Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.     

Energetics of RNA binding by the West Nile virus RNA triphosphatase

The West Nile virus (WNV) RNA genome harbors the characteristic methylated cap structure present at the 5' end of eukaryotic mRNAs. In the present study, we report a detailed study of the binding energetics and thermodynamic parameters involved in the interaction between RNA and the WNV RNA triphosphatase, an enzyme involved in the synthesis of the RNA cap structure. Fluorescence spectroscopy assays revealed that the initial interaction between RNA and the enzyme is characterized by a high enthalpy of association and that the minimal RNA binding site of NS3 is 13 nucleotides. In order to provide insight into the relationship between the enzyme structure and RNA binding, we also correlated the effect of RNA binding on protein structure using both circular dichroism and denaturation studies as structural indicators. Our data indicate that the protein undergoes structural modifications upon RNA binding, although the interaction does not significantly modify the stability of the protein.     

Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two majorcis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3 splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on thecis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.     

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z_eff) and nonelectrostatic (i.e., specific) component of binding, K_d(1M). Gag binds to Psi RNA with a dramatically reduced K_d(1M) and lower Z_eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z_eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z_eff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.     

Structural insights into small RNA sorting and mRNA target binding by Arabidopsis Argonaute Mid domains

The RISC-associated Argonaute (Ago) proteins play the catalytic role for RISC-mediated gene regulation by selecting small RNAs and subsequent targeting and cleavage of complementary mRNAs. Ago Mid domains are proposed to play essential roles in small RNA sorting. Here, we report the crystal structures of Arabidopsis Ago1 Mid domain and its chimera mutant with part of Ago1 replaced by Ago4. The structures demonstrate that a single amino insertion in the nucleotide specificity loop of AtAgo1 will change the nucleotide binding preference of AtAgo1 from “5′-U” to “5′-A”. Moreover, we identify a long positively charged groove located along the “5′-end-nucleotide specificity loop” and occupied by several sulfate ions with the distance of 9-11 Å distance, indicating a putative mRNA target binding groove.     

Human hnRNP protein A1: A model polypeptide for a structural and genetic investigation of a broad family of RNA binding proteins

The hnRNP fiber is the substrate on which pre-mRNA processing occurs. The protein moiety of the fiber (hnRNP proteins) constitutes a broad family of RNA binding proteins that revealed, upon molecular analysis, a number of interesting features.Heterogeneous nuclear ribonucleoprotein A1 is a major component of the human hnRNP complex. In recent years this protein has attracted great attention because of several emerging evidences of its direct involvement in pre-mRNA processing and it has become one of the best characterized RNA binding proteins. Detailed knowledge of the structure of protein A1 has laid the basis for the understanding of its function, and for this reason A1 can be considered as a model polypeptide for the investigation of a large number of RNA binding proteins.In this work we report recent findings regarding the binding properties of protein A1 as well as new data on the gene structure of A1 and of its closely related hnRNP protein A2. Our results show that a single A1 molecule contains the determinants for simultaneous binding of two single-stranded nucleic acid molecules and we demonstrate that the glycine-rich domain of A1, isolated from the rest of the molecule, is capable of sustaining protein-protein interactions. These features probably account for the reannealing activity of the protein and for its capacity to modulate the binding of snRNPs to intron sequencesin vitro. Comparison of A1 and A2 gene sequences revealed a remarkable conservation of the overall structural organization, suggesting important functions for the different structural elements.     

Modulation of ligand binding by alternative splicing of the alphaPS2 integrin subunit

The Drosophila alphaPS2 integrin subunit is found in two isoforms. alphaPS2C contains 25 residues not found in alphaPS2m8, encoded by the alternative eighth exon. Previously, it was shown that cells expressing alphaPS2C spread more effectively than alphaPS2m8 cells on fragments of the ECM protein Tiggrin, and that alphaPS2C-containing integrins are relatively insensitive to depletion of Ca(2+). Using a ligand mimetic probe for Tiggrin affinity (TWOW-1), we show that the affinity of alphaPS2CbetaPS for this ligand is much higher than that of alphaPS2m8betaPS. However, the two isoforms become more similar in the presence of activating levels of Mn(2+). Modeling indicates that the exon 8-encoded residues replace the third beta strand of the third blade of the alpha subunit beta-propeller structure, and generate an exaggerated loop between this and the fourth strand. alphaPS2 subunits with the extra loop structure but with an m8-like third strand, or subunits with a C-like strand but an m8-like short loop, both fail to show alphaPS2C-like affinity for TWOW-1. Surprisingly, a single C > m8-like change at the third strand-loop transition point is sufficient to make alphaPS2C require Ca(2+) for function, despite the absence of any known cation binding site in this region. These data indicate that alternative splicing in integrin alpha subunit extracellular domains may affect ligand affinity via relatively subtle alterations in integrin conformation. These results may have relevance for vertebrate alpha6 and alpha7, which are alternatively spliced at the same site.     

Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding

We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.     

Ankyrin-G in skeletal muscle: tissue-specific alternative splicing contributes to the complexity of the sarcolemmal cytoskeleton

Ankyrins are versatile adaptor proteins that join the spectrin-based cytoskeleton to transmembrane proteins, and have roles in organizing the microstructure of cell membranes. Molecular diversity of ankyrins in mammals arises from extensive alternative splicing of the products of three genes. There has been no systematic analysis of the diversity of expression of ankyrins-G, the widely expressed Ank3 gene products, in a complex tissue. We previously described Ank(G107), the first muscle-specific ankyrin-G. Here, we combined cDNA and database analyses to gain novel insight into the ankyrins-G of skeletal muscle. We find: (i) that Ank3 is composed of at least 53 exons, many of which are subject to tissue-specific splicing; (ii) five novel full-length cDNAs encoding two canonical (Ank(G197), Ank(G217)) and three small isoforms (Ank(G109), Ank(G128), Ank(G130)) bring to six the number of ankyrins-G expressed in skeletal muscle; (iii) a 76-residue insert in the C-terminal domain is a 'signature' for muscle ankyrins; (iv) variably spliced sequences of 17/18 and 195 residues increase diversity in the C-terminal domains. Comparison of endogenous ankyrins-G with in vitro translated cDNAs revealed that small ankyrins account for the majority of the immunoreactivity for ankyrin-G in soleus muscle. The small ankyrins, when expressed in vivo in the rat muscle, are all targeted to sarcolemmal costameres. Our results demonstrate the tissue-dependent alternative splicing of Ank3 in skeletal muscle and point to novel functions of small ankyrins-G in organizing microdomains of the plasma membrane.     

Tissue-Specific Expression of the Gene Encoding a Mouse RNA Binding Protein Homologous to Human HuD Antigen

We have cloned and sequenced cDNAs encoding a mouse RNA-bindingprotein that is homologous to human HuD antigen. The amino acidsequence deduced from the nucleotide sequence has revealed thatthe mouse HuD protein is identicalto the human counterpart exceptfor two amino-acid substitutions outside the three RNA recognitionmotifs (RRMs) and a difference in the N-terminus. The mouseHuD gene produces two major brain-specific mRNAs (3.7 kb and4.4 kb) and a minor testis-specific mRNA (1.3 kb), which isindicative of alternative RNA processing. These results suggestthat the mouse HuD homolog is a member of the tissue-specificRNA-binding protein family, possibly involved in RNA metabolismin the nervous system.