Investigation of a conserved stacking interaction in target site recognition by the U1A protein

Three highly conserved aromatic residues in RNA recognition motifs (RRM) participate in stacking interactions with RNA bases upon binding RNA. We have investigated the contribution of one of these aromatic residues, Phe56, to the complex formed between the N-terminal RRM of the spliceosomal protein U1A and stem–loop 2 of U1 snRNA. Previous work showed that the aromatic group is important for high affinity binding. Here we probe how mutation of Phe56 affects the kinetics of complex dissociation, the strength of the hydrogen bonds formed between U1A and the base that stacks with Phe56 (A6) and specific target site recognition. Substitution of Phe56 with Trp or Tyr increased the rate of dissociation of the complex, consistent with previously reported results. However, substitution of Phe56 with His decreased the rate of complex association, implying a change in the initial formation of the complex. Simultaneous modification of residue 56 and A6 revealed energetic coupling between the aromatic group and the functional groups of A6 that hydrogen bond to U1A. Finally, mutation of Phe56 to Leu reduced the ability of U1A to recognize stem–loop 2 correctly. Taken together, these experiments suggest that Phe56 contributes to binding affinity by stacking with A6 and participating in networks of energetically coupled interactions that enable this conserved aromatic amino acid to play a complex role in target site recognition.     

Molecular dynamics simulation studies of a protein-RNA complex with a selectively modified binding interface

The RNA recognition motif (RRM) is one of the most common RNA binding domains. We have investigated the contribution of three highly conserved aromatic amino acids to RNA binding by the N-terminal RRM of the U1A protein. Recently, we synthesized a modified base (A-4CPh) in which a phenyl group is tethered to adenine using a linker of 4 methylene groups. The substitution of this base for adenine in the target RNA selectively stabilizes the complex formed with a U1A protein in which one of the conserved aromatic amino acids is replaced with Ala (Phe56Ala). In this article, we report molecular dynamics (MD) simulations that probe the structural consequences of the substitution of A-4CPh for adenine in the wild type and Phe56Ala U1A-RNA complexes and in the free RNA. The simulations suggest that A-4CPh stabilizes the complex formed with Phe56Ala by adopting a folded conformation in which the tethered phenyl group fills the site occupied by the phenyl group of Phe56 in the wild-type complex. In contrast, an extended conformation of A-4CPh is predicted to be most stable in the complex formed with the wild-type protein. The calculations indicate A-4CPh is in an extended conformation in the free RNA. Therefore, preorganizing the structure of the phenyl-tethered base for binding may improve both the affinity and specificity of the RNA containing A-4CPh for the Phe56Ala U1A protein. Taken together, the previous experimental work and the calculations reported here suggest a general design strategy for altering RRM-RNA complex stability.     

U1A protein-stem loop 2 RNA recognition: prediction of structural differences from protein mutations

Molecular dynamics (MD) simulations were carried out to compare the free and bound structures of wild type U1A protein with several Phe56 mutant U1A proteins that bind the target stem loop 2 (SL2) RNA with a range of affinities. The simulations indicate the free U1A protein is more flexible than the U1A-RNA complex for both wild type and Phe56 mutant systems. A complete analysis of the hydrogen-bonding (HB) and non-bonded (VDW) interactions over the course of the MD simulations suggested that changes in the interactions in the free U1A protein caused by the Phe56Ala and Phe56Leu mutations may stabilize the helical character in loop 3, and contribute to the weak binding of these proteins to SL2 RNA. Compared with wild type, changes in HB and VDW interactions in Phe56 mutants of the free U1A protein are global, and include differences in β-sheet, loop 1 and loop 3 interactions. In the U1A-RNA complex, the Phe56Ala mutation leads to a series of differences in interactions that resonate through the complex, while the Phe56Leu and Phe56Trp mutations cause local differences around the site of mutation. The long-range networks of interactions identified in the simulations suggest that direct interactions and dynamic processes in both the free and bound forms contribute to complex stability.     

Interaction of N-terminal domain of U1A protein with an RNA stem/loop.

