NMR studies of U1 snRNA recognition by the N-terminal RNP domain of the human U1A protein.

The RNP domain is a very common motif found in hundreds of proteins, including many protein components of the RNA processing machinery. The 70-90 amino acid domain contains two highly conserved stretches of 6-8 amino acids (RNP-1 and RNP-2) in the central strands of a four-stranded antiparallel beta-sheet, packed against two alpha-helices by a conserved hydrophobic core. Using multidimensional heteronuclear NMR, we have mapped intermolecular contacts between the human U1A protein 102 amino acid N-terminal RNP domain and a 31-mer oligonucleotide derived from stem-loop II of U1 snRNA. Chemical shift changes induced on the protein by the RNA define the surface of the beta-sheet as the recognition interface. The reverse face of the protein, with the two alpha-helices, remains exposed to the solvent in the presence of the RNA, and is potentially available for protein-protein contacts in spliceosome assembly or splice site selection. Protein-RNA contacts occur at the single-stranded apical loop of the hairpin, but also in the major groove of the helical stem at neighbouring U.G and U.U non-Watson-Crick base pairs. Examination of a proposed model for the complex in the light of the present results reveals several features of RNA recognition by RNP proteins. The quality of the spectra for this complex of 22 kDa demonstrates the feasibility of NMR investigation of RNA-protein complexes.     

Recognition of U1 and U2 small nuclear RNAs can be altered by a 5-amino-acid segment in the U2 small nuclear ribonucleoprotein particle (snRNP) B" protein and through interactions with U2 snRNP-A'' protein.

We have investigated the sequence elements influencing RNA recognition in two closely related small nuclear ribonucleoprotein particle (snRNP) proteins, U1 snRNP-A and U2 snRNP-B". A 5-amino-acid segment in the RNA-binding domain of the U2 snRNP-B" protein was found to confer U2 RNA recognition when substituted into the corresponding position in the U1 snRNP-A protein. In addition, B", but not A, was found to require the U2 snRNP-A' protein as an accessory factor for high-affinity binding to U2 RNA. The pentamer segment in B" that conferred U2 RNA recognition was not sufficient to allow the A' enhancement of U2 RNA binding by B", thus implicating other sequences in this protein-protein interaction. Sequence elements involved in these interactions have been localized to variable loops of the RNA-binding domain as determined by nuclear magnetic resonance spectroscopy (D. Hoffman, C.C. Query, B. Golden, S.W. White, and J.D. Keene, Proc. Natl. Acad. Sci. USA, in press). These findings suggest a role for accessory proteins in the formation of RNP complexes and pinpoint amino acid sequences that affect the specificity of RNA recognition in two members of a large family of proteins involved in RNA processing.     

The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression

The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM-RNA or RRM-protein), we reviewed the structure-function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM-nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM beta-sheet, the two external beta-strands, the loops, the C- and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein-RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM-containing proteins.     

Structure and RNA binding of the third KH domain of poly(C)-binding protein 1

Poly(C)-binding proteins (CPs) are important regulators of mRNA stability and translational regulation. They recognize C-rich RNA through their triple KH (hn RNP K homology) domain structures and are thought to carry out their function though direct protection of mRNA sites as well as through interactions with other RNA-binding proteins. We report the crystallographically derived structure of the third domain of alphaCP1 to 2.1 A resolution. alphaCP1-KH3 assumes a classical type I KH domain fold with a triple-stranded beta-sheet held against a three-helix cluster in a betaalphaalphabetabetaalpha configuration. Its binding affinity to an RNA sequence from the 3'-untranslated region (3'-UTR) of androgen receptor mRNA was determined using surface plasmon resonance, giving a K(d) of 4.37 microM, which is indicative of intermediate binding. A model of alphaCP1-KH3 with poly(C)-RNA was generated by homology to a recently reported RNA-bound KH domain structure and suggests the molecular basis for oligonucleotide binding and poly(C)-RNA specificity.     

Molecular basis of RNA recognition by the human alternative splicing factor Fox-1

The Fox-1 protein regulates alternative splicing of tissue-specific exons by binding to GCAUG elements. Here, we report the solution structure of the Fox-1 RNA binding domain (RBD) in complex with UGCAUGU. The last three nucleotides, UGU, are recognized in a canonical way by the four-stranded beta-sheet of the RBD. In contrast, the first four nucleotides, UGCA, are bound by two loops of the protein in an unprecedented manner. Nucleotides U1, G2, and C3 are wrapped around a single phenylalanine, while G2 and A4 form a base-pair. This novel RNA binding site is independent from the beta-sheet binding interface. Surface plasmon resonance analyses were used to quantify the energetic contributions of electrostatic and hydrogen bond interactions to complex formation and support our structural findings. These results demonstrate the unusual molecular mechanism of sequence-specific RNA recognition by Fox-1, which is exceptional in its high affinity for a defined but short sequence element.     

