Extra double-stranded RNA binding domain (dsRBD) in a squid RNA editing enzyme confers resistance to high salt environment

A-to-I RNA editing is particularly common in coding regions of squid mRNAs. Previously, we isolated a squid editing enzyme (sqADAR2) that shows a unique structural feature when compared with other ADAR2 family members: an additional double-stranded RNA (dsRNA) binding domain (dsRBD). Alternative splicing includes or excludes this motif, generating a novel or a conventional variant termed sqADAR2a and sqADAR2b, respectively. The extra dsRBD of sqADAR2a increases its editing activity in vitro. We hypothesized that the high activity is due to an increase in the affinity of the enzyme for dsRNA. This may be important because protein-RNA interactions can be influenced by physical factors. We became particularly interested in analyzing the effects of salt on interactions between sqADAR2 and RNA because squid cells have a ～3-fold higher ionic strength and proportionally more Cl(-) than vertebrate cells. To date, in vitro biochemical analyses of adenosine deamination have been conducted using vertebrate-like ionic strength buffers containing chloride as the major anion, although the vast majority of cellular anions are known to be organic. We found that squid-like salt conditions severely impair the binding affinity of conventional ADAR2s for dsRNA, leading to a decrease in nonspecific and site-specific editing activity. Inhibition of editing was mostly due to high Cl(-) levels and not to the high concentrations of K(+), Na(+), and organic anions like glutamate. Interestingly, the extra dsRBD in sqADAR2a conferred resistance to the high Cl(-) levels found in squid neurons. It does so by increasing the affinity of sqADAR2 for dsRNA by 30- or 100-fold in vertebrate-like or squid-like conditions, respectively. Site-directed mutagenesis of squid ADAR2a showed that its increased affinity and editing activity are directly attributable to the RNA binding activity of the extra dsRBD.     

ARTIFICIAL SELECTION ON RELATIVE BRAIN SIZE REVEALS A POSITIVE GENETIC CORRELATION BETWEEN BRAIN SIZE AND PROACTIVE PERSONALITY IN THE GUPPY

Animal personalities range from individuals that are shy, cautious, and easily stressed (a “reactive” personality type) to individuals that are bold, innovative, and quick to learn novel tasks, but also prone to routine formation (a “proactive” personality type). Although personality differences should have important consequences for fitness, their underlying mechanisms remain poorly understood. Here, we investigated how genetic variation in brain size affects personality. We put selection lines of large‐ and small‐brained guppies (Poecilia reticulata), with known differences in cognitive ability, through three standard personality assays. First, we found that large‐brained animals were faster to habituate to, and more exploratory in, open field tests. Large‐brained females were also bolder. Second, large‐brained animals excreted less cortisol in a stressful situation (confinement). Third, large‐brained animals were slower to feed from a novel food source, which we interpret as being caused by reduced behavioral flexibility rather than lack of innovation in the large‐brained lines. Overall, the results point toward a more proactive personality type in large‐brained animals. Thus, this study provides the first experimental evidence linking brain size and personality, an interaction that may affect important fitness‐related aspects of ecology such as dispersal and niche exploration.     

A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate the prevalence of Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of the disease and hotspots of malaria transmission within the country. The prevalence of P. falciparum and P. vivax within the districts of the capital city and the rest of the Republic of Djibouti were assessed using 13 and 2 serological markers, respectively. The relationship between the immune humeral response to P. falciparum and P. vivax and variables such as age, gender, wealth status, urbanism, educational level, distance to rivers/lakes, living area, having fever in the last month, and staying in a malaria-endemic country more than one year was estimated and analysed by questionnaires administered to 1910 Djiboutians. Multivariate ordinal logistic regression models of the immune humeral response were obtained for P. falciparum and P. vivax. The P. falciparum and P. vivax seroprevalence rates were 31.5%, CI95% [29.4-33.7] and 17.5%, CI95% [15.8-19.3], respectively. Protective effects against P. falciparum and P. vivax were female gender, educational level, and never having visited a malaria-endemic area for more than one year. For P. falciparum only, a protective effect was observed for not having a fever in the last month, living more than 1.5 km away from lakes and rivers, and younger ages. This is the first study that assessed the seroprevalence of P. vivax in the Republic of Djibouti. It is necessary to improve knowledge of this pathogen in order to create an effective elimination programme. As supported by recent observations on the subject, the Republic of Djibouti has probably demonstrated a real decrease in the transmission of P. falciparum in the past seven years, which should encourage authorities to improve efforts toward elimination.     

Integrating sequence and structural biology with DAS

Background  

The Distributed Annotation System (DAS) is a network protocol for exchanging biological data. It is frequently used to share annotations of genomes and protein sequence.     

Glycosylation sites and site-specific glycosylation in human Tamm- Horsfall glycoprotein

The N-glycosylation sites of human Tamm-Horsfall glycoprotein from one healthy male donor have been characterized, based on an approach using endoproteinase Glu-C (V-8 protease, Staphylococcus aureus ) digestion and a combination of chromatographic techniques, automated Edman sequencing, and fast atom bombardment mass spectrometry. Seven out of the eight potential N-glycosylation sites, namely, Asn52, Asn56, Asn208, Asn251, Asn298, Asn372, and Asn489, turned out to be glycosylated, and the potential glycosylation site at Asn14, being close to the N-terminus, is not used. The carbohydrate microheterogeneity on three of the glycosylation sites was studied in more detail by high-pH anion-exchange chromatographic profiling and 500 MHz1H-NMR spectroscopy. Glycosylation site Asn489 contains mainly di- and tri-charged oligosaccharides which comprise, among others, the GalNAc4 S (beta1-4)GlcNAc terminal sequence. Only glycosylation site Asn251 bears oligomannose-type carbohydrate chains ranging from Man5GlcNAc2to Man8GlcNAc2, in addition to a small amount of complex- type structures. Profiling of the carbohydrate moieties of Asn208 indicates a large heterogeneity, similar to that established for native human Tamm-Horsfall glycoprotein, namely, multiply charged complex-type carbohydrate structures, terminated by sulfate groups, sialic acid residues, and/or the Sda-determinant.      

An extra double-stranded RNA binding domain confers high activity to a squid RNA editing enzyme

RNA editing by adenosine deamination is particularly prevalent in the squid nervous system. We hypothesized that the squid editing enzyme might contain structural differences that help explain this phenomenon. As a first step, a squid adenosine deaminase that acts on RNA (sqADAR2a) cDNA and the gene that encodes it were cloned from the giant axon system. PCR and RNase protection assays showed that a splice variant of this clone (sqADAR2b) was also expressed in this tissue. Both versions are homologous to the vertebrate ADAR2 family. sqADAR2b encodes a conventional ADAR2 family member with an evolutionarily conserved deaminase domain and two double-stranded RNA binding domains (dsRBD). sqADAR2a differs from sqADAR2b by containing an optional exon that encodes an “extra” dsRBD. Both splice variants are expressed at comparable levels and are extensively edited, each in a unique pattern. Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. Using a standard 48-h protein induction, both sqADAR2a and sqADAR2b exhibit promiscuous self-editing; however, this activity is particularly robust for sqADAR2a. By decreasing the induction time to 16 h, self-editing was mostly eliminated. We next tested the ability of sqADAR2a and sqADAR2b to edit two K⁺ channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. These data suggest that the “extra” dsRBD confers high activity on sqADAR2a.