The U1A protein is a sequence-specific RNA binding protein found in the U1 snRNP particle where it binds to stem/loop II of U1 snRNA. U1A contains two 'RNP' or 'RRM' (RNA Recognition Motif) domains, which are common to many RNA-binding proteins. The N-terminal RRM has been shown to bind specifically to the U1 RNA stem/loop, while the RNA target of the C-terminal domain is unknown. Here, we describe experiments using a 102 amino acid N-terminal RRM of U1A (102A) and a 25-nucleotide RNA stem/loop to measure the binding constants and thermodynamic parameters of this RNA:protein complex. Using nitrocellulose filter binding, we measure a dissociation constant KD = 2 x 10(-11) M in 250 mM NaCl, 2 mM MgC2, and 10 mM sodium cacodylate, pH 6 at room temperature, and a half-life for the complex of 5 minutes. The free energy of association (delta G degrees) of this complex is about -14 kcal/mol in these conditions. Determination of the salt dependence of the binding suggests that at least 8 ion-pairs are formed upon complex formation. A mutation in the RNA loop sequence reduces the affinity 10 x, or about 10% of the total free energy.     

Kinetic analysis of the role of the tyrosine 13, phenylalanine 56 and glutamine 54 network in the U1A/U1 hairpin II interaction

The A protein of the U1 small nuclear ribonucleoprotein particle, interacting with its stem–loop RNA target (U1hpII), is frequently used as a paradigm for RNA binding by recognition motif domains (RRMs). U1A/U1hpII complex formation has been proposed to consist of at least two steps: electrostatically mediated alignment of both molecules followed by locking into place, based on the establishment of close-range interactions. The sequence of events between alignment and locking remains obscure. Here we examine the roles of three critical residues, Tyr13, Phe56 and Gln54, in complex formation and stability using Biacore. Our mutational and kinetic data suggest that Tyr13 plays a more important role than Phe56 in complex formation. Mutational analysis of Gln54, combined with molecular dynamics studies, points to Arg52 as another key residue in association. Based on our data and previous structural and modeling studies, we propose that electrostatic alignment of the molecules is followed by hydrogen bond formation between the RNA and Arg52, and the sequential establishment of interactions with loop bases (including Tyr13). A quadruple stack, sandwiching two bases between Phe56 and Asp92, would occur last and coincide with the rearrangement of a C-terminal helix that partially occludes the RRM surface in the free protein.     

The N- and C-terminal RNA recognition motifs of splicing factor Prp24 have distinct functions in U6 RNA binding

Prp24 is an essential yeast U6 snRNP protein with four RNA recognition motifs (RRMs) that facilitates the association of U4 and U6 snRNPs during spliceosome assembly. Genetic interactions led to the proposal that RRMs 2 and 3 of Prp24 bind U6 RNA, while RRMs 1 and 4 bind U4 RNA. However, the function of each RRM has yet to be established through biochemical means. We compared the binding of recombinant full-length Prp24 and truncated forms lacking RRM 1 or RRM 4 with U6 RNA. Contrary to expectations, we found that the N-terminal segment containing RRM 1 is important for high-affinity binding to U6 RNA and for discrimination between wild-type U6 RNA and U6 with point mutations in the 3' intramolecular stem-loop. In contrast, deletion of RRM 4 and the C terminus did not significantly alter the affinity for U6 RNA, but resulted in the formation of higher order Prp24.U6 complexes. Truncation and internal deletion of U6 RNA mapped three Prp24-binding sites, with the central site providing most of the affinity for Prp24. A newly identified temperature-sensitive lethal point mutation in RRM 1 is exacerbated by mutations in the U6 RNA telestem, as is a mutation in RRM 2, but not one in RRM 3. We propose that RRMs 1 and 2 of yeast Prp24 bind the same central site in U6 RNA that is bound by the two RRMs of human Prp24, and that RRMs 3 and 4 bind lower affinity flanking sites, thereby restricting the stoichiometry of Prp24 binding.     

The role of the C-terminal helix of U1A protein in the interaction with U1hpII RNA

Previous kinetic investigations of the N-terminal RNA Recognition Motif (RRM) domain of spliceosomal A protein of the U1 small nuclear ribonucleoprotein particle (U1A) interacting with its RNA target U1 hairpin II (U1hpII) provided experimental evidence for a ‘lure and lock’ model of binding. The final step of locking has been proposed to involve conformational changes in an α-helix immediately C-terminal to the RRM domain (helix C), which occludes the RNA binding surface in the unbound protein. Helix C must shift its position to accommodate RNA binding in the RNA–protein complex. This results in a new hydrophobic core, an intraprotein hydrogen bond and a quadruple stacking interaction between U1A and U1hpII. Here, we used a surface plasmon resonance-based biosensor to gain mechanistic insight into the role of helix C in mediating the interaction with U1hpII. Truncation, removal or disruption of the helix exposes the RNA-binding surface, resulting in an increase in the association rate, while simultaneously reducing the ability of the complex to lock, reflected in a loss of complex stability. Disruption of the quadruple stacking interaction has minor kinetic effects when compared with removal of the intraprotein hydrogen bonds. These data provide new insights into the mechanism whereby sequences C-terminal to an RRM can influence RNA binding.     