Contribution of the C-terminal tail of U1A RBD1 to RNA recognition and protein stability.

The N-terminal RNA binding domain (RBD1) of the human U1A protein interacts specifically with a short RNA hairpin containing the U1 snRNA stem/loop II sequence. Previous RNA binding studies have suggested that the C-terminal tail of RBD1 contributes to RNA recognition in addition to interactions on the beta-sheet surface of the protein. To evaluate the contributions of these C-terminal residues in RBD1 to RNA binding affinity and specificity, as well as to study the thermodynamic stability of RBDs, a number of RBD1 mutants with truncated tails, with single amino acid substitutions, and with both a truncation and an amino acid substitution, have been constructed. The thermodynamic stabilities of these mutants have been measured and compared by GdnHCI unfolding experiments. The RNA binding affinity and specificity of these mutant proteins have been assessed by measuring the binding of each protein to the wild-type RNA hairpin and to selected RNA mutants with nucleotide substitutions in the RNA loop. The results demonstrate first that, although the C-terminal tail of RBD1 makes significant contributions to RNA binding affinity, it is not required for RNA binding, and second, its contributions to binding specificity are mediated only through selected nucleotides in the RNA loop, for in the absence of the tail, the protein continues to use other nucleotides to discriminate among RNAs. In these truncated proteins, the secondary structure intrinsic to the C-terminal tail is absent, yet their affinity and discrimination for RNAs are not lost. Thus, a structured tail is not required for RNA recognition.     

Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a new protein family with an RNA recognition motif-like domain

We have determined the crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 at 1.9 A resolution. This protein is a member of the Escherichia coli ygcH sequence family, which contains approximately 15 sequence homologs of bacterial origin. These homologs have a high isoelectric point. The crystal structure reveals that TTHB192 consists of two independently folded domains, and that each domain exhibits a ferredoxin-like fold with a four-stranded antiparallel beta-sheet packed on one side by alpha-helices. These two tandem domains face each other to generate a beta-sheet platform. TTHB192 displays overall structural similarity to Sex-lethal protein and poly(A)-binding protein fragments. These proteins have RNA binding activity which is supported by a beta-sheet platform formed by two tandem repeats of an RNA recognition motif domain with signature sequence motifs on the beta-sheet surface. Although TTHB192 does not have the same signature sequence motif as the RNA recognition motif domain, the presence of an evolutionarily conserved basic patch on the beta-sheet platform could be functionally relevant for nucleic acid-binding. This report shows that TTHB192 and its sequence homologs adopt an RNA recognition motif-like domain and provides the first testable functional hypothesis for this protein family.     

Prediction of a new class of RNA recognition motif

The observation that activation domains (AD) of procarboxypeptidases are rather long compared to the pro-regions of other zymogens raises the possibility that they could play additional roles apart from precluding enzymatic activity within the proenzyme and helping in its folding process. In the present work, we compared the overall pro-domain tertiary structure with several proteins belonging to the same fold in the structural classification of proteins (SCOP) database by using structure and sequence comparisons. The best score obtained was between the activation domain of human procarboxypeptidase A4 (ADA4h) and the human U1A protein from the U1 snRNP. Structural alignment revealed the existence of RNP1- and RNP2-related sequences in ADA4h. After modeling ADA4h on U1A, the new structure was used to extract a new sequence pattern characteristic for important residues at key positions. The new sequence pattern allowed scanning protein sequences to predict the RNA-binding function for 32 sequences undetected by PFAM. Unspecific RNA electrophoretic mobility shift assays experimentally supported the prediction that ADA4h binds an RNA motif similar to the U1A binding-motif of stem-loop II of U1 small nuclear RNA. The experiments carried out with ADA4h in the present work suggest the sharing of a common ancestor with other RNA recognition motifs. However, the fact that key residues preventing activity within the proenzyme are also key residues for RNA binding might have induced the activation domains of procarboxypeptidases to evolve from the canonical RNP1 and RNP2 sequences.     

