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.     

Multiple factors including the small nuclear ribonucleoproteins U1 and U2 are necessary for pre-mRNA splicing in vitro

We have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures. Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H. SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract. SF3 is a factor that can be preferentially inactivated by moderate heat treatment. Two additional factors (SF4A and SF4B) were identified by fractionation of the nuclear extract using spermine-agarose and CM-sepharose chromatography. SF1, SF2, and SF4B appear to be required for cleavage of the pre-mRNA at the 5' splice site and lariat formation, whereas SF3 and SF4A are only required for cleavage at the 3' splice site and exon ligation.     

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.     

Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.     

Sequence-specific affinity selection of mammalian splicing complexes.

Antisense oligonucleotides made of 2'-OMe RNA are shown to bind specifically and efficiently to targeted sites on pre-mRNA substrates, allowing affinity selection of splicing complexes using streptavidin/biotin chromatography. The position of probe binding to the pre-mRNA influences which type of splicing complex can be selected. The accessibility of pre-mRNA sequences to antisense probes changes during the course of the splicing reaction. U1, U2, U4, U5 and U6 snRNAs are all detected in affinity-selected mammalian splicing complexes. However, antisense oligonucleotides targeted to snRNAs can block the binding of specific snRNPs to pre-mRNA. Quantitative affinity selection analyses show that only a small fraction of snRNPs in a HeLa nuclear splicing extract participate in spliceosome formation.     

Roles of U4 and U6 snRNAs in the assembly of splicing complexes.

A series of U4 and U6 snRNA mutants was analysed in Xenopus oocytes to determine whether they block splicing complex assembly or splicing itself. All the U4 and U6 mutants found to be inactive in splicing complementation resulted in defects in assembly of either U4/U6 snRNP or of splicing complexes. No mutants were found to separate the entry of U5 and U6 snRNAs into splicing complexes and neither of these RNAs was able to associate with the pre-mRNA in the absence of U4. In the absence of U6 snRNA, however, U4 entered a complex containing pre-mRNA as well as the U1 and U2 snRNAs. U6 nucleotides whose mutation resulted in specific blockage of the second step of splicing in Saccharomyces cerevisiae are shown not to be essential for splicing in the oocyte assay. The results are discussed in terms of the roles of U4 and U6 in the assembly and catalytic steps of the splicing process.     

Different requirements of the kinase and UHM domains of KIS for its nuclear localization and binding to splicing factors

The protein kinase KIS is made by the juxtaposition of a unique kinase domain and a C-terminal domain with a U2AF homology motif (UHM), a sequence motif for protein interaction initially identified in the heterodimeric pre-mRNA splicing factor U2AF. This domain of KIS is closely related to the C-terminal UHM domain of the U2AF large subunit, U2AF⁶⁵. KIS phosphorylates the splicing factor SF1, which in turn enhances SF1 binding to U2AF⁶⁵ and the 3′ splice site, an event known to take place at an early step of spliceosome assembly. Here, the analysis of the subcellular localization of mutated forms of KIS indicates that the kinase domain of KIS is the necessary domain for its nuclear localization. As in the case of U2AF⁶⁵, the UHM-containing C-terminal domain of KIS is required for binding to the splicing factors SF1 and SF3b155. The efficiency of KIS binding to SF1 and SF3b155 is similar to that of U2AF⁶⁵ in pull-down assays. These results further support the functional link of KIS with splicing factors. Interestingly, when compared to other UHM-containing proteins, KIS presents a different specificity for the UHM docking sites that are present in the N-terminal region of SF3b155, thus providing a new insight into the variety of interactions mediated by UHM domains.     

Ser/Thr-specific protein phosphatases are required for both catalytic steps of pre-mRNA splicing.