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.     

Structural insights into RNA recognition by the alternate-splicing regulator CUG-binding protein 1

CUG-binding protein 1 (CUGBP1) regulates multiple aspects of nuclear and cytoplasmic mRNA processing, with implications for onset of myotonic dystrophy. CUGBP1 harbors three RRM domains and preferentially targets UGU-rich mRNA elements. We describe crystal structures of CUGBP1 RRM1 and tandem RRM1/2 domains bound to RNAs containing tandem UGU(U/G) elements. Both RRM1 in RRM1-RNA and RRM2 in RRM1/2-RNA complexes use similar principles to target UGU(U/G) elements, with recognition mediated by face-to-edge stacking and water-mediated hydrogen-bonding networks. The UG step adopts a left-handed Z-RNA conformation, with the syn guanine recognized through Hoogsteen edge-protein backbone hydrogen-bonding interactions. NMR studies on the RRM1/2-RNA complex establish that both RRM domains target tandem UGUU motifs in solution, whereas filter-binding assays identify a preference for recognition of GU over AU or GC steps. We discuss the implications of CUGBP1-mediated targeting and sequestration of UGU(U/G) elements on pre-mRNA alternative-splicing regulation, translational regulation, and mRNA decay.     

Large favorable enthalpy changes drive specific RNA recognition by RNA recognition motif proteins

The RNA recognition motif (RRM) is a prevalent class of RNA binding domains. Although a number of RRM/RNA structures have been determined, thermodynamic analyses are relatively uncommon. Here, we use isothermal titration calorimetry to characterize single-stranded (ss)RNA binding by four representative RRM-containing proteins: (i) U2AF(65), (ii) SXL, (iii) TIA-1, and (iv) PAB. In all cases, ssRNA binding is accompanied by remarkably large favorable enthalpy changes (-30 to -60 kcal mol(-1)) and unfavorable entropy changes. Alterations of key RRM residues and binding sites indicate that under the nearly physiological conditions of these studies, large thermodynamic changes represent a signature of specific ssRNA recognition by RRMs.     

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.     

The role of positively charged amino acids and electrostatic interactions in the complex of U1A protein and U1 hairpin II RNA

Previous kinetic investigations of the N-terminal RNA recognition motif (RRM) domain of spliceosomal protein U1A, interacting with its RNA target U1 hairpin II, provided experimental evidence for a ‘lure and lock’ model of binding in which electrostatic interactions first guide the RNA to the protein, and close range interactions then lock the two molecules together. To further investigate the ‘lure’ step, here we examined the electrostatic roles of two sets of positively charged amino acids in U1A that do not make hydrogen bonds to the RNA: Lys20, Lys22 and Lys23 close to the RNA-binding site, and Arg7, Lys60 and Arg70, located on ‘top’ of the RRM domain, away from the RNA. Surface plasmon resonance-based kinetic studies, supplemented with salt dependence experiments and molecular dynamics simulation, indicate that Lys20 predominantly plays a role in association, while nearby residues Lys22 and Lys23 appear to be at least as important for complex stability. In contrast, kinetic analyses of residues away from the RNA indicate that they have a minimal effect on association and stability. Thus, well-positioned positively charged residues can be important for both initial complex formation and complex maintenance, illustrating the multiple roles of electrostatic interactions in protein–RNA complexes.     

