Crystal structure of Cwc2 reveals a novel architecture of a multipartite RNA-binding protein

The yeast splicing factor Cwc2 contacts several catalytically important RNA elements in the active spliceosome, suggesting that Cwc2 is involved in determining their spatial arrangement at the spliceosome's catalytic centre. We have determined the crystal structure of the Cwc2 functional core, revealing how a previously uncharacterized Torus domain, an RNA recognition motif (RRM) and a zinc finger (ZnF) are tightly integrated in a compact folding unit. The ZnF plays a pivotal role in the architecture of the whole assembly. UV-induced crosslinking of Cwc2-U6 snRNA allowed the identification by mass spectrometry of six RNA-contacting sites: four in or close to the RRM domain, one in the ZnF and one on a protruding element connecting the Torus and RRM domains. The three distinct regions contacting RNA are connected by a contiguous and conserved positively charged surface, suggesting an expanded interface for RNA accommodation. Cwc2 mutations confirmed that the connector element plays a crucial role in splicing. We conclude that Cwc2 acts as a multipartite RNA-binding platform to bring RNA elements of the spliceosome's catalytic centre into an active conformation.     

The RNA binding protein Cwc2 interacts directly with the U6 snRNA to link the nineteen complex to the spliceosome during pre-mRNA splicing

Intron removal during pre-messenger RNA (pre-mRNA) splicing involves arrangement of snRNAs into conformations that promote the two catalytic steps. The Prp19 complex [nineteen complex (NTC)] can specify U5 and U6 snRNA interactions with pre-mRNA during spliceosome activation. A candidate for linking the NTC to the snRNAs is the NTC protein Cwc2, which contains motifs known to bind RNA, a zinc finger and RNA recognition motif (RRM). In yeast cells mutation of either the zinc finger or RRM destabilize Cwc2 and are lethal. Yeast cells depleted of Cwc2 accumulate pre-mRNA and display reduced levels of U1, U4, U5 and U6 snRNAs. Cwc2 depletion also reduces U4/U6 snRNA complex levels, as found with depletion of other NTC proteins, but without increase in free U4. Purified Cwc2 displays general RNA binding properties and can bind both snRNAs and pre-mRNA in vitro. A Cwc2 RRM fragment alone can bind RNA but with reduced efficiency. Under splicing conditions Cwc2 can associate with U2, U5 and U6 snRNAs, but can only be crosslinked directly to the U6 snRNA. Cwc2 associates with U6 both before and after the first step of splicing. We propose that Cwc2 links the NTC to the spliceosome during pre-mRNA splicing through the U6 snRNA.     

Remodeling of U2-U6 snRNA helix I during pre-mRNA splicing by Prp16 and the NineTeen Complex protein Cwc2

Removal of intron regions from pre-messenger RNA (pre-mRNA) requires spliceosome assembly with pre-mRNA, then subsequent spliceosome remodeling to allow activation for the two steps of intron removal. Spliceosome remodeling is carried out through the action of DExD/H-box ATPases that modulate RNA–RNA and protein–RNA interactions. The ATPase Prp16 remodels the spliceosome between the first and second steps of splicing by catalyzing release of first step factors Yju2 and Cwc25 as well as destabilizing U2-U6 snRNA helix I. How Prp16 destabilizes U2-U6 helix I is not clear. We show that the NineTeen Complex (NTC) protein Cwc2 displays genetic interactions with the U6 ACAGAGA, the U6 internal stem loop (ISL) and the U2-U6 helix I, all RNA elements that form the spliceosome active site. We find that one function of Cwc2 is to stabilize U2-U6 snRNA helix I during splicing. Cwc2 also functionally cooperates with the NTC protein Isy1/NTC30. Mutation in Cwc2 can suppress the cold sensitive phenotype of the prp16-302 mutation indicating a functional link between Cwc2 and Prp16. Specifically the prp16-302 mutation in Prp16 stabilizes Cwc2 interactions with U6 snRNA and destabilizes Cwc2 interactions with pre-mRNA, indicating antagonistic functions of Cwc2 and Prp16. We propose that Cwc2 is a target for Prp16-mediated spliceosome remodeling during pre-mRNA splicing.     

The structure and selectivity of the SR protein SRSF2 RRM domain with RNA

SRSF2 is a prototypical SR protein which plays important roles in the alternative splicing of pre-mRNA. It has been shown to be involved in regulatory pathways for maintaining genomic stability and play important roles in regulating key receptors in the heart. We report here the solution structure of the RNA recognition motifs (RRM) domain of free human SRSF2 (residues 9-101). Compared with other members of the SR protein family, SRSF2 structure has a longer L3 loop region. The conserved aromatic residue in the RNP2 motif is absent in SRSF2. Calorimetric titration shows that the RNA sequence 5'AGCAGAGUA3' binds SRSF2 with a K(d) of 61 ± 1 nM and a 1:1 stoichiometry. NMR and mutagenesis experiments reveal that for SFSF2, the canonical β1 and β3 interactions are themselves not sufficient for effective RNA binding; the additional loop L3 is crucial for RNA complex formation. A comparison is made between the structures of SRSF2-RNA complex with other known RNA complexes of SR proteins. We conclude that interactions involving the L3 loop, N- and C-termini of the RRM domain are collectively important for determining selectivity between the protein and RNA.     

Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.     

Structural and functional analysis of RNA and TAP binding to SF2/ASF

The serine/arginine-rich (SR) protein splicing factor 2/alternative splicing factor (SF2/ASF) has a role in splicing, stability, export and translation of messenger RNA. Here, we present the structure of the RNA recognition motif (RRM) 2 from SF2/ASF, which has an RRM fold with a considerably extended loop 5 region, containing a two-stranded beta-sheet. The loop 5 extension places the previously identified SR protein kinase 1 docking sequence largely within the RRM fold. We show that RRM2 binds to RNA in a new way, by using a tryptophan within a conserved SWQLKD motif that resides on helix alpha1, together with amino acids from strand beta2 and a histidine on loop 5. The linker connecting RRM1 and RRM2 contains arginine residues, which provide a binding site for the mRNA export factor TAP, and when TAP binds to this region it displaces RNA bound to RRM2.     

Splicing factor Cwc22 is required for the function of Prp2 and for the spliceosome to escape from a futile pathway

Cwc22 was previously identified to associate with the pre-mRNA splicing factor Cef1/Ntc85, a component of the Prp19-associated complex (nineteen complex [NTC]) involved in spliceosome activation. We show here that Cwc22 is required for pre-mRNA splicing both in vivo and in vitro but is neither tightly associated with the NTC nor required for spliceosome activation. Cwc22 is associated with the spliceosome prior to catalytic steps and remains associated throughout the reaction. The stable association of Cwc22 with the spliceosome requires the presence of the NTC but is independent of Prp2. Although Cwc22 is not required for the recruitment of Prp2 to the spliceosome, it is essential for the function of Prp2 in promoting the release of the U2 components SF3a and SF3b. In the absence of Cwc22, Prp2 can bind to the spliceosome but is dissociated upon ATP hydrolysis without promoting the release of SF3a/b. Thus, Cwc22 represents a novel ATP-dependent step one factor besides Prp2 and Spp2 and has a distinct role from that of Spp2 in mediating the function of Prp2.     

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.     

Cwc2 and its human homologue RBM22 promote an active conformation of the spliceosome catalytic centre

RNA-structural elements play key roles in pre-mRNA splicing catalysis; yet, the formation of catalytically competent RNA structures requires the assistance of spliceosomal proteins. We show that the S. cerevisiae Cwc2 protein functions prior to step 1 of splicing, and it is not required for the Prp2-mediated spliceosome remodelling that generates the catalytically active B complex, suggesting that Cwc2 plays a more sophisticated role in the generation of a functional catalytic centre. In active spliceosomes, Cwc2 contacts catalytically important RNA elements, including the U6 internal stem-loop (ISL), and regions of U6 and the pre-mRNA intron near the 5' splice site, placing Cwc2 at/near the spliceosome's catalytic centre. These interactions are evolutionarily conserved, as shown by studies with Cwc2's human counterpart RBM22, indicating that Cwc2/RBM22-RNA contacts are functionally important. We propose that Cwc2 induces an active conformation of the spliceosome's catalytic RNA elements. Thus, the function of RNA-RNA tertiary interactions within group II introns, namely to induce an active conformation of domain V, may be fulfilled by proteins that contact the functionally analogous U6-ISL, within the spliceosome.     

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.     

Regiospecific Phosphorylation Control of the SR Protein ASF/SF2 by SRPK1

SR proteins (splicing factors containing arginine-serine repeats) are essential factors that control the splicing of precursor mRNA by regulating multiple steps in spliceosome development. The prototypical SR protein ASF/SF2 (human alternative splicing factor) contains two N-terminal RNA recognition motifs (RRMs) (RRM1 and RRM2) and a 50-residue C-terminal RS (arginine-serine-rich) domain that can be phosphorylated at numerous serines by the protein kinase SR-specific protein kinase (SRPK) 1. The RS domain [C-terminal domain that is rich in arginine-serine repeats (residues 198-248)] is further divided into N-terminal [RS1: N-terminal portion of the RS domain (residues 198-227)] and C-terminal [RS2: C-terminal portion of the RS domain (residues 228-248)] segments whose modification guides the nuclear localization of ASF/SF2. While previous studies revealed that SRPK1 phosphorylates RS1, regiospecific and temporal-specific control within the largely redundant RS domain is not well understood. To address this issue, we performed engineered footprinting and single-turnover experiments to determine where and how SRPK1 initiates phosphorylation within the RS domain. The data show that local sequence elements in the RS domain control the strong kinetic preference for RS1 phosphorylation. SRPK1 initiates phosphorylation in a small region of serines (initiation box) in the middle of the RS domain at the C-terminal end of RS1 and then proceeds in an N-terminal direction. This initiation process requires both a viable docking groove in the large lobe of SRPK1 and one RRM (RRM2) on the N-terminal flank of the RS domain. Thus, while local RS/SR content steers regional preferences in the RS domain, distal contacts with SRPK1 guide initiation and directional phosphorylation within these regions.     

