Mutagenesis of a stacking contact in the MS2 coat protein-RNA complex.

The thermodynamic contribution of a stacking interaction between Tyr85 in MS2 coat protein and a single-stranded pyrimidine in its RNA binding site has been examined. Mutation of Tyr85 to Phe, His, Cys, Ser and Ala decreased the RNA affinity by 1-3 kcal/mol under standard binding conditions. Since the Phe, His and Cys 85 proteins formed UV photocrosslinks with iodouracil-containing RNA at the same rate as the wild-type protein, the mutant proteins interact with RNA in a similar manner. The pH dependence of KD for the Phe and His proteins differs substantially from the wild-type protein, suggesting that the titration of position 85 contributes substantially to the binding properties. Experiments with specifically substituted phosphorothioate RNAs confirm a hydrogen bond between the hydroxyl group of tyrosine and a phosphate predicted by the crystal structure.     

Saccharomyces SRP RNA secondary structures: a conserved S-domain and extended Alu-domain

The contribution made by the RNA component of signal recognition particle (SRP) to its function in protein targeting is poorly understood. We have generated a complete secondary structure for Saccharomyces cerevisiae SRP RNA, scR1. The structure conforms to that of other eukaryotic SRP RNAs. It is rod-shaped with, at opposite ends, binding sites for proteins required for the SRP functions of signal sequence recognition (S-domain) and translational elongation arrest (Alu-domain). Micrococcal nuclease digestion of purified S. cerevisiae SRP separated the S-domain of the RNA from the Alu-domain as a discrete fragment. The Alu-domain resolved into several stable fragments indicating a compact structure. Comparison of scR1 with SRP RNAs of five yeast species related to S. cerevisiae revealed the S-domain to be the most conserved region of the RNA. Extending data from nuclease digestion with phylogenetic comparison, we built the secondary structure model for scR1. The Alu-domain contains large extensions, including a sequence with hallmarks of an expansion segment. Evolutionarily conserved bases are placed in the Alu- and S-domains as in other SRP RNAs, the exception being an unusual GU(4)A loop closing the helix onto which the signal sequence binding Srp54p assembles (domain IV). Surprisingly, several mutations within the predicted Srp54p binding site failed to disrupt SRP function in vivo. However, the strength of the Srp54p-scR1 and, to a lesser extent, Sec65p-scR1 interaction was decreased in these mutant particles. The availability of a secondary structure for scR1 will facilitate interpretation of data from genetic analysis of the RNA.     

Using RNA secondary structures to guide sequence motif finding towards single-stranded regions

RNA binding proteins recognize RNA targets in a sequence specific manner. Apart from the sequence, the secondary structure context of the binding site also affects the binding affinity. Binding sites are often located in single-stranded RNA regions and it was shown that the sequestration of a binding motif in a double-strand abolishes protein binding. Thus, it is desirable to include knowledge about RNA secondary structures when searching for the binding motif of a protein. We present the approach MEMERIS for searching sequence motifs in a set of RNA sequences and simultaneously integrating information about secondary structures. To abstract from specific structural elements, we precompute position-specific values measuring the single-strandedness of all substrings of an RNA sequence. These values are used as prior knowledge about the motif starts to guide the motif search. Extensive tests with artificial and biological data demonstrate that MEMERIS is able to identify motifs in single-stranded regions even if a stronger motif located in double-strand parts exists. The discovered motif occurrences in biological datasets mostly coincide with known protein-binding sites. This algorithm can be used for finding the binding motif of single-stranded RNA-binding proteins in SELEX or other biological sequence data.     

Protein-RNA sequence covariation in a ribosomal protein-rRNA complex

Comparative sequence analysis has successfully predicted secondary structure and tertiary interactions in ribosomal and other RNAs. Experiments presented here ask whether the scope of comparative sequence-based predictions can be extended to specific interactions between proteins and RNA, using as a system the well-characterized C-terminal RNA binding domain of ribosomal protein L11 (L11-C76) and its 58 nucleotide binding region in 23S rRNA. The surface of L11-C76 alpha-helix 3 is known to contact RNA; position 69 in this helix is conserved as serine in most organisms but varies to asparagine (all plastids) or glutamine (Mycoplasma). RNA sequence substitutions unique to these groups of organisms occur at base pairs 1062/1076 or 1058/1080, respectively. The possibility that rRNA base pair substitutions compensate for variants in L11 alpha-helix 3 has been tested by measuring binding affinities between sets of protein and RNA sequence variants. Stability of the RNA tertiary structure, as measured by UV melting experiments, was unexpectedly affected by a 1062/1076 base pair substitution; additional mutations were required to restore a stably folded structure to this RNA. The results show that the asparagine variant of L11-C76 residue 69 has been compensated by substitution of a 1062/1076 base pair, and plausibly suggest a direct contact between the amino acid and base pair. For some of the protein and RNA mutations studied, changes in binding affinity probably reflect longer-range adjustments of the protein-RNA contact surface.     

