RNA 沉默的病毒抑制子

RNA 沉默是一种在真核生物体内普遍保守的、通过核酸序列特异性的相互作用来抑制基因表达的调控机制 . RNA 沉默的一种重要生物学效应是防御病毒的侵染,而针对寄主的这种防御机制,许多植物病毒已演化通过编码 RNA 沉默的抑制子来克服这种防御反应 . 目前,已从植物、动物和人类病毒中鉴定了 20 多种 RNA 沉默的抑制子,围绕抑制子的鉴定和作用机理研究已成为病毒学研究的一个热点 . 对 RNA 沉默抑制子的发现、鉴定方法、作用机理及与病毒病症状形成的关系、动物病毒的沉默抑制子等方面的最新进展做了综述,并对沉默抑制子的应用和存在的问题进行了讨论 .     

Endogenous Small RNA Clusters in Plants

In plants, small RNAs（sRNAs） usually refer to non-coding RNAs（ncRNAs） with lengths of 20–24 nucleotides. sRNAs are involved in the regulation of many essential processes related to plant development and environmental responses. sRNAs in plants are mainly grouped into microRNAs（miRNAs） and small interfering RNAs（siRNAs）, and the latter can be further classified into trans-acting siRNAs（ta-siRNAs）, repeat-associated siRNAs（ra-siRNAs）, natural anti-sense siRNAs（nat-siRNAs）, etc. Many sRNAs exhibit a clustered distribution pattern in the genome. Here, we summarize the features and functions of cluster-distributed sRNAs, aimed to not only provide a thorough picture of sRNA clusters（SRCs） in plants, but also shed light on the identification of new classes of functional sRNAs.     

A Small Subset of Fruitless Subesophageal Neurons Modulate Early Courtship in Drosophila

We show that a small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitless^Male (fru^M), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fru^M expression or inhibition of synaptic transmission in these fru^M(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. We identify a fru^M-dependent sexually dimorphic arborization in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship.     

Supervised Learning Classification Models for Prediction of Plant Virus Encoded RNA Silencing Suppressors

Viral encoded RNA silencing suppressor proteins interfere with the host RNA silencing machinery, facilitating viral infection by evading host immunity. In plant hosts, the viral proteins have several basic science implications and biotechnology applications. However in silico identification of these proteins is limited by their high sequence diversity. In this study we developed supervised learning based classification models for plant viral RNA silencing suppressor proteins in plant viruses. We developed four classifiers based on supervised learning algorithms: J48, Random Forest, LibSVM and Naïve Bayes algorithms, with enriched model learning by correlation based feature selection. Structural and physicochemical features calculated for experimentally verified primary protein sequences were used to train the classifiers. The training features include amino acid composition; auto correlation coefficients; composition, transition, and distribution of various physicochemical properties; and pseudo amino acid composition. Performance analysis of predictive models based on 10 fold cross-validation and independent data testing revealed that the Random Forest based model was the best and achieved 86.11% overall accuracy and 86.22% balanced accuracy with a remarkably high area under the Receivers Operating Characteristic curve of 0.95 to predict viral RNA silencing suppressor proteins. The prediction models for plant viral RNA silencing suppressors can potentially aid identification of novel viral RNA silencing suppressors, which will provide valuable insights into the mechanism of RNA silencing and could be further explored as potential targets for designing novel antiviral therapeutics. Also, the key subset of identified optimal features may help in determining compositional patterns in the viral proteins which are important determinants for RNA silencing suppressor activities. The best prediction model developed in the study is available as a freely accessible web server pVsupPred at http://bioinfo.icgeb.res.in/pvsup/.     