Specific and modular binding code for cytosine recognition in Pumilio/FBF (PUF) RNA-binding domains

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.     

RNA recognition motifs: boring? Not quite

The RNA recognition motif (RRM) is one of the most abundant protein domains in eukaryotes. While the structure of this domain is well characterized by the packing of two alpha-helices on a four-stranded beta-sheet, the mode of protein and RNA recognition by RRMs is not clear owing to the high variability of these interactions. Here we report recent structural data on RRM-RNA and RRM-protein interactions showing the ability of this domain to modulate its binding affinity and specificity using each of its constitutive elements (beta-strands, loops, alpha-helices). The extreme structural versatility of the RRM interactions explains why RRM-containing proteins have so diverse biological functions.     

The association of the U1-specific 70K and C proteins with U1 snRNPs is mediated in part by common U snRNP proteins.

The U1 small nuclear ribonucleoprotein particle (snRNP)-specific 70K and A proteins are known to bind directly to stem-loops of the U1 snRNA, whereas the U1-C protein does not bind to naked U1 snRNA, but depends on other U1 snRNP protein components for its association. Focusing on the U1-70K and U1-C proteins, protein-protein interactions contributing to the association of these particle-specific proteins with the U1 snRNP were studied. Immunoprecipitation of complexes formed after incubation of naked U1 snRNA or purified U1 snRNPs lacking their specific proteins (core U1 snRNP) with in vitro translated U1-C protein, revealed that both common snRNP proteins and the U1-70K protein are required for the association of U1-C with the U1 snRNP. Binding studies with various in vitro translated U1-70K mutants demonstrated that the U1-70K N-terminal domain is necessary and sufficient for the interaction of U1-C with core U1 snRNPs. Surprisingly, several N-terminal fragments of the U1-70K protein, which lacked the U1-70K RNP-80 motif and did not bind naked U1 RNA, associated stably with core U1 snRNPs. This suggests that a new U1-70K binding site is generated upon association of common U1 snRNP proteins with U1 RNA. The interaction between the N-terminal domain of U1-70K and the core RNP domain was specific for the U1 snRNP; stable binding was not observed with core U2 or U5 snRNPs, suggesting essential structural differences among snRNP core domains. Evidence for direct protein-protein interactions between U1-specific proteins and common snRNP proteins was supported by chemical crosslinking experiments using purified U1 snRNPs. Individual crosslinks between the U1-70K and the common D2 or B'/B protein, as well as between U1-C and B'/B, were detected. A model for the assembly of U1 snRNP is presented in which the complex of common proteins on the RNA backbone functions as a platform for the association of the U1-specific proteins.     

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.     

Molecular dynamics simulation of the RNA complex of a double-stranded RNA-binding domain reveals dynamic features of the intermolecular interface and its hydration

The interaction between double-stranded RNA (dsRNA) and the third double-stranded domain (dsRBD) from Drosophila Staufen protein represents a paradigm to understand how the dsRBD protein family, one of the most common RNA-binding protein units, binds dsRNA. The nuclear magnetic resonance (NMR) structure of this complex and the x-ray structure of another family member revealed the stereochemical basis for recognition, but also raised new questions. Although the crystallographic studies revealed a highly ordered interface containing numerous water-mediated contacts, NMR suggested extensive residual motion at the interface. To address how interfacial motion contributes to molecular recognition in the dsRBD-dsRNA system, we conducted a 2-ns molecular dynamics simulation of the complex derived from Staufen protein and of the separate protein and RNA components. The results support the observation that a high degree of conformational flexibility is retained upon complex formation and that this involves interfacial residues that are critical for dsRBD-dsRNA binding. The structural origin of this residual flexibility is revealed by the analysis of the trajectory of motion. Individual basic side chains switch continuously from one RNA polar group to another with a residence time seldom exceeding 100 ps, while retaining favorable interaction with RNA throughout much of the simulation. Short-lived water molecules mediate some of these interactions for a large fraction of the trajectory studied here. This result indicates that water molecules are not statically associated with the interface, but continuously exchange with the bulk solvent on a 1-10-ps time scale. This work provides new insight into dsRBD-dsRNA recognition and builds upon a growing body of evidence, suggesting that short-lived dynamic interactions play important roles in protein-nucleic acid interactions.     

Analysis of in vitro binding of U1-A protein mutants to U1 snRNA.