Pre-mRNA secondary structures influence exon recognition

The secondary structure of a pre-mRNA influences a number of processing steps including alternative splicing. Since most splicing regulatory proteins bind to single-stranded RNA, the sequestration of RNA into double strands could prevent their binding. Here, we analyzed the secondary structure context of experimentally determined splicing enhancer and silencer motifs in their natural pre-mRNA context. We found that these splicing motifs are significantly more single-stranded than controls. These findings were validated by transfection experiments, where the effect of enhancer or silencer motifs on exon skipping was much more pronounced in single-stranded conformation. We also found that the structural context of predicted splicing motifs is under selection, suggesting a general importance of secondary structures on splicing and adding another level of evolutionary constraints on pre-mRNAs. Our results explain the action of mutations that affect splicing and indicate that the structural context of splicing motifs is part of the mRNA splicing code.     

The determinants for Sm protein binding to Xenopus U1 and U5 snRNAs are complex and non-identical.

The Sm binding sites of different spliceosomal U small nuclear RNAs (snRNAs), the RNA structural elements required for interaction with common snRNP proteins, have been considered to be similar or identical. Here we show that this is not the case. Instead, structural and sequence features unique to U1 or U5 snRNAs that contribute to common protein binding are identified. The determinants of Sm protein binding in both RNAs are complex, consisting in U5 of minimally two and in U1 of minimally four separate structural elements. Even the most conserved features of the two RNAs, single-stranded regions whose generalized sequence is PuA(U)nGPu, are not functionally interchangeable in protein binding. At least one of the newly defined RNA elements functions in assembly with the common proteins, but is not required for their stable binding thereafter. U1, but not U5, snRNP requires a trimethyl guanosine cap structure for its transport to the nucleus. This is not a consequence of the differences in common snRNP binding to the two RNAs, but is due to structural features of U1 RNA that do not contribute to Sm protein binding.     

Themes in RNA-protein recognition.

Atomic resolution structures are now available for more than 20 complexes of proteins with specific RNAs. This review examines two main themes that appear in this set of structures. A "groove binder" class of proteins places a protein structure (alpha-helix, 310-helix, beta-ribbon, or irregular loop) in the groove of an RNA helix, recognizing both the specific sequence of bases and the shape or dimensions of the groove, which are sometimes distorted from the normal A-form. A second class of proteins uses beta-sheet surfaces to create pockets that examine single-stranded RNA bases. Some of these proteins recognize completely unstructured RNA, and in others RNA secondary structure indirectly promotes binding by constraining bases in an appropriate orientation. Thermodynamic studies have shown that binding specificity is generally a function of several factors, including base-specific hydrogen bonds, non-polar contacts, and mutual accommodation of the protein and RNA-binding surfaces. The recognition strategies and structural frameworks used by RNA binding proteins are not exotically different from those employed by DNA-binding proteins, suggesting that the two kinds of nucleic acid-binding proteins have not evolved independently.     

