RNA binding in an Sm core domain: X-ray structure and functional analysis of an archaeal Sm protein complex

Eukaryotic Sm and Sm-like proteins associate with RNA to form the core domain of ribonucleoprotein particles involved in pre-mRNA splicing and other processes. Recently, putative Sm proteins of unknown function have been identified in ARCHAEA: We show by immunoprecipitation experiments that the two Sm proteins present in Archaeoglobus fulgidus (AF-Sm1 and AF-Sm2) associate with RNase P RNA in vivo, suggesting a role in tRNA processing. The AF-Sm1 protein also interacts specifically with oligouridylate in vitro. We have solved the crystal structures of this protein and a complex with RNA. AF-Sm1 forms a seven-membered ring, with the RNA interacting inside the central cavity on one face of the doughnut-shaped complex. The bases are bound via stacking and specific hydrogen bonding contacts in pockets lined by residues highly conserved in archaeal and eukaryotic Sm proteins, while the phosphates remain solvent accessible. A comparison with the structures of human Sm protein dimers reveals closely related monomer folds and intersubunit contacts, indicating that the architecture of the Sm core domain and RNA binding have been conserved during evolution.     

Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction.

The bacterial Hfq protein modulates the stability or the translation of mRNAs and has recently been shown to interact with small regulatory RNAs in E. coli. Here we show that Hfq belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif, known as the Sm1 motif, forms a doughnut-shaped structure, and has RNA binding specificity very similar to the Sm proteins. Moreover, we provide evidence that Hfq strongly cooperates in intermolecular base pairing between the antisense regulator Spot 42 RNA and its target RNA. We speculate that Sm proteins in general cooperate in bimolecular RNA-RNA interaction and that protein-mediated complex formation permits small RNAs to interact with a broad range of target RNAs.     

An Hfq-like protein in archaea: crystal structure and functional characterization of the Sm protein from Methanococcus jannaschii

The Sm and Sm-like proteins are conserved in all three domains of life and have emerged as important players in many different RNA-processing reactions. Their proposed role is to mediate RNA-RNA and/or RNA-protein interactions. In marked contrast to eukaryotes, bacteria appear to contain only one distinct Sm-like protein belonging to the Hfq family of proteins. Similarly, there are generally only one or two subtypes of Sm-related proteins in archaea, but at least one archaeon, Methanococcus jannaschii, encodes a protein that is related to Hfq. This archaeon does not contain any gene encoding a conventional archaeal Sm-type protein, suggesting that Hfq proteins and archaeal Sm-homologs can complement each other functionally. Here, we report the functional characterization of M. jannaschii Hfq and its crystal structure at 2.5 A resolution. The protein forms a hexameric ring. The monomer fold, as well as the overall structure of the complex is similar to that found for the bacterial Hfq proteins. However, clear differences are seen in the charge distribution on the distal face of the ring, which is unusually negative in M. jannaschii Hfq. Moreover, owing to a very short N-terminal alpha-helix, the overall diameter of the archaeal Hfq hexamer is significantly smaller than its bacterial counterparts. Functional analysis reveals that Escherichia coli and M. jannaschii Hfqs display very similar biochemical and biological properties. It thus appears that the archaeal and bacterial Hfq proteins are largely functionally interchangeable.     

Archaeal Sm proteins form heptameric and hexameric complexes: crystal structures of the Sm1 and Sm2 proteins from the hyperthermophile Archaeoglobus fulgidus

Proteins of largely unknown function related to the Sm proteins present in the core domain of eukaryotic small nuclear ribonucleoprotein particles have recently been detected in Archaea. In contrast to eukaryotes, Archaea contain maximally two distinct Sm-related proteins belonging to different subfamilies, we refer to as Sm1 and Sm2. Here we report the crystal structures of the Sm1- and Sm2-type proteins from the hyperthermophilic euryarchaeon Archaeoglobus fulgidus (AF-Sm1 and AF-Sm2) at a resolution of 2.5 and 1.95 A, respectively. While the AF-Sm1 protein forms a heptameric ring structure similar to that found in other archaeal Sm1-type proteins, the AF-Sm2 protein unexpectedly forms a homo-hexamer in the crystals, and, as is evident from the mass spectrometric analysis, also in solution. Both proteins have essentially the same monomer fold and inter-subunit beta-sheet hydrogen bonding giving rise to a similar overall architecture of the doughnut-shaped six and seven-membered rings. In addition, a conserved uracil-binding pocket identified previously in an AF-Sm1/RNA complex, suggests a common RNA-binding mode for the AF-Sm1 and AF-Sm2 proteins, in line with solution studies showing preferential binding to U-rich oligonucleotides for both proteins. Clear differences are however seen in the charge distribution within the two structures. The rough faces of the rings, i.e. the faces not containing the base binding pockets, have opposite charges in the two structures, being predominantly positive in AF-Sm1 and negative in AF-Sm2. Differences in the ionic interactions between subunits provide an explanation for the distinctly different oligomerisation behaviour of the AF-Sm1 and AF-Sm2 proteins and of Sm1- and Sm2-type proteins in general, as well as the stability of their complexes. Implications for the functions of archaeal Sm proteins are being discussed.     

