Andrew G. Cridge

Comparison of characteristics and function of translation termination signals between and within prokaryotic and eukaryotic organisms

Six diverse prokaryotic and five eukaryotic genomes were compared to deduce whether the protein synthesis termination signal has common determinants within and across both kingdoms. Four of the six prokaryotic and all of the eukaryotic genomes investigated demonstrated a similar pattern of nucleotide bias both 5′ and 3′ of the stop codon. A preferred core signal of 4 nt was evident, encompassing the stop codon and the following nucleotide. Codons decoded by hyper-modified tRNAs were over-represented in the region 5′ to the stop codon in genes from both kingdoms. The origin of the 3′ bias was more variable particularly among the prokaryotic organisms. In both kingdoms, genes with the highest expression index exhibited a strong bias but genes with the lowest expression showed none. Absence of bias in parasitic prokaryotes may reflect an absence of pressure to evolve more efficient translation. Experiments were undertaken to determine if a correlation existed between bias in signal abundance and termination efficiency. In Escherichia coli signal abundance correlated with termination efficiency for UAA and UGA stop codons, but not in mammalian cells. Termination signals that were highly inefficient could be made more efficient by increasing the concentration of the cognate decoding release factor.

Identifying eIF4E-binding protein translationally- controlled transcripts reveals links to mRNAs bound by specific PUF proteins

eIF4E-binding proteins (4E-BPs) regulate translation of mRNAs in eukaryotes. However the extent to which specific mRNA targets are regulated by 4E-BPs remains unknown. We performed translational profiling by microarray analysis of polysome and monosome associated mRNAs in wild-type and mutant cells to identify mRNAs in yeast regulated by the 4E-BPs Caf20p and Eap1p; the first-global comparison of 4E-BP target mRNAs. We find that yeast 4E-BPs modulate the translation of >1000 genes. Most target mRNAs differ between the 4E-BPs revealing mRNA specificity for translational control by each 4E-BP. This is supported by observations that eap1Δ and caf20Δ cells have different nitrogen source utilization defects, implying different mRNA targets. To account for the mRNA specificity shown by each 4E-BP, we found correlations between our data sets and previously determined targets of yeast mRNA-binding proteins. We used affinity chromatography experiments to uncover specific RNA-stabilized complexes formed between Caf20p and Puf4p/Puf5p and between Eap1p and Puf1p/Puf2p. Thus the combined action of each 4E-BP with specific 3′-UTR-binding proteins mediates mRNA-specific translational control in yeast, showing that this form of translational control is more widely employed than previously thought.

Improved annotation of the insect vector of citrus greening disease: biocuration by a diverse genomics community

Abstract The Asian citrus psyllid (Diaphorina citri Kuwayama) is the insect vector of the bacterium Candidatus Liberibacter asiaticus (CLas), the pathogen associated with citrus Huanglongbing (HLB, citrus greening). HLB threatens citrus production worldwide. Suppression or reduction of the insect vector using chemical insecticides has been the primary method to inhibit the spread of citrus greening disease. Accurate structural and functional annotation of the Asian citrus psyllid genome, as well as a clear understanding of the interactions between the insect and CLas, are required for development of new molecular-based HLB control methods. A draft assembly of the D. citri genome has been generated and annotated with automated pipelines. However, knowledge transfer from well-curated reference genomes such as that of Drosophila melanogaster to newly sequenced ones is challenging due to the complexity and diversity of insect genomes. To identify and improve gene models as potential targets for pest control, we manually curated several gene families with a focus on genes that have key functional roles in D. citri biology and CLas interactions. This community effort produced 530 manually curated gene models across developmental, physiological, RNAi regulatory and immunity-related pathways. As previously shown in the pea aphid, RNAi machinery genes putatively involved in the microRNA pathway have been specifically duplicated. A comprehensive transcriptome enabled us to identify a number of gene families that are either missing or misassembled in the draft genome. In order to develop biocuration as a training experience, we included undergraduate and graduate students from multiple institutions, as well as experienced annotators from the insect genomics research community. The resulting gene set (OGS v1.0) combines both automatically predicted and manually curated gene models.

What Do Studies of Insect Polyphenisms Tell Us about Nutritionally-Triggered Epigenomic Changes and Their Consequences?

Many insects are capable of remarkable changes in biology and form in response to their environment or diet. The most extreme example of these are polyphenisms, which are when two or more different phenotypes are produced from a single genotype in response to the environment. Polyphenisms provide a fascinating opportunity to study how the environment affects an animal’s genome, and how this produces changes in form. Here we review the current state of knowledge of the molecular basis of polyphenisms and what can be learnt from them to understand how nutrition may influence our own genomes.

The nucleolus: a raft adrift in the nuclear sea or the keystone in nuclear structure?

Abstract The nucleolus is a prominent nuclear structure that is the site of ribosomal RNA (rRNA) transcription, and hence ribosome biogenesis. Cellular demand for ribosomes, and hence rRNA, is tightly linked to cell growth and the rRNA makes up the majority of all the RNA within a cell. To fulfill the cellular demand for rRNA, the ribosomal RNA (rDNA) genes are amplified to high copy number and transcribed at very high rates. As such, understanding the rDNA has profound consequences for our comprehension of genome and transcriptional organization in cells. In this review, we address the question of whether the nucleolus is a raft adrift the sea of nuclear DNA, or actively contributes to genome organization. We present evidence supporting the idea that the nucleolus, and the rDNA contained therein, play more roles in the biology of the cell than simply ribosome biogenesis. We propose that the nucleolus and the rDNA are central factors in the spatial organization of the genome, and that rapid alterations in nucleolar structure in response to changing conditions manifest themselves in altered genomic structures that have functional consequences. Finally, we discuss some predictions that result from the nucleolus having a central role in nuclear organization.