Selective binding by the RNA binding domain of PKR revealed by affinity cleavage

The RNA-dependent protein kinase (PKR) is regulated by the binding of double-stranded RNA (dsRNA) or single-stranded RNAs with extensive duplex secondary structure. PKR has an RNA binding domain (RBD) composed of two copies of the dsRNA binding motif (dsRBM). The dsRBM is an alpha-beta-beta-beta-alpha structure present in a number of proteins that bind RNA, and the selectivity demonstrated by these proteins is currently not well understood. We have used affinity cleavage to study the binding of PKR's RBD to RNA. In this study, we site-specifically modified the first dsRBM of PKR's RBD at two different amino acid positions with the hydroxyl radical generator EDTA.Fe. Cleavage by these proteins of a synthetic stem-loop ligand of PKR indicates that PKR's dsRBMI binds the RNA in a preferred orientation, placing the loop between strands beta1 and beta2 near the single-stranded RNA loop. Additional cleavage experiments demonstrated that defects in the RNA stem, such as an A bulge and two GA mismatches, do not dictate dsRBMI's binding orientation preference. Cleavage of VA(I) RNA, an adenoviral RNA inhibitor of PKR, indicates that dsRBMI is bound near the loop of the apical stem of this RNA in the same orientation as observed with the synthetic stem-loop RNA ligands. This work, along with an NMR study of the binding of a dsRBM derived from the Drosophila protein Staufen, indicates that dsRBMs can bind stem-loop RNAs in distinct ways. In addition, the successful application of the affinity cleavage technique to localizing dsRBMI of PKR on stem-loop RNAs and defining its orientation suggests this approach could be applied to dsRBMs found in other proteins.     

The effect of RNA secondary structures on RNA-ligand binding and the modifier RNA mechanism: a quantitative model

RNA-ligand binding often depends crucially on the local RNA secondary structure at the binding site. We develop here a model that quantitatively predicts the effect of RNA secondary structure on effective RNA-ligand binding activities based on equilibrium thermodynamics and the explicit computations of partition functions for the RNA structures. A statistical test for the impact of a particular structural feature on the binding affinities follows directly from this approach. The formalism is extended to describing the effects of hybridizing small "modifier RNAs" to a target RNA molecule outside its ligand binding site. We illustrate the applicability of our approach by quantitatively describing the interaction of the mRNA stabilizing protein HuR with AU-rich elements. We discuss our model and recent experimental findings demonstrating the effectivity of modifier RNAs in vitro in the context of the current research activities in the field of non-coding RNAs. We speculate that modifier RNAs might also exist in nature; if so, they present an additional regulatory layer for fine-tuning gene expression that could evolve rapidly, leaving no obvious traces in the genomic DNA sequences.     

H/ACA guide RNAs, proteins and complexes

H/ACA guide RNAs direct site-specific pseudouridylation of substrate RNAs by forming ribonucleoprotein (RNP) complexes with pseudouridine synthase Cbf5 and three accessory proteins. Recently determined crystal structures of H/ACA protein complexes and a fully assembled H/ACA RNP complex have provided significant insights into the architecture, assembly and mechanism of action of RNA-guided pseudouridine synthase. The binding of guide RNA is directed by its conserved secondary structure and sequence motifs, which enables guide RNA with different sequences to be incorporated into the same protein complex. Accessory proteins and peripheral domains crucially coordinate the position of guide RNA, and possibly regulate the reaction process.     

Specific interaction of HeLa cell proteins with coxsackievirus B3 3'UTR: La autoantigen binds the 3' and 5'UTR independently of the poly(A) tail

Coxsackievirus B3 (CVB3) is a positive, single-stranded RNA virus. The secondary structure of the 3' untranslated region (3'UTR) of CVB3 RNA consists of three stem-loops and is followed by a poly(A) tail sequence. These stem-loop structures have been suggested to participate in the regulation of viral replication through interaction with cellular proteins that are yet to be identified. In this study, by competitive UV cross-linking using mutated 3'UTR probes we have demonstrated that the poly(A) tail is essential for promoting HeLa cell protein interactions with the 3'UTR because deletion of this sequence abolished most of the protein interactions. Unexpectedly, mutations that disrupted the tertiary loop-loop interactions without affecting the stem-loops did not apparently affect these protein interactions, indicating that secondary structure rather than the high-order structure may play a major role in recruiting these RNA binding proteins. Among the observed 3'UTR RNA binding proteins, we have confirmed a 52 kDa protein as the human La autoantigen by using purified recombinant protein and a polyclonal La antibody. This protein can interact with both the 3' and 5'UTRs independently of the poly(A) tail. Further analysis by two-stage UV cross-linking, we found that the 3' and 5'UTR sequences may share the same binding site on the La protein.     

