No abstract

Plant viral movement proteins (MPs) participate actively in the intra-and intercellular movement of RNA plant viruses to such an extent that MP dysfunction impairs viral infection. However, the molecular mechanism(s) of their interaction with cognate nucleic acids are not well understood, partly due to the lack of structural information. In this work, a protein dissection approach was used to gain information on the structural and RNA-binding properties of this class of proteins, as exemplified by the 61-amino acid residue p7 MP from carnation mottle virus (CarMV). Circular dichroism spectroscopy showed that CarMV p7 is an ␣/␤ RNA-binding soluble protein. Using synthetic peptides derived from the p7 sequence, we have identified three distinct putative domains within the protein. EMSA showed that the central region, from residue 17 to 35 (represented by peptide p7 17-35 ), is responsible for the RNA binding properties of CarMV p7. This binding peptide populates a nascent ␣-helix in water solution that is further stabilized in the presence of either secondary structure inducers, such as trifluoroethanol and monomeric SDS, or RNA (which also changes its conformation upon binding to the peptide). Thus, the RNA recognition appears to occur via an "adaptive binding" mechanism. Interestingly, the amino acid sequence and structural properties of the RNA-binding domain of p7 seem to be conserved among carmoviruses and some other RNAbinding proteins and peptides. The low conserved N terminus of p7 (peptide p7 1-16 ) is unstructured in solution. In contrast, the highly conserved C terminus motif (peptide p7 40 -61 ) adopts a ␤-sheet conformation in aqueous solution. Alanine scanning mutagenesis of the RNAbinding motif showed how selected positive charged amino acids are more relevant than others in the RNA binding process and how hydrophobic amino acid side chains would participate in the stabilization of the protein-RNA complex.Infection of plants by viruses requires viral genome cell-tocell movement through plasmodesmata (the plant intercellular symplastic connections), which is mediated by virus-encoded so-called movement proteins (MPs) 1 (1, 2). MPs participate actively in the intra-and intercellular movement of plant viruses, and mutant virus analyses, reverse genetics, and plant transformation have demonstrated that MP dysfunction impairs viral infection (3, 4). MP properties are best described in the tobacco mosaic tobamovirus (TMV) model system. The 30-kDa TMV MP binds single-stranded (viral) RNA in vitro with no sequence specificity and high cooperativity (5), co-localizes with the cytoskeleton and cell wall (6 -8), is required for the association of viral RNA with endoplasmic reticulum (9), increases the size exclusion limit of plasmodesmata (10), and mediates an active transport of the viral genome to the adjacent cell (4). Sequence deletion and mutagenesis studies have located some of these functions in separate motifs/domains of the protein (11-13). These properties define the tobamo-like model of viral cell t...

The environment of the oocyte during its in vivo maturation consists of follicular fluid (FF) and is surrounded by granulosa cells. The FF is derived from the sanguineous plasma and secretions, synthesised in the follicle wall, that contain a large variety of growth factors, cytokines, amino acids, and other metabolites. These metabolites are presumably involved in the physiology of the oocyte. The identification, quantification and study of FF metabolites can provide additional information about the oocyte state which can be helpful in distinguishing those oocytes that have a greater capacity to be fertilised and to develop properly. The aim of this work is to identify the metabolic profile of FF samples exhaustively using High Resolution Nuclear Magnetic Resonance (NMR). A total of 30 FF samples from oocyte donors (<35 years) were analysed. Different monodimensional (1D) and bidimensional (2D) (homo and heteronuclear) NMR experiments were acquired. A total of 131 chemical shifts were assigned and 42 metabolites, including as example glucose, lactate, acetate, acetoacetate, pyruvate and b-hydroxybutyrate, were identified. High correlations were found between these important intermediaries of the energetic metabolic pathways of the follicle which can indicate the importance of these pathways in oocyte development. Some of these identified metabolites might be useful as biomarkers of the follicular maturation state, allowing oocytes with a higher fertilisation potential to be selected, thereby increasing pregnancy rates in women following in vitro fertilisation (IVF) treatments.