AtRLI2 is an Endogenous Suppressor of RNA Silencing

RNA silencing is a mechanism involved in gene regulation during development and anti-viral defense in plants and animals. Although many viral suppressors of this mechanism have been described up to now, this is not the case for endogenous suppressors. We have identified a novel endogenous suppressor in plants: RNase L inhibitor (RLI) of Arabidopsis thaliana. RLI is a very conserved protein among eukaryotes and archaea. It was first known as component of the interferon-induced mammalian 2′–5′ oligoadenylate (2–5A) anti-viral pathway. This protein is in several organisms responsible for essential functions, which are not related to the 2–5A pathway, like ribosome biogenesis and translation initiation. Arabidopsis has two RLI paralogs. We have described in detail the expression pattern of one of these paralogs (AtRLI2), which is ubiquitously expressed in all plant organs during different developmental stages. Infiltrating Nicotiana benthamiana green fluorescent protein (GFP)-transgenic line with Agrobacterium strains harboring GFP and AtRLI2, we proved that AtRLI2 suppresses silencing at the local and at the systemic level, reducing drastically the amount of GFP small interfering RNAs.     

A Small Genomic Region Containing Several Loci Required for Gastrulation in Drosophila

Genetic screens in Drosophila designed to search for loci involved in gastrulation have identified four regions of the genome that are required zygotically for the formation of the ventral furrow. For three of these, the genes responsible for the mutant phenotypes have been found. We now describe a genetic characterization of the fourth region, which encompasses the cytogenetic interval 24C3-25B, and the mapping of genes involved in gastrulation in this region. We have determined the precise breakpoints of several existing deficiencies and have generated new deficiencies. Our results show that the region contains at least three different loci associated with gastrulation effects. One maternal effect gene involved in ventral furrow formation maps at 24F but could not be identified. For a second maternal effect gene which is required for germ band extension, we identify a candidate gene, CG31660, which encodes a G protein coupled receptor. Finally, one gene acts zygotically in ventral furrow formation and we identify it as Traf4.     

Quantification of Food Intake in Drosophila

Measurement of food intake in the fruit fly Drosophila melanogaster is often necessary for studies of behaviour, nutrition and drug administration. There is no reliable and agreed method for measuring food intake of flies in undisturbed, steady state, and normal culture conditions. We report such a method, based on measurement of feeding frequency by proboscis-extension, validated by short-term measurements of food dye intake. We used the method to demonstrate that (a) female flies feed more frequently than males, (b) flies feed more often when housed in larger groups and (c) fly feeding varies at different times of the day. We also show that alterations in food intake are not induced by dietary restriction or by a null mutation of the fly insulin receptor substrate chico. In contrast, mutation of takeout increases food intake by increasing feeding frequency while mutation of ovo^D increases food intake by increasing the volume of food consumed per proboscis-extension. This approach provides a practical and reliable method for quantification of food intake in Drosophila under normal, undisturbed culture conditions.     

Reinforcement of Gametic Isolation in Drosophila

Reinforcement, a process by which natural selection increases reproductive isolation between populations, has been suggested to be an important force in the formation of new species. However, all existing cases of reinforcement involve an increase in mate discrimination between species. Here, I report the first case of reinforcement of postmating prezygotic isolation (i.e., barriers that act after mating but before fertilization) in animals. On the slopes of the African island of São Tomé, Drosophila yakuba and its endemic sister species D. santomea hybridize within a well-demarcated hybrid zone. I find that D. yakuba females from within this zone, but not from outside it, show an increase in gametic isolation from males of D. santomea, an apparent result of natural selection acting to reduce maladaptive hybridization between species. To determine whether such a barrier could evolve under laboratory conditions, I exposed D. yakuba lines derived from allopatric populations to experimental sympatry with D. santomea, and found that both behavioral and gametic isolation become stronger after only four generations. Reinforcement thus appears to be the best explanation for the heightened gametic isolation seen in sympatry. This appears to be the first example in animals in which natural selection has promoted the evolution of stronger interspecific genetic barriers that act after mating but before fertilization. This suggests that many other genetic barriers between species have been increased by natural selection but have been overlooked because they are difficult to study.     