Recognition of diverse RNAs by a single protein structural framework

The coat proteins of different single-strand RNA phages utilize a common structural framework to recognize different RNA targets, making them suitable models for studies of RNA-protein recognition generally, especially for the class of proteins that bind RNA on a beta-sheet surface. Here we show that structurally distinct molecules are capable of satisfying the requirements for binding to Qbeta coat protein. Although the predicted secondary structures of the RNAs differ markedly, we contend that they are approximately equivalent structurally in their complexes with coat protein. Based on our prior observations that the RNA-binding specificities of Qbeta and MS2 coat proteins can be interconverted with as few as one amino acid substitution each, and taking into account details of the structures of complexes of MS2 coat protein with wild-type and aptamer RNAs, we propose a model for the Qbeta coat protein-RNA complex.     

OB-fold domain of KREPA4 mediates high-affinity interaction with guide RNA and possesses annealing activity

KREPA4, also called MP24, is an essential mitochondrial guide RNA (gRNA)-binding protein with a preference for the 3′ oligo(U) tail in trypanosomes. Structural prediction and compositional analysis of KREPA4 have identified a conserved OB (oligonucleotide/oligosaccharide-binding)-fold at the C-terminal end and two low compositional complexity regions (LCRs) at its N terminus. Concurrent with these predictions, one or both of these regions in KREPA4 protein may be involved in gRNA binding. To test this possibility, deletion mutants of KREPA4 were made and the effects on the gRNA-binding affinities were measured by quantitative electrophoretic mobility shift assays. The gRNA-binding specificities of these mutants were evaluated by competition experiments using gRNAs with U-tail deletions or stem–loop modifications and uridylated nonguide RNAs or heterologous RNA. Our results identified the predicted OB-fold as the functional domain of KREPA4 that mediates a high-affinity interaction with the gRNA oligo(U) tail. An additional contribution toward RNA-binding function was localized to LCRs that further stabilize the binding through sequence-specific interactions with the guide secondary structure. In this study we also found that the predicted OB-fold has an RNA annealing activity, representing the first report of such activity for a core component of the RNA editing complex.     

Interaction of rice and human SRP19 polypeptides with signal recognition particle RNA

The signal recognition particle (SRP) controls the transport of secretory proteins into and across lipid bilayers. SRP-like ribonucleoprotein complexes exist in all organisms, including plants. We characterized the rice SRP RNA and its primary RNA binding protein, SRP19. The secondary structure of the rice SRP RNA was similar to that found in other eukaryotes; however, as in other plant SRP RNAs, a GUUUCA hexamer sequence replaced the highly conserved GNRA-tetranucleotide loop motif at the apex of helix 8. The small domain of the rice SRP RNA was reduced considerably. Structurally, rice SRP19 lacked two small regions that can be present in other SRP19 homologues. Conservative structure prediction and site-directed mutagenesis of rice and human SRP19 polypeptides indicated that binding to the SRP RNAs occurred via a loop that is present in the N-domain of both proteins. Rice SRP19 protein was able to form a stable complex with the rice SRP RNA in vitro. Furthermore, heterologous ribonucleoprotein complexes with components of the human SRP were assembled, thus confirming a high degree of structural and functional conservation between plant and mammalian SRP components.     

Evolutionary conserved nucleotides within the E.coli 4.5S RNA are required for association with P48 in vitro and for optimal function in vivo.

E.coli 4.5S RNA is homologous to domain IV of eukaryotic SPR7S RNA, the RNA component of the signal recognition particle. The 4.5S RNA is associated in vivo with a 48kD protein (P48), which is homologous to a protein component of the signal recognition particle, SRP54. In addition to secondary structural features, a number of nucleotides are conserved between the 4.5S RNA and domain IV of all other characterised SRP-like RNAs from eubacteria, arachaebacteria and eukaryotes. This domain consists of an extended stem-loop structure; conserved nucleotides lie within the terminal loop and within single-stranded regions bulged from the stem immediately preceding the loop. This conserved region is a candidate for the SRP54/P48 binding site. To determine the functional importance of this region within the 4.5S RNA, mutations were introduced into the 4.5S RNA coding sequence. Mutated alleles were tested for their function in vivo and for the ability of the corresponding RNAs to bind P48 in vitro. Single point mutations in conserved nucleotides within the terminal tetranucleotide loop do not affect P48 binding in vitro and produce only slight growth defects. This suggests that the sequence of the loop may be important for the structure of the molecule rather than for specific interactions with P48. On the other hand, nucleotides within the single-stranded regions bulged from the stem were found to be important both for the binding of P48 to the RNA and for optimal function of the RNA in vivo.