Physical and genetic interactions of yeast Cwc21p,an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome

In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors—namely, Prp8p and Snu114p—and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu114p. Complementary chemical cross-linking experiments reveal reciprocal protein footprints between the interacting Prp8 and Cwc21 proteins, identifying the conserved cwf21 domain in Cwc21p as a Prp8p binding site. Genetic and functional interactions between Cwc21p and Isy1p indicate that they have related functions at or prior to the first catalytic step of splicing, and suggest that Cwc21p functions at the catalytic center of the spliceosome, possibly in response to environmental or metabolic changes. We demonstrate that SRm300, the only SR-related protein known to be at the core of human catalytic spliceosomes, is a functional ortholog of Cwc21p, also interacting directly with Prp8p and Snu114p. Thus, the function of Cwc21p is likely conserved from yeast to humans.     

RNA Binding of T-cell Intracellular Antigen-1 (TIA-1) C-terminal RNA Recognition Motif Is Modified by pH Conditions

T-cell intracellular antigen-1 (TIA-1) is a DNA/RNA-binding protein that regulates critical events in cell physiology by the regulation of pre-mRNA splicing and mRNA translation. TIA-1 is composed of three RNA recognition motifs (RRMs) and a glutamine-rich domain and binds to uridine-rich RNA sequences through its C-terminal RRM2 and RRM3 domains. Here, we show that RNA binding mediated by either isolated RRM3 or the RRM23 construct is controlled by slight environmental pH changes due to the protonation/deprotonation of TIA-1 RRM3 histidine residues. The auxiliary role of the C-terminal RRM3 domain in TIA-1 RNA recognition is poorly understood, and this work provides insight into its binding mechanisms.     

Apo raver1 structure reveals distinct RRM domain orientations

Raver1 is a multifunctional protein that modulates both alternative splicing and focal adhesion assembly by binding to the nucleoplasmic splicing repressor polypyrimidine tract protein (PTB) or to the cytoskeletal proteins vinculin and α‐actinin. The amino‐terminal region of raver1 has three RNA recognition motif (RRM1, RRM2, and RRM3) domains, and RRM1 interacts with the vinculin tail (Vt) domain and vinculin mRNA. We previously determined the crystal structure of the raver1 RRM1–3 domains in complex with Vt at 2.75 Å resolution. Here, we report crystal structure of the unbound raver1 RRM1–3 domains at 2 Å resolution. The apo structure reveals that a bound sulfate ion disrupts an electrostatic interaction between the RRM1 and RRM2 domains, triggering a large relative domain movement of over 30°. Superposition with other RNA‐bound RRM structures places the sulfate ion near the superposed RNA phosphate group suggesting that this is the raver1 RNA binding site. While several single and some tandem RRM domain structures have been described, to the best of our knowledge, this is the second report of a three‐tandem RRM domain structure.     

Solution structure and RNA interactions of the RNA recognition motif from eukaryotic translation initiation factor 4B

Eukaryotic initiation factor 4B (eIF4B) is a multidomain protein with a range of activities that serves primarily to promote association of messenger RNA to the 40S ribosomal subunit during translation initiation. We report here the solution structure of the eIF4B RNA recognition motif (RRM) domain. It adopts a classical RRM fold, with a beta alpha beta beta alpha beta topology. The most striking difference with other RRM structures is in the disposition of loop 3, which connects the beta 2 and beta 3 strands and is implicated in RNA recognition. This loop folds down against the body of the RRM and exhibits restricted motion on a milli- to microsecond time scale. Although it contributes to a large basic patch on the RNA binding surface, it does not protrude out from the domain as observed in other RRM structures, possibly implying a different mode of RNA binding. On its own, the core RRM domain provides only a relative weak interaction with RNA targets and appears to require extensions at the N- and C-terminus for high-affinity binding.     

A splicing repressor domain in polypyrimidine tract-binding protein

Polypyrimidine tract-binding protein (PTB) is an hnRNP with four RRM type domains. It plays roles as a repressive alternative splicing regulator of multilple target genes, as well as being involved in pre-mRNA 3' end processing, mRNA localization, stability, and internal ribosome entry site-mediated translation. Here we have used a tethered function assay, in which a fusion protein of PTB and the bacteriophage MS2 coat protein is recruited to a splicing regulatory site by binding to an artificially inserted MS2 binding site. Deletion mutations of PTB in this system allowed us to identify RRM2 and the following inter-RRM linker region as the minimal region of PTB that can act as splicing repressor domain when recruited to RNA. Splicing repression by the minimal repressor domain remained cell type-specific and dependent upon other defined regulatory elements in the alpha-tropomyosin test minigene. Our results highlight the fact that splicing repression by PTB can be uncoupled from the mode by which it binds to RNA.     

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.