Mapping the binding site of snurportin 1 on native U1 snRNP by cross-linking and mass spectrometry

Mass spectrometry allows the elucidation of molecular details of the interaction domains of the individual components in macromolecular complexes subsequent to cross-linking of the individual components. Here, we applied chemical and UV cross-linking combined with tandem mass-spectrometric analysis to identify contact sites of the nuclear import adaptor snurportin 1 to the small ribonucleoprotein particle U1 snRNP in addition to the known interaction of m₃G cap and snurportin 1. We were able to define previously unknown sites of protein–protein and protein–RNA interactions on the molecular level within U1 snRNP. We show that snurportin 1 interacts with its central m₃G-cap-binding domain with Sm proteins and with its extreme C-terminus with stem-loop III of U1 snRNA. The crosslinking data support the idea of a larger interaction area between snurportin 1 and U snRNPs and the contact sites identified prove useful for modeling the spatial arrangement of snurportin 1 domains when bound to U1 snRNP. Moreover, this suggests a functional nuclear import complex that assembles around the m₃G cap and the Sm proteins only when the Sm proteins are bound and arranged in the proper orientation to the cognate Sm site in U snRNA.     

Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G-cap-dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G-cap is hypermethylated to the 2,2,7-trimethylguanosine (m3G)-cap. The import adaptor snurportin1 recognises the m3G-cap and facilitates the nuclear import of the UsnRNPs by binding to importin-beta. Here we report the crystal structure of the m3G-cap-binding domain of snurportin1 with bound m3GpppG at 2.4 A resolution, revealing a structural similarity to the mRNA-guanyly-transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G-cap-binding proteins Cap-binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G-cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G-capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap-binding activity tests using several snurportin1 mutants.     

The importin-beta binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro

The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNA's 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-beta binding (IBB) domain and a COOH-terminal m3G-cap--binding region that bears no structural similarity to known import adaptors like importin-alpha (impalpha). Here, we show that recombinant snurportin1 and importin-beta (impbeta) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impalpha-dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1-impbeta, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impbeta and a snurportin1 mutant containing an impalpha IBB domain (IBBimpalpha), nuclear U1 snRNP import was Ran dependent. Furthermore, beta-galactosidase (betaGal) containing a snurportin1 IBB domain, but not IBBimpalpha-betaGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impbeta with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions.     

Designing cyclopentapeptide inhibitor as potential antiviral drug for dengue virus ns5 methyltransferase

NS5 methyltransferase (Mtase) has a crucial role in the replication of dengue virus. There are two active sites on NS5 Mtase i.e., SAM and RNA-cap binding sites. Inhibition of the NS5 Mtase activity is expected to prevent the propagation of dengue virus. This study was conducted to design cyclic peptide ligands as enzyme inhibitors of dengue virus NS5 Mtase through computational approach. Cyclopentapeptides were designed as ligand of SAM binding site as much as 1635 and 736 cyclopentpeptides were designed as ligand of RNA-cap binding site. Interaction between ligand and NS5 Mtase has been conducted on the Docking simulation. The result shows that cyclopentapeptide CTWYC was the best peptide candidate on SAM binding site, with estimated free binding energy -30.72 kca/mol. Cyclopentapeptide CYEFC was the best peptide on RNA-cap binding site with estimated free binding energy -22.89 kcal/mol. Both peptides did not have tendency toward toxicity properties. So it is expected that both CTWYC and CYEFC ligands could be used as a potential antiviral drug candidates, which can inhibit the SAM and RNA-cap binding sites of dengue virus NS5 Mtase.     

CRM1-mediated recycling of snurportin 1 to the cytoplasm

Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin alpha. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.     

RACK1 is a ribosome scaffold protein for β-actin mRNA/ZBP1 complex

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.     

