Predicted structure and phyletic distribution of the RNA-binding protein Hfq

Hfq, a bacterial RNA-binding protein, was recently shown to contain the Sm1 motif, a characteristic of Sm and LSm proteins that function in RNA processing events in archaea and eukaryotes. In this report, comparative structural modeling was used to predict a three-dimensional structure of the Hfq core sequence. The predicted structure aligns with most major features of the Methanobacterium thermoautotrophicum LSm protein structure. Conserved residues in Hfq are positioned at the same structural locations responsible for subunit assembly and RNA interaction in Sm proteins. A highly conserved portion of Hfq assumes a structural fold similar to the Sm2 motif of Sm proteins. The evolution of the Hfq protein was explored by conducting a BLAST search of microbial genomes followed by phylogenetic analysis. Approximately half of the 140 complete or nearly complete genomes examined contain at least one gene coding for Hfq. The presence or absence of Hfq closely followed major bacterial clades. It is absent from high-level clades and present in the ancient Thermotogales-Aquificales clade and all proteobacteria except for those that have undergone major reduction in genome size. Residues at three positions in Hfq form signatures for the beta/gamma proteobacteria, alpha proteobacteria and low GC Gram-positive bacteria groups.     

Hfq affects mRNA levels independently of degradation

Background  

The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account.     

Eukaryotic Lsm proteins: lessons from bacteria

Over the last five years Sm-like (Lsm) proteins have emerged as important players in many aspects of RNA metabolism, including splicing, nuclear RNA processing and messenger RNA decay. However, their precise function in these pathways remains somewhat obscure. In contrast, the role of the bacterial Lsm protein Hfq, which bears striking similarities in both structure and function to Lsm proteins, is much better characterized. In this perspective, we have highlighted several functions that Hfq shares with Lsm proteins and put forward hypotheses based on parallels between the two that might further the understanding of Lsm function.     

Global analysis of small RNA and mRNA targets of Hfq

Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs. Hfq binds this family of small RNAs efficiently. We have used co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family. This approach was extremely sensitive; even Hfq-binding small RNAs expressed at low levels were readily detected. At least 15 of 46 known small RNAs in E. coli interact with Hfq. In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis. Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets. Within the argX-hisR-leuT-proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation. Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.     

The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation

The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3′-terminal A-residues. Structural analysis of the adenylated RNA–Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3′–5′ exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.     

Novel Sm-like proteins with long C-terminal tails and associated methyltransferases

Sm and Sm-like proteins of the Lsm (like Sm) domain family are generally involved in essential RNA-processing tasks. While recent research has focused on the function and structure of small family members, little is known about Lsm domain proteins carrying additional domains. Using an integrative bioinformatics approach, we discovered five novel groups of Lsm domain proteins (Lsm12-16) with long C-terminal tails and investigated their functions. All of them are evolutionarily conserved in eukaryotes with an N-terminal Lsm domain to bind nucleic acids followed by as yet uncharacterized C-terminal domains and sequence motifs. Based on known yeast interaction partners, Lsm12-16 may play important roles in RNA metabolism. Particularly, Lsm12 is possibly involved in mRNA degradation or tRNA splicing, and Lsm13-16 in the regulation of the mitotic G2/M phase. Lsm16 proteins have an additional C-terminal YjeF_N domain of as yet unknown function. The identification of an additional methyltransferase domain at the C-terminus of one of the Lsm12 proteins also led to the recognition of three new groups of methyltransferases, presumably dependent on S-adenosyl-l-methionine. Further computational analyses revealed that some methyltransferases contain putative RNA-binding helix-turn-helix domains and zinc fingers.     

Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli

The Hfq protein was discovered in Escherichia coli in the early seventies as a host factor for the Qbeta phage RNA replication. During the last decade, it was shown to be involved in many RNA processing events and remote sequence homology indicated a link to spliceosomal Sm proteins. We report the crystal structure of the E.coli Hfq protein showing that its monomer displays a characteristic Sm-fold and forms a homo-hexamer, in agreement with former biochemical data. Overall, the structure of the E.coli Hfq ring is similar to the one recently described for Staphylococcus aureus. This confirms that bacteria contain a hexameric Sm-like protein which is likely to be an ancient and less specialized form characterized by a relaxed RNA binding specificity. In addition, we identified an Hfq ortholog in the archaeon Methanococcus jannaschii which lacks a classical Sm/Lsm gene. Finally, a detailed structural comparison shows that the Sm-fold is remarkably well conserved in bacteria, Archaea and Eukarya, and represents a universal and modular building unit for oligomeric RNA binding proteins.     