The role of RNA structure in the interaction of U1A protein with U1 hairpin II RNA

The N-terminal RNA Recognition Motif (RRM1) of the spliceosomal protein U1A interacting with its target U1 hairpin II (U1hpII) has been used as a paradigm for RRM-containing proteins interacting with their RNA targets. U1A binds to U1hpII via direct interactions with a 7-nucleotide (nt) consensus binding sequence at the 5' end of a 10-nt loop, and via hydrogen bonds with the closing C-G base pair at the top of the RNA stem. Using surface plasmon resonance (Biacore), we have examined the role of structural features of U1hpII in binding to U1A RRM1. Mutational analysis of the closing base pair suggests it plays a minor role in binding and mainly prevents "breathing" of the loop. Lengthening the stem and nontarget part of the loop suggests that the increased negative charge of the RNA might slightly aid association. However, this is offset by an increase in dissociation, which may be caused by attraction of the RRM to nontarget parts of the RNA. Studies of a single stranded target and RNAs with untethered loops indicate that structure is not very relevant for association but is important for complex stability. In particular, breaking the link between the stem and the 5' side of the loop greatly increases complex dissociation, presumably by hindering simultaneous contacts between the RRM and stem and loop nucleotides. While binding of U1A to a single stranded target is much weaker than to U1hpII, it occurs with nanomolar affinity, supporting recent evidence that binding of unstructured RNA by U1A has physiological significance.     

Functional analysis of SNF, the Drosophila U1A/U2B" homolog: identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-UlA and U2 snRNP-U2B". The structure and sequence of SNF, U1A, and U2B" are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B" binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the "RNA-specificity" domain is necessary for SNF's dual function whereas the mixed motif in the U2A'-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.     

Spliceosome assembly in the absence of stable U4/U6 RNA pairing

The cycle of spliceosome assembly, intron excision, and spliceosome disassembly involves large-scale structural rearrangements of U6 snRNA that are functionally important. U6 enters the splicing pathway bound to the Prp24 protein, which chaperones annealing of U6 to U4 RNA to form a U4/U6 di-snRNP. During catalytic activation of the assembled spliceosome, U4 snRNP is released and U6 is paired to U2 snRNA. Here we show that point mutations in U4 and U6 that decrease U4/U6 base-pairing in vivo are lethal in combination. However, this synthetic phenotype is rescued by a mutation in U6 that alters a U6–Prp24 contact and stabilizes U2/U6. Remarkably, the resulting viable triple mutant strain lacks detectable U4/U6 base-pairing and U4/U6 di-snRNP. Instead, this strain accumulates free U4 snRNP, protein-free U6 RNA, and a novel complex containing U2/U6 di-snRNP. Further mutational analysis indicates that disruption of the U6–Prp24 interaction rather than stabilization of U2/U6 renders stable U4/U6 di-snRNP assembly nonessential. We propose that an essential function of U4/U6 pairing is to displace Prp24 from U6 RNA, and thus a destabilized U6–Prp24 complex renders stable U4/U6 pairing nonessential.     

Three RNA recognition motifs participate in RNA recognition and structural organization by the pro-apoptotic factor TIA-1

T-cell intracellular antigen-1 (TIA-1) regulates developmental and stress-responsive pathways through distinct activities at the levels of alternative pre-mRNA splicing and mRNA translation. The TIA-1 polypeptide contains three RNA recognition motifs (RRMs). The central RRM2 and C-terminal RRM3 associate with cellular mRNAs. The N-terminal RRM1 enhances interactions of a C-terminal Q-rich domain of TIA-1 with the U1-C splicing factor, despite linear separation of the domains in the TIA-1 sequence. Given the expanded functional repertoire of the RRM family, it was unknown whether TIA-1 RRM1 contributes to RNA binding as well as documented protein interactions. To address this question, we used isothermal titration calorimetry and small-angle X-ray scattering to dissect the roles of the TIA-1 RRMs in RNA recognition. Notably, the fas RNA exhibited two binding sites with indistinguishable affinities for TIA-1. Analyses of TIA-1 variants established that RRM1 was dispensable for binding AU-rich fas sites, yet all three RRMs were required to bind a polyU RNA with high affinity. Small-angle X-ray scattering analyses demonstrated a "V" shape for a TIA-1 construct comprising the three RRMs and revealed that its dimensions became more compact in the RNA-bound state. The sequence-selective involvement of TIA-1 RRM1 in RNA recognition suggests a possible role for RNA sequences in regulating the distinct functions of TIA-1. Further implications for U1-C recruitment by the adjacent TIA-1 binding sites of the fas pre-mRNA and the bent TIA-1 shape, which organizes the N- and C-termini on the same side of the protein, are discussed.     

The crystal structure of human isopentenyl diphosphate isomerase at 1.7 A resolution reveals its catalytic mechanism in isoprenoid biosynthesis

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.