Mechanism of Mss116 ATPase reveals functional diversity of DEAD-Box proteins

Mss116 is a Saccharomyces cerevisiae mitochondrial DEAD-box RNA helicase protein that is essential for efficient in vivo splicing of all group I and group II introns and for activation of mRNA translation. Catalysis of intron splicing by Mss116 is coupled to its ATPase activity. Knowledge of the kinetic pathway(s) and biochemical intermediates populated during RNA-stimulated Mss116 ATPase is fundamental for defining how Mss116 ATP utilization is linked to in vivo function. We therefore measured the rate and equilibrium constants underlying Mss116 ATP utilization and nucleotide-linked RNA binding. RNA accelerates the Mss116 steady-state ATPase ∼ 7-fold by promoting rate-limiting ATP hydrolysis such that inorganic phosphate (P_i) release becomes (partially) rate-limiting. RNA binding displays strong thermodynamic coupling to the chemical states of the Mss116-bound nucleotide such that Mss116 with bound ADP-P_i binds RNA more strongly than Mss116 with bound ADP or in the absence of nucleotide. The predominant biochemical intermediate populated during in vivo steady-state cycling is the strong RNA-binding Mss116-ADP-P_i state. Strong RNA binding allows Mss116 to fulfill its biological role in the stabilization of group II intron folding intermediates. ATPase cycling allows for transient population of the weak RNA-binding ADP state of Mss116 and linked dissociation from RNA, which is required for the final stages of intron folding. In cases where Mss116 functions as a helicase, the data collectively favor a model in which ATP hydrolysis promotes a weak-to-strong RNA binding transition that disrupts stable RNA duplexes. The subsequent strong-to-weak RNA binding transition associated with P_i release dissociates Mss116-RNA complexes, regenerating free Mss116.     

Cross-talk between snurportin1 subdomains

The initial steps of spliceosomal small nuclear ribonucleoprotein (snRNP) maturation take place in the cytoplasm. After formation of an Sm-core and a trimethylguanosine (TMG) cap, the RNPs are transported into the nucleus via the import adaptor snurportin1 (SPN) and the import receptor importin-beta. To better understand this process, we identified SPN residues that are required to mediate interactions with TMG caps, importin-beta, and the export receptor, exportin1 (Xpo1/Crm1). Mutation of a single arginine residue within the importin-beta binding domain (IBB) disrupted the interaction with importin-beta, but preserved the ability of SPN to bind Xpo1 or TMG caps. Nuclear transport assays showed that this IBB mutant is deficient for snRNP import but that import can be rescued by addition of purified survival of motor neurons (SMN) protein complexes. Conserved tryptophan residues outside of the IBB are required for TMG binding. However, SPN can be imported into the nucleus without cargo. Interestingly, SPN targets to Cajal bodies when U2 but not U1 snRNPs are imported as cargo. SPN also relocalizes to Cajal bodies upon treatment with leptomycin B. Finally, we uncovered an interaction between the N- and C-terminal domains of SPN, suggesting an autoregulatory function similar to that of importin-alpha.     

Molecular basis for the recognition of snurportin 1 by importin beta

The nuclear import of uridine-rich ribonucleoproteins is mediated by the transport adaptor snurportin 1 (SNP1). Similar to importin alpha, SNP1 uses an N-terminal importin beta binding (sIBB) domain to recruit the receptor importin beta and gain access to the nucleus. In this study, we demonstrate that the sIBB domain has a bipartite nature, which contains two distinct binding determinants for importin beta. The first determinant spans residues 25-65 and includes the previously identified importin alpha IBB (alphaIBB) region of homology. The second binding determinant encompasses residues 1-24 and resembles region 1011-1035 of the nucleoporin 153 (Nup153). The two binding determinants synergize within the sIBB domain to confer a low nanomolar binding affinity for importin beta (K(d) approximately 2 nm) in an interaction that, in vitro, is displaced by RanGTP. We propose that in vivo the synergy of Nup153 and nuclear RanGTP promotes translocation of uridine-rich ribonucleoproteins into the nucleus.     

Potato virus X: structure, disassembly and reconstitution

This paper summarizes some structural characteristics of Potato virus X (PVX), the flexuous filamentous plant potexvirus. A model of PVX coat protein (CP) tertiary structure in the virion proposed on the basis of tritium planigraphy combined with predictions of the protein tertiary structure is described. A possible role of glycosylation and phosphorylation in the CP structure and function is discussed. Two forms of PVX virion disassembly are discussed: (i) the virion co-translational disassembly after PVX CP in situ phosphorylation and (ii) disassembly of PVX triggered by different factors after linear destabilization of the virion by binding of the PVX-coded movement protein (TGBp1) to one end of the polar CP-helix. Special emphasis was placed on a translational activation of encapsidated PVX RNA and rapid disassembly of TGBp1-PVX complexes into free RNA and CP. The results of experiments on the PVX CP repolymerization and PVX reconstitution are considered. In particular, the products assembled from PVX RNA, CP and TGBp1 were examined. Single-tailed particles were found with a helical, head-like structure consisting of helically arranged CP subunits located at the 5'-tail of RNA; the TGBp1 was bound to the end of the head. Translatable 'RNA-CP-TGBp1' complexes may represent the transport form of the PVX infection.     