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.     

The Archaeal Lsm Protein Binds to Small RNAs

Proteins of the Lsm family, including eukaryotic Sm proteins and bacterial Hfq, are key players in RNA metabolism. Little is known about the archaeal homologues of these proteins. Therefore, we characterized the Lsm protein from the haloarchaeon Haloferax volcanii using in vitro and in vivo approaches. H. volcanii encodes a single Lsm protein, which belongs to the Lsm1 subfamily. The lsm gene is co-transcribed and overlaps with the gene for the ribosomal protein L37e. Northern blot analysis shows that the lsm gene is differentially transcribed. The Lsm protein forms homoheptameric complexes and has a copy number of 4000 molecules/cell. In vitro analyses using electrophoretic mobility shift assays and ultrasoft mass spectrometry (laser-induced liquid bead ion desorption) showed a complex formation of the recombinant Lsm protein with oligo(U)-RNA, tRNAs, and an small RNA. Co-immunoprecipitation with a FLAG-tagged Lsm protein produced in vivo confirmed that the protein binds to small RNAs. Furthermore, the co-immunoprecipitation revealed several protein interaction partners, suggesting its involvement in different cellular pathways. The deletion of the lsm gene is viable, resulting in a pleiotropic phenotype, indicating that the haloarchaeal Lsm is involved in many cellular processes, which is in congruence with the number of protein interaction partners.     

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.     

Both Sm-domain and C-terminal extension of Lsm1 are important for the RNA-binding activity of the Lsm1-7-Pat1 complex

Lsm proteins are a ubiquitous family of proteins characterized by the Sm-domain. They exist as hexa- or heptameric RNA-binding complexes and carry out RNA-related functions. The Sm-domain is thought to be sufficient for the RNA-binding activity of these proteins. The highly conserved eukaryotic Lsm1 through Lsm7 proteins are part of the cytoplasmic Lsm1-7-Pat1 complex, which is an activator of decapping in the conserved 5'-3' mRNA decay pathway. This complex also protects mRNA 3'-ends from trimming in vivo. Purified Lsm1-7-Pat1 complex is able to bind RNA in vitro and exhibits a unique binding preference for oligoadenylated RNA (over polyadenylated and unadenylated RNA). Lsm1 is a key subunit that determines the RNA-binding properties of this complex. The normal RNA-binding activity of this complex is crucial for mRNA decay and 3'-end protection in vivo and requires the intact Sm-domain of Lsm1. Here, we show that though necessary, the Sm-domain of Lsm1 is not sufficient for the normal RNA-binding ability of the Lsm1-7-Pat1 complex. Deletion of the C-terminal domain (CTD) of Lsm1 (while keeping the Sm-domain intact) impairs mRNA decay in vivo and results in Lsm1-7-Pat1 complexes that are severely impaired in RNA binding in vitro. Interestingly, the mRNA decay and 3'-end protection defects of such CTD-truncated lsm1 mutants could be suppressed in trans by overexpression of the CTD polypeptide. Thus, unlike most Sm-like proteins, Lsm1 uniquely requires both its Sm-domain and CTD for its normal RNA-binding function.     

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.     

建立基于RNA免疫共沉淀技术的鼠疫耶尔森菌Hfq蛋白相关sRNA的体内验证方法

目的：建立RNA免疫共沉淀方法,为鼠疫耶尔森菌Hfq蛋白相关非编码小RNA（sRNA）提供体内验证方法。方法：首先在RNA结合蛋白Hfq下游加入Flag标签,用Flag标签抗体进行免疫共沉淀,获得蛋白-RNA复合物,然后从沉淀的蛋白-RNA复合物中分离得到纯化的RNA;通过Western印迹检测各步骤Hfq蛋白的表达,再利用Northern印迹检测目的sRNA--RyhB1和RyhB2。结果：构建了带有Flag标签的RNA结合蛋白Hfq的载体,此载体转导入hfq缺失株后与鼠疫菌野生株的生长曲线无明显差异;通过RNA-蛋白免疫共沉淀技术鉴定出已知与鼠疫菌Hfq蛋白结合的2个sRNA--RyhB1和RyhB2。结论：建立了利用RNA-蛋白免疫共沉淀鉴定与鼠疫菌Hfq蛋白结合的sRNA的技术,为细菌sRNA的验证、功能研究和体内蛋白质与RNA相互作用研究提供了有利工具。     

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.     

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.