Spliceosomal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA nonanucleotide in a specific and thermodynamically stable manner.

The association of Sm proteins with U small nuclear RNA (snRNA) requires the single-stranded Sm site (PuAU(4-6)GPu) but also is influenced by nonconserved flanking RNA structural elements. Here we demonstrate that a nonameric Sm site RNA oligonucleotide sufficed for sequence-specific assembly of a minimal core ribonucleoprotein (RNP), which contained all seven Sm proteins. The minimal core RNP displayed several conserved biochemical features of native U snRNP core particles, including a similar morphology in electron micrographs. This minimal system allowed us to study in detail the RNA requirements for Sm protein-Sm site interactions as well as the kinetics of core RNP assembly. In addition to the uridine bases, the 2' hydroxyl moieties were important for stable RNP formation, indicating that both the sugar backbone and the bases are intimately involved in RNA-protein interactions. Moreover, our data imply that an initial phase of core RNP assembly is mediated by a high affinity of the Sm proteins for the single-stranded uridine tract but that the presence of the conserved adenosine (PuAU.) is essential to commit the RNP particle to thermodynamic stability. Comparison of intact U4 and U5 snRNAs with the Sm site oligonucleotide in core RNP assembly revealed that the regions flanking the Sm site within the U snRNAs facilitate the kinetics of core RNP assembly by increasing the rate of Sm protein association and by decreasing the activation energy.     

Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis.     

The Sm-like Hfq protein increases OxyS RNA interaction with target mRNAs.

The Escherichia coli host factor I, Hfq, binds to many small regulatory RNAs and is required for OxyS RNA repression of fhlA and rpoS mRNA translation. Here we report that Hfq is a bacterial homolog of the Sm and Sm-like proteins integral to RNA processing and mRNA degradation complexes in eukaryotic cells. Hfq exhibits the hallmark features of Sm and Sm-like proteins: the Sm1 sequence motif, a multisubunit ring structure (in this case a homomeric hexamer), and preferential binding to polyU. We also show that Hfq increases the OxyS RNA interaction with its target messages and propose that the enhancement of RNA-RNA pairing may be a general function of Hfq, Sm, and Sm-like proteins.     

Dissection of Prp8 protein defines multiple interactions with crucial RNA sequences in the catalytic core of the spliceosome

Current models of the core of the spliceosome include a network of RNA-RNA interactions involving the pre-mRNA and the U2, U5, and U6 snRNAs. The essential spliceosomal protein Prp8 interacts with U5 and U6 snRNAs and with specific pre-mRNA sequences that participate in catalysis. This close association with crucial RNA sequences, together with extensive genetic evidence, suggests that Prp8 could directly affect the function of the catalytic core, perhaps acting as a splicing cofactor. However, the sequence of Prp8 is almost entirely novel, and it offers few clues to the molecular basis of Prp8-RNA interactions. We have used an innovative transposon-based strategy to establish that catalytic core RNAs make multiple contacts in the central region of Prp8, underscoring the intimate relationship between this protein and the catalytic center of the spliceosome. Our analysis of RNA interactions identifies a discrete, highly conserved region of Prp8 as a prime candidate for the role of cofactor for the spliceosome's RNA core.     

Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA. Crystal structure of an oligomeric complex of yeast SmF

Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.     

snRNP Sm proteins share two evolutionarily conserved sequence motifs which are involved in Sm protein-protein interactions.

The spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/U6 and U5 share eight proteins B', B, D1, D2, D3, E, F and G which form the structural core of the snRNPs. This class of common proteins plays an essential role in the biogenesis of the snRNPs. In addition, these proteins represent the major targets for the so-called anti-Sm auto-antibodies which are diagnostic for systemic lupus erythematosus (SLE). We have characterized the proteins F and G from HeLa cells by cDNA cloning, and, thus, all human Sm protein sequences are now available for comparison. Similar to the D, B/B' and E proteins, the F and G proteins do not possess any of the known RNA binding motifs, suggesting that other types of RNA-protein interactions occur in the snRNP core. Strikingly, the eight human Sm proteins possess mutual homology in two regions, 32 and 14 amino acids long, that we term Sm motifs 1 and 2. The Sm motifs are evolutionarily highly conserved in all of the putative homologues of the human Sm proteins identified in the data base. These results suggest that the Sm proteins may have arisen from a single common ancestor. Several hypothetical proteins, mainly of plant origin, that clearly contain the conserved Sm motifs but exhibit only comparatively low overall homology to one of the human Sm proteins, were identified in the data base. This suggests that the Sm motifs may also be shared by non-spliceosomal proteins. Further, we provide experimental evidence that the Sm motifs are involved, at least in part, in Sm protein-protein interactions. Specifically, we show by co-immunoprecipitation analyses of in vitro translated B' and D3 that the Sm motifs are essential for complex formation between B' and D3. Our finding that the Sm proteins share conserved sequence motifs may help to explain the frequent occurrence in patient sera of anti-Sm antibodies that cross-react with multiple Sm proteins and may ultimately further our understanding of how the snRNPs act as auto-antigens and immunogens in SLE.     

Sm protein-Sm site RNA interactions within the inner ring of the spliceosomal snRNP core structure

Seven Sm proteins, E, F, G, D1, D2, D3 and B/B', assemble in a stepwise manner onto the single-stranded Sm site element (PuAU(4-6)GPu) of the U1, U2, U4 and U5 spliceosomal snRNAs, resulting in a doughnut-shaped core RNP structure. Here we show by UV cross-linking experiments using an Sm site RNA oligonucleotide (AAUUUUUGA) that several Sm proteins contact the Sm site RNA, with the most efficient cross-links observed for the G and B/B' proteins. Site-specific photo-cross-linking revealed that the G and B/B' proteins contact distinct uridines (in the first and third positions, respectively) in a highly position-specific manner. Amino acids involved in contacting the RNA are located at equivalent regions in both proteins, namely in loop L3 of the Sm1 motif, which has been predicted to jut into the hole of the Sm ring. Our results thus provide the first evidence that, within the core snRNP, multiple Sm protein-Sm site RNA contacts occur on the inner surface of the heptameric Sm protein ring.     

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.     

Stereospecific interactions of proline residues in protein structures and complexes

The constrained backbone torsion angle of a proline (Pro) residue has usually been invoked to explain its three-dimensional context in proteins. Here we show that specific interactions involving the pyrrolidine ring atoms also contribute to its location in a given secondary structure and its binding to another molecule. It is adept at participating in two rather non-conventional interactions, C-H...pi and C-H...O. The geometry of interaction between the pyrrolidine and aromatic rings, vis-à-vis the occurrence of the C-H...pi interactions has been elucidated. Some of the secondary structural elements stabilized by Pro-aromatic interactions are beta-turns, where a Pro can interact with an adjacent aromatic residue, and in antiparallel beta-sheet, where a Pro in an edge strand can interact with an aromatic residue in the adjacent strand at a non-hydrogen-bonded site. The C-H groups at the Calpha and Cdelta positions can form strong C-H...O interactions (as seen from the clustering of points) and such interactions involving a Pro residue at C' position relative to an alpha-helix can cap the hydrogen bond forming potentials of the free carbonyl groups at the helix C terminus. Functionally important Pro residues occurring at the binding site of a protein almost invariably engage aromatic residues (with one of them being held by C-H...pi interaction) from the partner molecule in the complex, and such aromatic residues are highly conserved during evolution.     

Crystal structure of Lsm3 octamer from Saccharomyces cerevisiae: implications for Lsm ring organisation and recruitment

Sm and Sm-like (Lsm) proteins are core components of the ribonucleoprotein complexes essential to key nucleic acid processing events within the eukaryotic cell. They assemble as polyprotein ring scaffolds that have the capacity to bind RNA substrates and other necessary protein factors. The crystal structure of yeast Lsm3 reveals a new organisation of the L/Sm β-propeller ring, containing eight protein subunits. Little distortion of the characteristic L/Sm fold is required to form the octamer, indicating that the eukaryotic Lsm ring may be more pliable than previously thought. The homomeric Lsm3 octamer is found to successfully recruit Lsm6, Lsm2 and Lsm5 directly from yeast lysate. Our crystal structure shows the C-terminal tail of each Lsm3 subunit to be engaged in connections across rings through specific β-sheet interactions with elongated loops protruding from neighbouring octamers. While these loops are of distinct length for each Lsm protein and generally comprise low-complexity polar sequences, several Lsm C-termini comprise hydrophobic sequences suitable for β-sheet interactions. The Lsm3 structure thus provides evidence for protein-protein interactions likely utilised by the highly variable Lsm loops and termini in the recruitment of RNA processing factors to mixed Lsm ring scaffolds. Our coordinates also provide updated homology models for the active Lsm[1-7] and Lsm[2-8] heptameric rings.     