Despite the great sequence similarity between U1A and U2B", both proteins do have a difference in RNA binding specificity and in the way they bind to their cognate RNAs. The U1A protein is able to bind in vitro U1 RNA independently of other factors. The U2B" protein binds specifically to U2 RNA in the presence of the U2A' protein only. We have compared the effect on RNA binding of multiple double point mutations at analogous positions in the U1A and U2B" protein. The results obtained show that amino acids at almost all of the analogous positions tested in U1A and U2B" have a comparable qualitative effect on RNA binding although the quantitative effect of mutations on U2B" is more severe than on U1A. Using U1A mutants with internal duplications a distinct area of the RNP motif of the U1A protein was identified which appears not to be directly involved in U1 RNA binding. In addition, roles of the highly conserved RNP1 and RNP2 sequences of the N-terminal RNP motif of the U1A protein, are investigated by replacing them with the analogous U1-70K sequences.     

G-quartet-dependent recognition between the FMRP RGG box and RNA

Fragile-X syndrome, the most common monogenic form of mental retardation, is caused by down-regulation of the expression of Fragile X Mental Retardation Protein (FMRP). FMRP is a multifunctional, multidomain RNA-binding protein that acts as a translational repressor in neuronal cells. Interaction between FMRP and mRNA targets involves an RGG box, a protein motif commonly thought to mediate unspecific interactions with nucleic acids. Instead, FMRP RGG box has been shown to recognize RNA G-quartet structures specifically and to be necessary in neurons for RNP particle formation and dendritic mRNA localization. In the present study, we have characterized structurally three representative RNA targets of FMRP in their unbound form and in complex with the RGG box. We observe a large heterogeneity in the conformation of the RNA targets and in their RGG binding mode, which could be the basis of recognition specificity. We also found that G-quartet formation occurs not only intramolecularly but can also be mediated by RNA dimerization. These findings suggest a potential role of RNA:RNA interactions in protein:RNA complexes and in RNP particle assembly.     

Protein-protein and protein-RNA contacts both contribute to the 15.5K-mediated assembly of the U4/U6 snRNP and the box C/D snoRNPs

The k-turn-binding protein 15.5K is unique in that it is essential for the hierarchical assembly of three RNP complexes distinct in both composition and function, namely, the U4/U6 snRNP, the box C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. 15.5K interacts with the cognate RNAs via an induced fit mechanism, which results in the folding of the surrounding RNA to create a binding site(s) for the RNP-specific proteins. However, it is possible that 15.5K also mediates RNP formation via protein-protein interactions with the complex-specific proteins. To investigate this possibility, we created a series of 15.5K mutations in which the surface properties of the protein had been changed. We assessed their ability to support the formation of the three distinct RNP complexes and found that the formation of each RNP requires a distinct set of regions on the surface of 15.5K. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein-protein interaction could be observed between hU3-55K and 15.5K. In conclusion, our data suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA.     

Stacking interactions in PUF-RNA complexes

Stacking interactions between amino acids and bases are common in RNA-protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 3' UTR (3'-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA-protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stacking amino acids in FBF affect both the affinity and specificity of the RNA-protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to "target" versus "off-target" interactions, and thus be an important consideration in the design of proteins with new specificities.     

Small nuclear ribonucleoproteins of Drosophila: identification of U1 RNA-associated proteins and their behavior during heat shock.

In Drosophila, two nuclear proteins of approximately 26,000 and 14,000 molecular weight are recognized by a human autoimmune antibody for mammalian ribonucleoprotein (RNP) particles that contain U1 small nuclear RNA. The antibody-selected Drosophila RNP contains, in addition to these two proteins, a single RNA species that has been identified as U1 by hybridization with a cloned Drosophila U1 DNA probe. Small nuclear RNP isolated from human cells under the same conditions as used for Drosophila and selected by the anti-U1 RNP-specific antibody contains eight proteins, two of which are similar in molecular weight to the two Drosophila U1 RNP proteins. Thus, even though the nucleotide sequences of Drosophila and human U1 RNA are about 72% homologous, and the corresponding RNPs are both recognized by the same human autoantibody, Drosophila U1 RNP appears to have a simpler protein complement than its mammalian counterpart. The two Drosophila U1 RNA-associated proteins are synthesized at normal or slightly increased rates during the heat shock response and are incorporated into antibody-recognizable RNP complexes. This raises the possibility that U1 RNP is an indispensable nuclear element for cell survival during heat shock.