Strong Binding of Single-stranded DNA by Stem-loop Oligonucleotides

Abstract

We report that oligodeoxynucleotides which form stem-loop hairpin structures and which have pyrimidine-rich loops can form strong complexes with complementary single-stranded DNA sequences. Stem-loop oligonucleotides were constructed with a 25-nt T-rich loop and with variable Watson-Crick stems. The complexes of these oligomers with the sequence dA_gwere studied by thermal denaturation. Evidence is presented that the complexes are one-to-one, bimolecular complexes in which the pyrimidine loop bases comprise the outer strands in a pyr · pur · pyr triplex, in effect chelating the purine strand in the center of the loop. Melting temperatures for the loop complexes are shown to be up to 29 °C higher than Watson- Crick duplex of the same length. It is shown that the presence of a stem increases stability of the triplex relative to an analogous oligomer without a stem. The effect of stem length on the stability of such a complex is examined. Such hairpin oligomers represent a new approach to the sequence-specific binding of single-stranded RNA and DNA. In addition, the finding raises the possibility that such a complex may exist in natural RNA folded sequences.     

Identification of the nucleotide sequences involved in the interaction between Escherichia coli 5 RNA and specific 50 S subunit proteins

Pancreatic RNase partial digests of ³²P-labelled 5 S RNA-protein complexes have been fractionated by electrophoresis on polyacrylamide gels. Specific fragments of the 5 S RNA molecule have been recovered from electrophoresis bands containing polynucleotide-protein complexes. These digestion-resistant complexes are only found if RNase treatment is carried out in the presence of at least one of the two 50 S subunit proteins L18 and L25, which are able to bind to 5 S RNA individually and specifically. The sequences of the isolated fragments have been determined. From the results, it can be concluded that sequence 69 to 120 and, possibly, sequence 1 to 11, are involved in the 5 S RNA-protein interactions which are responsible for the insertion of 5 S RNA in the 50 S subunit structure. Sequence 12 to 68, on the other hand, has no strong interactions with proteins L18 and L25. Each protein certainly binds to several nucleotide residues, which are not contiguous in the primary structure. In particular, good experimental evidence has been obtained in favour of the binding of protein L25 to two distant regions of the 5 S RNA molecule, which must have a bihelical secondary structure. The importance of the 5 S RNA conformation for its proper insertion in the 50 S subunit is thus confirmed.     

Molecular dynamics studies of U1A-RNA complexes

The U1A protein binds to a hairpin RNA and an internal-loop RNA with picomolar affinities. To probe the molecular basis of U1A binding, we performed state-of-the-art nanosecond molecular dynamics simulations on both complexes. The good agreement with experimental structures supports the protocols used in the simulations. We compare the dynamics, hydrogen-bonding occupancies, and interfacial flexibility of both complexes and also describe a rigid-body motion in the U1A-internal loop complex that is not observed in the U1A-hairpin simulation. We relate these observations to experimental mutational studies and highlight their significance in U1A binding affinity and specificity.     

Phenomenological theory of gel electrophoresis of protein-nucleic acid complexes

A phenomenological theory of gel electrophoresis is elaborated for protein-DNA complexes involving one, two, or three binding sites on the DNA molecule. The computed electrophoretic patterns simulate experimental patterns shown by both prokaryotic and eukaryotic systems. The mechanism whereby the electrophoretic protein-DNA ladder is generated upon titration of the operator with repressor is embodied in theory of mass transport coupled to reversible interactions under chemical kinetic control. In contrast to strong interactions (association constant greater than 10(12) M-1), patterns observed with weak complexes (K less than 10(10) M-1) could be simulated only by applying the cage effect, a model of which is formulated. Theoretical underpinning is provided for the electrophoretic estimation of equilibrium association constants, and requisite chemical kinetic conditions are elucidated for direct estimation of the rate constant for dissociation of the protein-DNA complex from gel patterns. The theory thus affords an experimenter with a means for determining the conditions required to render the gel retardation method a valid procedure for evaluating equilibrium constants and/or kinetic parameters for the particular protein-nucleic acid system under investigation. These several considerations apply not only to interactions of proteins with nucleic acids (DNA or RNA) but also to a wide range of macromolecular interactions involving peptides, drugs, and other ligands as well as large assemblies such as multienzyme complexes.     