Specific alterations of U1-C protein or U1 small nuclear RNA can eliminate the requirement of Prp28p, an essential DEAD box splicing factor

While some members of the ubiquitous DExD/H box family of proteins have RNA helicase activity in vitro, their roles in vivo remain virtually unknown. Here, we show that the function of an otherwise essential DEAD box protein, Prp28p, can be bypassed by mutations that alter either the protein U1-C or the U1 small nuclear RNA. Further analysis suggests that the conserved L13 residue in the U1-C protein makes specific contact to stabilize the U1 snRNA/5' splice site duplex in the prespliceosome, and that Prp28p functions to counteract the stabilizing effect of the U1-C protein, thereby promoting the dissociation of the U1 small nuclear ribonucleoprotein particle from the 5' splice site. Thus, in addition to unwinding RNA, the DExD/H box proteins may affect RNA-RNA rearrangements by antagonizing specific RNA-stabilizing proteins.     

Protein-DNA interactions: amino acid conservation and the effects of mutations on binding specificity

We investigate the conservation of amino acid residue sequences in 21 DNA-binding protein families and study the effects that mutations have on DNA-sequence recognition. The observations are best understood by assigning each protein family to one of three classes: (i) non-specific, where binding is independent of DNA sequence; (ii) highly specific, where binding is specific and all members of the family target the same DNA sequence; and (iii) multi-specific, where binding is also specific, but individual family members target different DNA sequences. Overall, protein residues in contact with the DNA are better conserved than the rest of the protein surface, but there is a complex underlying trend of conservation for individual residue positions. Amino acid residues that interact with the DNA backbone are well conserved across all protein families and provide a core of stabilising contacts for homologous protein-DNA complexes. In contrast, amino acid residues that interact with DNA bases have variable levels of conservation depending on the family classification. In non-specific families, base-contacting residues are well conserved and interactions are always found in the minor groove where there is little discrimination between base types. In highly specific families, base-contacting residues are highly conserved and allow member proteins to recognise the same target sequence. In multi-specific families, base-contacting residues undergo frequent mutations and enable different proteins to recognise distinct target sequences. Finally, we report that interactions with bases in the target sequence often follow (though not always) a universal code of amino acid-base recognition and the effects of amino acid mutations can be most easily understood for these interactions.     

Structural insights into the interaction of the Nip7 PUA domain with polyuridine RNA

The conserved protein Nip7 is involved in ribosome biogenesis, being required for proper 27S pre-rRNA processing and 60S ribosome subunit assembly in Saccharomyces cerevisiae. Yeast Nip7p interacts with nucleolar proteins and with the exosome subunit Rrp43p, but its molecular function remains to be determined. Solution of the Pyrococcus abyssi Nip7 (PaNip7) crystal structure revealed a monomeric protein composed by two alpha-beta domains. The N-terminal domain is formed by a five-stranded antiparallel beta-sheet surrounded by three alpha-helices and a 310 helix while the C-terminal, a mixed beta-sheet domain composed by strands beta8 to beta12, one alpha-helix, and a 310 helix, corresponds to the conserved PUA domain (after Pseudo-Uridine synthases and Archaeosine-specific transglycosylases). By combining structural analyses and RNA interaction assays, we assessed the ability of both yeast and archaeal Nip7 orthologues to interact with RNA. Structural alignment of the PaNip7 PUA domain with the RNA-interacting surface of the ArcTGT (archaeosine tRNA-guanine transglycosylase) PUA domain indicated that in the archaeal PUA domain positively charged residues (R151, R152, K155, and K158) are involved in RNA interaction. However, equivalent positions are occupied by mostly hydrophobic residues (A/G160, I161, F164, and A167) in eukaryotic Nip7 orthologues. Both proteins can bind specifically to polyuridine, and RNA interaction requires specific residues of the PUA domain as determined by site-directed mutagenesis. This work provides experimental verification that the PUA domain mediates Nip7 interaction with RNA and reveals that the preference for interaction with polyuridine sequences is conserved in Archaea and eukaryotic Nip7 proteins.