeast spliceosomal B complex

The spliceosome assembles on a pre‐mRNA intron by binding of five snRNPs and numerous proteins, leading to the formation of the pre‐catalytic B complex. While the general morphology of the B complex is known, the spatial arrangement of proteins and snRNP subunits within it remain to be elucidated. To shed light on the architecture of the yeast B complex, we immuno‐labelled selected proteins and located them by negative‐stain electron microscopy. The B complex exhibited a triangular shape with main body, head and neck domains. We located the U5 snRNP components Brr2 at the top and Prp8 and Snu114 in the centre of the main body. We found several U2 SF3a (Prp9 and Prp11) and SF3b (Hsh155 and Cus1) proteins in the head domain and two U4/U6 snRNP proteins (Prp3 and Lsm4) in the neck domain that connects the main body with the head. Thus, we could assign distinct domains of the B complex to the respective snRNPs and provide the first detailed picture of the subunit architecture and protein arrangements of the B complex.     

FRET analyses of the U2AF complex localize the U2AF35/U2AF65 interaction in vivo and reveal a novel self-interaction of U2AF35

We have analyzed the interaction between the U2AF subunits U2AF35 and U2AF65 in vivo using fluorescence resonance energy transfer (FRET) microscopy. U2 snRNP Auxiliary Factor (U2AF) is an essential pre-mRNA splicing factor complex, comprising 35-kDa (U2AF35) and 65-kDa (U2AF65) subunits. U2AF65 interacts directly with the polypyrimidine tract and promotes binding of U2 snRNP to the pre-mRNA branchpoint, while U2AF35 associates with the conserved AG dinucleotide at the 3' end of the intron and has multiple functions in the splicing process. Using two different approaches for measuring FRET, we have identified and spatially localized sites of direct interaction between U2AF35 and U2AF65 in vivo in live cell nuclei. While U2AF is thought to function as a heterodimeric complex, the FRET data have also revealed a novel U2AF35 self-interaction in vivo, which is confirmed in vitro using biochemical assays. These results suggest that the stoichiometry of the U2AF complex may, at least in part, differ in vivo from the expected heterodimeric complex. The data show that FRET studies offer a valuable approach for probing interactions between pre-mRNA splicing factors in vivo.     

A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.     

Structural basis for recognition of intron branchpoint RNA by yeast Msl5 and selective effects of interfacial mutations on splicing of yeast pre-mRNAs

Saccharomyces cerevisiae Msl5 orchestrates spliceosome assembly by binding the intron branchpoint sequence 5′-UACUAAC and, with its heterodimer partner protein Mud2, establishing cross intron-bridging interactions with the U1 snRNP at the 5′ splice site. Here we define the central Msl5 KH-QUA2 domain as sufficient for branchpoint RNA recognition. The 1.8 Å crystal structure of Msl5-(KH-QUA2) bound to the branchpoint highlights an extensive network of direct and water-mediated protein–RNA and intra-RNA atomic contacts at the interface that illuminate how Msl5 recognizes each nucleobase of the UACUAAC element. The Msl5 structure rationalizes a large body of mutational data and inspires new functional studies herein, which reveal how perturbations of the Msl5·RNA interface impede the splicing of specific yeast pre-mRNAs. We also identify interfacial mutations in Msl5 that bypass the essentiality of Sub2, a DExD-box ATPase implicated in displacing Msl5 from the branchpoint in exchange for the U2 snRNP. These studies establish an atomic resolution framework for understanding splice site selection and early spliceosome dynamics.     

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.     

A yeast intronic splicing enhancer and Nam8p are required for Mer1p-activated splicing

Three introns whose splicing is activated during meiosis in S. cerevisiae contain a Mer1p-dependent splicing enhancer. The enhancer can impose Mer1p-activated splicing upon the constitutively spliced actin intron provided the basal splicing efficiency of actin is first reduced. Of several nonessential splicing factors tested, only the U1 snRNP protein Nam8p is indispensable for Mer1 p-activated splicing. We show that Mer1p associates with the U1 snRNP even in the absence of Nam8p or pre-mRNA. This work defines a yeast splicing enhancer and shows that constitutively expressed and cell type-specific factors combine to regulate splicing of a specific subset of pre-mRNAs including SPO70, MER2, and MER3.     

PUF60: a novel U2AF65-related splicing activity

We have identified a new pyrimidine-tract binding factor, PUF, that is required, together with U2AF, for efficient reconstitution of RNA splicing in vitro. The activity has been purified and consists of two proteins, PUF60 and the previously described splicing factor p54. p54 and PUF60 form a stable complex in vitro when cotranslated in a reaction mixture. PUF activity, in conjunction with U2AF, facilitates the association of U2 snRNP with the pre-mRNA. This reaction is dependent upon the presence of the large subunit of U2AF, U2AF65, but not the small subunit U2AF35. PUF60 is homologous to both U2AF65 and the yeast splicing factor Mud2p. The C-terminal domain of PUF60, the PUMP domain, is distantly related to the RNA-recognition motif domain, and is probably important in protein-protein interactions.