The honeybee as a model insect for developmental genetics

Honeybees are an important component of modern agricultural systems, and a fascinating and scientifically engrossing insect. Honeybees are not commonly used as model systems for understanding development in insects despite their importance in agriculture. Honeybee embryogenesis, while being superficially similar to Drosophila, is molecularly very different, especially in axis formation and sex determination. In later development, much of honeybee biology is modified by caste development, an as yet poorly understood, but excellent, system to study developmental plasticity. In adult stages, developmental plasticity of the ovaries, related to reproductive constraint exhibits another aspect of plasticity. Here they review the tools, current knowledge and opportunities in honeybee developmental biology, and provide an updated embryonic staging scheme to support future studies.

Molecular evolutionary trends and feeding ecology diversification in the Hemiptera, anchored by the milkweed bug genome

Background The Hemiptera (aphids, cicadas, and true bugs) are a key insect order whose members offer a close outgroup to the Holometabola, with high diversity within the order for feeding ecology and excellent experimental tractability for molecular genetics. Sequenced genomes have recently become available for hemipteran pest species such as phloem-feeding aphids and blood-feeding bed bugs. To complement and build upon these resources, we present the genome sequence and comparative analyses centered on the large milkweed bug, Oncopeltus fasciatus, a seed feeder of the family Lygaeidae. Results The 926-Mb genome of Oncopeltus is relatively well represented by the current assembly and official gene set, which supports Oncopeltus as a fairly conservative hemipteran species for anchoring molecular comparisons. We use our genomic and RNA-seq data not only to characterize features of the protein-coding gene repertoire and perform isoform-specific RNAi, but also to elucidate patterns of molecular evolution and physiology. We find ongoing, lineage-specific expansion and diversification of repressive C2H2 zinc finger proteins and of intron gain and turnover in the Hemiptera. These analyses also weigh the relative importance of lineage and genome size as predictors of gene structure evolution in insects. Furthermore, we identify enzymatic gains and losses that correlate with hemipteran feeding biology, particularly for reductions in chemoreceptor family size and loss of metabolic reactions within species with derived, fluid-nutrition feeding modes. Conclusions With the milkweed bug genome, for the first time we have a critical mass of sequenced species representing a hemimetabolous insect order, substantially improving the diversity of insect genomics beyond holometabolans such as flies and ants. We use this addition to define commonalities among the Hemiptera and then delve into how hemipteran species’ genomes reflect their feeding ecology types. Our novel and detailed analyses integrate global and rigorous manual approaches, generating hypotheses and identifying specific sets of genes for future investigation. Given Oncopeltus’s strength as an experimental research model, we take particular care to evaluate the sequence resources presented here, augmenting its foundation for molecular research and highlighting potentially general considerations exemplified in the assembly and annotation of this medium-sized genome.

Convergent occurrence of the developmental hourglass in plant and animal embryogenesis

Background The remarkable similarity of animal embryos at particular stages of development led to the proposal of a developmental hourglass. In this model, early events in development are less conserved across species but lead to a highly conserved ‘phylotypic period’. Beyond this stage, the model suggests that development once again becomes less conserved, leading to the diversity of forms. Recent comparative studies of gene expression in animal groups have provided strong support for the hourglass model. How and why might such an hourglass pattern be generated? More importantly, how might early acting events in development evolve while still maintaining a later conserved stage? Scope The discovery that an hourglass pattern may also exist in the embryogenesis of plants provides comparative data that may help us explain this phenomenon. Whether the developmental hourglass occurs in plants, and what this means for our understanding of embryogenesis in plants and animals is discussed. Models by which conserved early-acting genes might change their functional role in the evolution of gene networks, how networks buffer these changes, and how that might constrain, or confer diversity, of the body plan are also discused. Conclusions Evidence of a morphological and molecular hourglass in plant and animal embryogenesis suggests convergent evolution. This convergence is likely due to developmental constraints imposed upon embryogenesis by the need to produce a viable embryo with an established body plan, controlled by the architecture of the underlying gene regulatory networks. As the body plan is largely laid down during the middle phases of embryo development in plants and animals, then it is perhaps not surprising this stage represents the narrow waist of the hourglass where the gene regulatory networks are the oldest and most robust and integrated, limiting species diversity and constraining morphological space.

Eukaryotic translational termination efficiency is influenced by the 3′ nucleotides within the ribosomal mRNA channel

Abstract When a stop codon is at the 80S ribosomal A site, there are six nucleotides (+4 to +9) downstream that are inferred to be occupying the mRNA channel. We examined the influence of these downstream nucleotides on translation termination success or failure in mammalian cells at the three stop codons. The expected hierarchy in the intrinsic fidelity of the stop codons (UAA>UAG>>UGA) was observed, with highly influential effects on termination readthrough mediated by nucleotides at position +4 and position +8. A more complex influence was observed from the nucleotides at positions +5 and +6. The weakest termination contexts were most affected by increases or decreases in the concentration of the decoding release factor (eRF1), indicating that eRF1 binding to these signals was rate-limiting. When termination efficiency was significantly reduced by cognate suppressor tRNAs, the observed influence of downstream nucleotides was maintained. There was a positive correlation between experimentally measured signal strength and frequency of the signal in eukaryotic genomes, particularly in Saccharomyces cerevisiae and Drosophila melanogaster. We propose that termination efficiency is not only influenced by interrogation of the stop signal directly by the release factor, but also by downstream ribosomal interactions with the mRNA nucleotides in the entry channel.

The Toxicogenome of Hyalella azteca: a model for sediment ecotoxicology and evolutionary toxicology

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.