We have used a combination of highly specific protein phosphatase inhibitors and purified mammalian protein phosphatases to show that at least two separate Ser/Thr protein phosphatase activities are required for pre-mRNA splicing, but not for spliceosome assembly. Okadaic acid, tautomycin, and microcystin-LR, which are potent and specific inhibitors of PP1 and PP2A, two of the four major types of Ser/Thr-specific phosphatase catalytic subunits, block both catalytic steps of the pre-mRNA splicing mechanism in HeLa nuclear extracts. Inhibition of PP2A inhibits the second step of splicing predominantly while inhibition of both PP1 and PP2A blocks both steps, indicating a differential contribution of PP1 and PP2A activities to the two separate catalytic steps of splicing. Splicing activity is restored to toxin-inhibited extracts by the addition of highly purified mammalian PP1 or PP2A. Protein phosphatase activity was not required for efficient assembly of splicing complexes containing each of the U1, U2, U4/U6 and U5 snRNPs. The data indicate that reversible protein phosphorylation may play an important role in regulating the pre-mRNA splicing mechanism.     

Factors associated with a purine-rich exonic splicing enhancer sequence in Xenopus oocyte nucleus

Purine-rich exonic splicing enhancers (ESEs) stimulate splicing of the adjacent introns with suboptimal splice sites. To elucidate the mechanism regarding ESEs, factors specifically associated with ESEs in HeLa cell nuclear extracts were previously investigated, and shown to include SR (serine/arginine-rich) proteins. However, factors associated with ESEs in vivo have not yet been explored. Here we show that a GAA repeat RNA sequence, a typical ESE, is associated in Xenopus oocyte nuclei with at least one SR protein, SF2/ASF, as was expected. Moreover, components of SF3a/b complexes, U2 snRNA, and U2AF(65) were also found to be associated with the ESE in the nucleus. Since SF3a/b complexes are the constituents of the 17S U2 snRNP, these results suggest that the 17S U2 snRNP is associated with the ESE in the nucleus, probably through bridging interactions of U2AF and SR proteins. The identified factors may represent a functional splicing enhancer complex in vivo.     

Multiple U2AF65 binding sites within SF3b155: thermodynamic and spectroscopic characterization of protein-protein interactions among pre-mRNA splicing factors

Essential, protein-protein complexes between the large subunit of the U2 small nuclear RNA auxiliary factor (U2AF65) with the splicing factor 1 (SF1) or the spliceosomal component SF3b155 are exchanged during a critical, ATP-dependent step of pre-mRNA splicing. Both SF1 and the N-terminal domain of SF3b155 interact with a U2AF homology motif (UHM) of U2AF65. SF3b155 contains seven tryptophan-containing sites with sequence similarity to the previously characterized U2AF65-binding domain of SF1. We show that the SF3b155 domain lacks detectable secondary structure using circular dichroism spectroscopy, and demonstrate that five of the tryptophan-containing SF3b155 sites are recognized by the U2AF65-UHM using intrinsic tryptophan fluorescence experiments with SF3b155 variants. When compared with SF1, similar spectral shifts and sequence requirements indicate that U2AF65 interactions with each of the SF3b155 sites are similar to the minimal SF1 site. However, thermodynamic comparison of SF1 or SF3b155 proteins with minimal peptides demonstrates that formation the SF1/U2AF65 complex is likely to affect regions of SF1 beyond the previously identified, linear interaction site, in a remarkably distinct manner from the local U2AF65 binding mode of SF3b155. Furthermore, the complex of the SF1/U2AF65 interacting domains is stabilized by 3.3 kcal mol-1 relative to the complex of the SF3b155/U2AF65 interacting domains, consistent with the need for ATP hydrolysis to drive exchange of these partners during pre-mRNA splicing. We propose that the multiple U2AF65 binding sites within SF3b155 regulate conformational rearrangements during spliceosome assembly. Comparison of the SF3b155 sites defines an (R/K)nXRW(DE) consensus sequence for predicting U2AF65-UHM ligands from genomic sequences, where parentheses denote residues that contribute to, but are not required for binding.     

Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly.

Splicing factor 1 (SF1) functions at early stages of pre-mRNA splicing and contributes to splice site recognition by interacting with the essential splicing factor U2AF65 and binding to the intron branch site. We have identified an 80 kDa substrate of cGMP-dependent protein kinase-I (PKG-I) isolated from rat brain, which is identical to SF1. PKG phosphorylates SF1 at Ser20, which inhibits the SF1-U2AF65 interaction leading to a block of pre-spliceosome assembly. Mutation of Ser20 to Ala or Thr also inhibits the interaction with U2AF65, indicating that Ser20 is essential for binding. SF1 is phosphorylated in vitro by PKG, but not by cAMP-dependent protein kinase A (PKA). Phosphorylation of SF1 also occurs in cultured neuronal cells and is increased on Ser20 in response to a cGMP analogue. These results suggest a new role for PKG in mammalian pre-mRNA splicing by regulating in a phosphorylation-dependent manner the association of SF1 with U2AF65 and spliceosome assembly.     