Key Importance of Small RNA Binding for the Activity of a Glycine-Tryptophan (GW) Motif-containing Viral Suppressor of RNA Silencing

Viruses express viral suppressors of RNA silencing (VSRs) to counteract RNA silencing-based host defenses. Although virtually all stages of the antiviral silencing pathway can be inhibited by VSRs, small RNAs (sRNAs) and Argonaute (AGO) proteins seem to be the most frequent targets. Recently, GW/WG motifs of some VSRs have been proposed to dictate their suppressor function by mediating interaction with AGO(s). Here we have studied the VSR encoded by Pelargonium line pattern virus (family Tombusviridae). The results show that p37, the viral coat protein, blocks RNA silencing. Site-directed mutagenesis of some p37 sequence traits, including a conserved GW motif, allowed generation of suppressor-competent and -incompetent molecules and uncoupling of the VSR and particle assembly capacities. The engineered mutants were used to assess the importance of p37 functions for viral infection and the relative contribution of diverse molecular interactions to suppressor activity. Two main conclusions can be drawn: (i) the silencing suppression and encapsidation functions of p37 are both required for systemic Pelargonium line pattern virus infection, and (ii) the suppressor activity of p37 relies on the ability to bind sRNAs rather than on interaction with AGOs. The data also caution against potential misinterpretations of results due to overlap of sequence signals related to distinct protein properties. This is well illustrated by mutation of the GW motif in p37 that concurrently affects nucleolar localization, efficient interaction with AGO1, and sRNA binding capability. These concomitant effects could have been overlooked in other GW motif-containing suppressors, as we exemplify with the orthologous p38 of turnip crinkle virus.     

The Small GTPase Rheb Affects Central Brain Neuronal Morphology and Memory Formation in Drosophila

Mutations in either of two tumor suppressor genes, TSC1 or TSC2, cause tuberous sclerosis complex (TSC), a syndrome resulting in benign hamartomatous tumors and neurological disorders. Cellular growth defects and neuronal disorganization associated with TSC are believed to be due to upregulated TOR signaling. We overexpressed Rheb, an upstream regulator of TOR, in two different subsets of D. melanogaster central brain neurons in order to upregulate the Tsc-Rheb-TOR pathway. Overexpression of Rheb in either the mushroom bodies or the insulin producing cells resulted in enlarged axon projections and cell bodies, which continued to increase in size with prolonged Rheb expression as the animals aged. Additionally, Rheb overexpression in the mushroom bodies resulted in deficiencies in 3 hr but not immediate appetitive memory. Thus, Rheb overexpression in the central brain neurons of flies causes not only morphological phenotypes, but behavioral and aging phenotypes that may mirror symptoms of TSC.     

Chromosomal localization of microsatellite loci in Drosophila mediopunctata

Drosophila mediopunctata has been used as a model organism forgenetics and evolutionary studies in the last three decades. A linkage map with 48microsatellite loci recently published for this species showed five syntenic groups,which had their homology determined to Drosophila melanogasterchromosomes. Then, by inference, each of the groups was associated with one of thefive major chromosomes of D. mediopunctata. Our objective was tocarry out a genetic (chromosomal) analysis to increase the number of available lociwith known chromosomal location. We made a simultaneous analysis of visible mutantphenotypes and microsatellite genotypes in a backcross of a standard strain and amutant strain, which had each major autosome marked. Hence, we could establish thechromosomal location of seventeen loci; including one from each of the five majorlinkage groups previously published, and twelve new loci. Our results were congruentwith the previous location and they open new possibilities to future work integratingmicrosatellites, chromosomal inversions, and genetic determinants of physiologicaland morphological variation.     

How Food Controls Aggression in Drosophila

How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. For instance, it is unclear whether food increases aggression directly, or as a secondary consequence of increased social interactions caused by attraction to food. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner by which food influences aggression. We show that food promotes aggression in flies, and that it does so independently of any effect on frequency of contact between males, increase in locomotor activity or general enhancement of social interactions. Importantly, the level of aggression depends on the absolute amount of food, rather than on its surface area or concentration. When food resources exceed a certain level, aggression is diminished, suggestive of reduced competition. Finally, we show that detection of sugar via Gr5a⁺ gustatory receptor neurons (GRNs) is necessary for food-promoted aggression. These data demonstrate that food exerts a specific effect to promote aggression in male flies, and that this effect is mediated, at least in part, by sweet-sensing GRNs.