Electronic effects on the interactions of complexes [Ru(phen)2(p-L)] (L=MOPIP, HPIP, and NPIP) with DNA

In order to explore the electronic effects of Ru(II) complexes binding to DNA, a series of Ru(II) complexes [Ru(phen)₂ (p-MOPIP)]²⁺ (1), [Ru(phen)₂ (p-HPIP)]²⁺ (2), and [Ru(phen)₂(p-NPIP)]²⁺ (3) were synthesized and characterized by elementary, ¹H NMR, and ES-MS analysis. The binding properties of these complexes to CT-DNA were investigated with spectroscopic methods and viscosity experiments. Furthermore, the computations for these complexes applying the density functional theory (DFT) method have also been performed. The results show that all of these complexes can well bind to DNA in intercalation mode and DNA-binding affinity of these complexes is greatly influenced by electronic effects of intercalating ligands. The intrinsic binding constants for 1, 2, and 3 are 0.20, 0.69, and 1.56 × 10⁵ M⁻¹, respectively. This order is in accordance with that of the electron-withdrawing ability of substituent [-OR < -OH < -NO₂]. Such a trend in electronic effects of Ru(II) complexes binding to DNA can be reasonably explained by the DFT calculations.     

Analysis of the gel electrophoresis of looped protein-DNA complexes by computer simulation

The theory of mass transport coupled to reversible interactions under chemical kinetic control forms the basis of a numerical model that has been applied to systems such as lac repressor-lac operator DNA, in which a protein binds in two different modes to linear DNA carrying two specific binding sites. Three complexes may be formed: (1) a linear 1:1 complex with one protein molecule bound to one site on the DNA molecule; (2) a 1:1 complex in which a single protein molecule is bound to both sites simultaneously, thereby inducing a large DNA loop; and (3) a 2:1 linear complex in which two protein molecules are bound in tandem, each occupying a single site. The computational model affords a quantitative numerical simulation of the observed gel electrophoretic patterns produced by titration of the DNA with protein and provides new insights into the shape and nature of the patterns. In particular, the patterns may represent unimodal or bimodal reaction zones. Nevertheless, analysis of the peaks in the patterns obtained at low DNA and high protein concentration provides essential information as to the stoichiometry of the complexes and satisfactory estimates of association constants. The theory thus provides the experimenter with guidelines for quantitative evaluation of the results of gel retardation assays of the particular system under investigation, once protein-induced DNA (or RNA) loops have been established by independent physical or chemical methods. It is suggested that these insights might also find application to systems involving the binding of two or three different proteins to DNA with loop formation.     

Ribonucleoprotein complexes in neurologic diseases

Ribonucleoprotein (RNP) complexes regulate the tissue-specific RNA processing and transport that increases the coding capacity of our genome and the ability to respond quickly and precisely to the diverse set of signals. This review focuses on three proteins that are part of RNP complexes in most cells of our body: TAR DNA-binding protein (TDP-43), the survival motor neuron protein (SMN), and fragile-X mental retardation protein (FMRP). In particular, the review asks the question why these ubiquitous proteins are primarily associated with defects in specific regions of the central nervous system? To understand this question, it is important to understand the role of genetic and cellular environment in causing the defect in the protein, as well as how the defective protein leads to misregulation of specific target RNAs. Two approaches for comprehensive analysis of defective RNA-protein interactions are presented. The first approach defines the RNA code or the collection of proteins that bind to a certain cis-acting RNA site in order to lead to a predictable outcome. The second approach defines the RNA map or the summary of positions on target RNAs where binding of a particular RNA-binding protein leads to a predictable outcome. As we learn more about the RNA codes and maps that guide the action of the dynamic RNP world in our brain, possibilities for new treatments of neurologic diseases are bound to emerge.