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.     

Transient interaction of BBP/ScSF1 and Mud2 with the splicing machinery affects the kinetics of spliceosome assembly.

Removal of introns from pre-mRNA is an essential step of gene expression. The splicing reaction is catalyzed in a large complex termed the spliceosome. Introns are recognized during the early steps of spliceosome assembly with the formation of commitment complexes. Intron recognition is mediated by the interaction of splicing factors with conserved sequences present in the pre-mRNA. BBP/SF1 participates in this recognition by interacting with the pre-mRNA branch point in both yeast and mammals. This protein, which is essential in yeast, also interacts with the U2AF65/Mud2 splicing factor. However, its precise role in splicing complex formation is still unclear. We have now analyzed the presence of BBP and Mud2 in yeast splicing complexes using supershift and coprecipitation assays. We found that BBP is present together with Mud2 in commitment complex 2 (CC2), but is not detectable in commitment complex 1 (CC1). Furthermore, genetic and biochemical depletion of BBP demonstrated that it is required for CC2 formation. In addition we observed that BBP and Mud2 are not detectable in pre-spliceosomes. These are the first commitment complex components that are shown to be released during or immediately after pre-spliceosome formation. Interestingly, depletion of BBP or disruption of MUD2 had no significant effect on pre-spliceosome formation and splicing in vitro but led to a transient accumulation of CC1. These observations support a model in which BBP and Mud2 are recycled during transition from CC2 to pre-spliceosome.     

Crooked neck is a component of the human spliceosome and implicated in the splicing process

The Drosophila crooked neck (crn) gene is essential for embryogenesis and has been implicated in cell cycle progression and in pre-mRNA splicing although a direct role in either process has not been established. Here we report isolation of the human crooked neck homolog, HCRN, and provide evidence for its function in splicing. HCRN encodes an unusual protein composed largely of tetratricopeptide repeat (TPR) elements. The crooked neck protein co-localizes with the SR and Sm protein splicing factors in discrete subnuclear domains implicated in snRNP biogenesis. In vitro assembly experiments show that an 83 kDa hcrn isoform is stably recruited to splicing complexes coincident with the addition of the U4/U6.U5 tri-snRNP particle. Crooked neck activity appears essential as extracts depleted of hcrn fail to splice pre-mRNA. These and related data support the view that crooked neck is a phylogenetically conserved pre-mRNA splicing factor.     

The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities.

ASF/SF2 and SC35 belong to a highly conserved family of nuclear proteins that are both essential for splicing of pre-mRNA in vitro and are able to influence selection of alternative splice sites. An important question is whether these proteins display distinct RNA binding specificities and, if so, whether this influences their functional interactions with pre-mRNA. To address these issues, we first performed selection/amplification from pools of random RNA sequences (SELEX) with portions of the two proteins comprising the RNA binding domains (RBDs). Although both molecules selected mainly purine-rich sequences, comparison of individual sequences indicated that the motifs recognized are different. Binding assays performed with the full-length proteins confirmed that ASF/SF2 and SC35 indeed have distinct specificities, and at the same time provided evidence that the highly charged arginine-serine region of each protein is not a major determinant of specificity. In the case of ASF/SF2, evidence is presented that binding specificity involves cooperation between the protein's two RBDs. Finally, we demonstrate that an element containing three copies of a high-affinity ASF/SF2 binding site constitutes a powerful splicing enhancer. In contrast, a similar element consisting of three SC35 sites was inactive. The ASF/SF2 enhancer can be activated specifically in splicing-deficient S100 extracts by recombinant ASF/SF2 in conjunction with one or more additional protein factors. These and other results suggest a central role for ASF/SF2 in the function of purine-rich splicing enhancers.     

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).