Solution structure of the Vts1 SAM domain in the presence of RNA

The yeast Vts1 SAM (sterile alpha motif) domain is a member of a new class of SAM domains that specifically bind RNA. To elucidate the structural basis for RNA binding, the solution structure of the Vts1 SAM domain, in the presence of a specific target RNA, has been solved by multidimensional heteronuclear NMR spectroscopy. The Vts1 SAM domain retains the "core" five-helix-bundle architecture of traditional SAM domains, but has additional short helices at N and C termini, comprising a small substructure that caps the core helices. The RNA-binding surface of Vts1, determined by chemical shift perturbation, maps near the ends of three of the core helices, in agreement with mutational data and the electrostatic properties of the molecule. These results provide a structural basis for the versatility of the SAM domain in protein and RNA-recognition.     

Crystallization and characterization of Smaug: a novel RNA-binding motif

During Drosophila embryogenesis, Smaug protein represses translation of Nanos through an interaction with a specific element in its 3(')UTR. The repression occurs in the bulk cytoplasm of the embryo; Nanos is, however, successfully translated in the specialized cytoplasm of the posterior pole. This generates a gradient of Nanos emanating from the posterior pole that is essential for organizing proper abdominal segmentation. To understand the structural basis of RNA binding and translational control, we have crystallized a domain of Drosophila Smaug that binds RNA. The crystals belong to the space group R3 with unit cell dimensions of a=b=129.3A, c=33.1A, alpha=beta=90 degrees, gamma=120 degrees and diffract to 1.80A with synchrotron radiation. Initial characterization of this domain suggests that it encodes a novel RNA-binding motif.     

RNA recognition via the SAM domain of Smaug

The Nanos protein gradient in Drosophila, required for proper abdominal segmentation, is generated in part via translational repression of its mRNA by Smaug. We report here the crystal structure of the Smaug RNA binding domain, which shows no sequence homology to any previously characterized RNA binding motif. The structure reveals an unusual makeup in which a SAM domain, a common protein-protein interaction module, is affixed to a pseudo-HEAT repeat analogous topology (PHAT) domain. Unexpectedly, we find through a combination of structural and genetic analysis that it is primarily the SAM domain that interacts specifically with the appropriate nanos mRNA regulatory sequence. Therefore, in addition to their previously characterized roles in protein-protein interactions, some SAM domains play crucial roles in RNA binding.     

Coordinated control of lumen size and collective migration in the salivary gland

Drosophila Smaug is a sequence-specific RNA-binding protein that can repress the translation and induce the degradation of target mRNAs in the early Drosophila embryo. Our recent work has uncovered a new mechanism of Smaug-mediated translational repression whereby it interacts with and recruits the Argonaute 1 (Ago1) protein to an mRNA. Argonaute proteins are typically recruited to mRNAs through an associated small RNA, such as a microRNA (miRNA). Surprisingly, we found that Smaug is able to recruit Ago1 to an mRNA in a miRNA-independent manner. This work suggests that other RNA-binding proteins are likely to employ a similar mechanism of miRNA-independent Ago recruitment to control mRNA expression. Our work also adds yet another mechanism to the list that Smaug can use to regulate its targets and here we discuss some of the issues that are raised by Smaug’s multi-functional nature.     

Crystal structure of zinc-finger domain of Nanos and its functional implications

Nanos is an RNA-binding protein that is involved in the development and maintenance of germ cells. In combination with Pumilio, Nanos binds to the 3' untranslated region of a messenger RNA and represses its translation. Nanos has two conserved Cys-Cys-His-Cys zinc-finger motifs that are indispensable for its function. In this study, we have determined the crystal structure of the zinc-finger domain of zebrafish Nanos, for the first time revealing that Nanos adopts a novel zinc-finger structure. In addition, Nanos has a conserved basic surface that is directly involved in RNA binding. Our results provide the structural basis for further studies to clarify Nanos function.     

Visinin-like protein (VILIP) is a neuron-specific calcium-dependent double-stranded RNA-binding protein.

Double-stranded RNA-binding proteins function in regulating the stability, translation, and localization of specific mRNAs. In this study, we have demonstrated that the neuron-specific, calcium-binding protein, visinin-like protein (VILIP) contains one double-stranded RNA-binding domain, a protein motif conserved among many double-stranded RNA-binding proteins. We showed that VILIP can specifically bind double-stranded RNA, and this interaction specifically requires the presence of calcium. Mobility shift studies indicated that VILIP binds double-stranded RNA as a single protein-RNA complex with an apparent equilibrium dissociation constant of 9.0 x 10(-6) M. To our knowledge, VILIP is the first double-stranded RNA-binding protein shown to be calcium-dependent. Furthermore, VILIP specifically binds the 3'-untranslated region of the neurotrophin receptor, trkB, an mRNA localized to hippocampal dendrites in an activity-dependent manner. Given that VILIP is also expressed in the hippocampus, these data suggest that VILIP may employ a novel, calcium-dependent mechanism to regulate its binding to important localized mRNAs in the central nervous system.     

Smaug, a novel and conserved protein, contributes to repression of nanos mRNA translation in vitro

Proper deployment of Nanos protein at the posterior of the Drosophila embryo, where it directs posterior development, requires a combination of RNA localization and translational controls. These controls ensure that only the posteriorly-localized nanos mRNA is translated, whereas unlocalized nanos mRNA is translationally repressed. Here we describe cloning of the gene encoding Smaug, an RNA-binding protein that interacts with the sequences, SREs, in the nanos mRNA that mediate translational repression. Using an in vitro translation assay, we demonstrate that SRE-dependent repression occurs in extracts from early stage embryos. Immunodepletion of Smaug from the extracts eliminates repression, consistent with the notion that Smaug is involved. Smaug is a novel gene and the existence of potential mammalian Smaug homologs raises the possibility that Smaug represents a new class of conserved translational repressor.     

RNA recognition by a Staufen double-stranded RNA-binding domain

The double-stranded RNA-binding domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The domain binds optimally to RNA stem–loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem–loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix α1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix α1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.     

The widely conserved Era G-protein contains an RNA-binding domain required for Era function in vivo

Era is a small G-protein widely conserved in eubacteria and eukaryotes. Although essential for bacterial growth and implicated in diverse cellular processes, its actual function remains unclear. Several lines of evidence suggest that Era may be involved in some aspect of RNA biology. The GTPase domain contains features in common with all G-proteins and is required for Era function in vivo. The C-terminal domain (EraCTD) bears scant similarity to proteins outside the Era subfamily. On the basis of sequence comparisons, we argue that the EraCTD is similar to, but distinct from, the KH RNA-binding domain. Although both contain the consensus VIGxxGxxI RNA-binding motif, the protein folds are probably different. We show that bacterial Era binds RNA in vitro and can form higher-order RNA-protein complexes. Mutations in the VIGxxGxxI motif and other conserved residues of the Escherichia coli EraCTD decrease RNA binding in vitro and have corresponding effects on Era function in vivo, including previously described effects on cell division and chromosome partitioning. Importantly, mutations in L-66, located in the predicted switch II region of the E. coli Era GTPase domain, also perturb binding, leading us to propose that the GTPase domain regulates RNA binding in response to unknown cellular cues. The possible biological significance of Era RNA binding is discussed.     

Characterization of the RNA-binding domains in the replicase proteins of tomato bushy stunt virus

Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.     

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.