comprehensive-workflow-for-genome-wide-identification-expression-meta

Comprehensive Workflow for the Genome-wide Identification and Expression Meta-analysis of the ATL E3 Ubiquitin Ligase Gene Family in Grapevine

Classification and nomenclature of genes in a family can significantly contribute to the description of the diversity of encoded proteins and to the ...

Università degli Studi di Sassari

Recently features of gene expression profiles have been associated with structural parameters of gene sequences in organisms representing a diverse set of taxa. The emerging picture indicates that natural selection, mediated by gene expression profiles, has a significant role in determining genic structures. However the current situation is less clear in plants as the available data indicates that the effect of natural selection mediated by gene expression is very weak. Moreover, the direction of the patterns in plants appears to contradict those observed in animal genomes. In the present work we analized expression data for >18000 Arabidopsis genes retrieved from public datasets obtained with different technologies (MPSS and high density chip arrays) and compared them with gene parameters. Our results show that the impact of natural selection mediated by expression on genes sequences is significant and distinguishable from the effects of regional mutational biases. In addition, we provide evidence that the level and the breadth of gene expression are related in opposite ways to many structural parameters of gene sequences. Higher levels of expression abundance are associated with smaller transcripts, consistent with the need to reduce costs of both transcription and translation. Expression breadth, however, shows a contrasting pattern, i.e. longer genes have higher breadth of expression, possibly to ensure those structural features associated with gene plasticity. Based on these results, we propose that the specific balance between these two selective forces play a significant role in shaping the structure of Arabidopsis genes.

Mutational biases and selective forces shaping the structure of Arabidopsis genes.

Genomic DNA sequences display compositional heterogeneity on many scales. In this paper we analyzed tendencies and anomalies in the occurence of mono, di and trinucleotides in structural regions of plant genes. Representation of these trends as a function of position along genic sequences highlighted compositional features peculiar of either monocots or eudicots that were remarkably uniform within these two evolutionary clades. The most evident of these features appeared in the form of gradient of base content along the direction of transcription. The robustness of such a representation was validated in sequences sub-datasets generated considering structural and compositional features such as total length of cds, overall GC content and genic orientation in the genome. Piecewise regression analyses indicated that the gradients could be conveniently approximated to a two segmented model where a first region featuring a steep slope is followed by a second segment fitting a milder variation. In general, monocots species showed steeper segments than eudicots. The guanine gradient was the most distinctive feature between the two evolutionary clades, being moderately increasing in eudicots and firmly decreasing in monocots. Single gene investigation revealed that a high proportion of genes show compositional trends compatible with a segmented model suggesting that these features are essential attributes of gene organization. Dinucleotide and trinucleotide biases were referred to expectation based on a random union of the component elements. The average bias at dinucleotide level identified a significant undererpresentation of some dinucleotide and the overrepresention of others. The bias at trinucleotide level was on average low. Finally, the analysis of bryophyte coding sequences showed mononucleotide, dinucleotide and trinucleotide compositional trends resembling those of higher plants. This finding suggested that the emergenge of compositional bias is an ancient event in evolution which was already present at the time of land conquest by green plants.

Spatial analyses of mono, di and trinucleotide trends in plant genes.

The codon composition of coding sequences plays an important role in the regulation of gene expression. Herein, we report systematic differences in the usage of synonymous codons among Arabidopsis thaliana genes that are expressed specifically in distinct tissues. Although we observed that both regionally and transcriptionally associated mutational biases were associated significantly with codon bias, they could not explain the observed differences fully. Similarly, given that transcript abundances did not account for the differences in codon usage, it is unlikely that selection for translational efficiency can account exclusively for the observed codon bias. Thus, we considered the possible evolution of codon bias as an adaptive response to the different abundances of tRNAs in different tissues. Our analysis demonstrated that in some cases, codon usage in genes that were expressed in a broad range of tissues was influenced primarily by the tissue in which the gene was expressed maximally. On the basis of this finding we propose that genes that are expressed in certain tissues might show a tissue-specific compositional signature in relation to codon usage. These findings might have implications for the design of transgenes in relation to optimizing their expression.

The relation of codon bias to tissue-specific gene expression in Arabidopsis thaliana.

Little is known about the natural selection of synonymous codons within the coding sequences of plant genes. We analyzed the distribution of synonymous codons within plant coding sequences and found that preferred codons tend to encode the more conserved and functionally important residues of plant proteins. This was consistent among several synonymous codon families and applied to genes with different expression profiles and functions. Most of the randomly chosen alternative sets of codons scored weaker associations than the actual sets of preferred codons, suggesting that codon position within plant genes and codon usage bias have coevolved to maximize translational accuracy. All these findings are consistent with the mistranslation-induced protein misfolding theory, which predicts the natural selection of highly preferred codons more frequently at sites where translation errors could compromise protein folding or functionality. Our results will provide an important insight in future studies of protein folding, molecular evolution, and transgene design for optimal expression.

The signatures of selection for translational accuracy in plant genes.

General Feature Format (GFF) files are used to store genome features such as genes, exons, introns, primary transcripts etc. Although many software packages (i.e. ab initio gene prediction programs) can annotate features by using such a standard, a small number of tools have been developed to extract the corresponding sequence information from the original genome. However the present tools do not execute either a quality control or a customizable filter of the annotated features is available.

gff2sequence, a new user friendly tool for the generation of genomic sequences.

The majority of amino acid residues are encoded by more than one codon, and a bias in the usage of such synonymous codons has been repeatedly demonstrated. One assumption is that this phenomenon has evolved to improve the efficiency of translation by reducing the time required for the recruitment of isoacceptors. The most abundant tRNA species are preferred at sites on the protein which are key for its functionality, a behavior which has been termed "translational accuracy". Although observed in many species, as yet no public domain software has been made available for its quantification.

Seforta, an integrated tool for detecting the signature of selection in coding sequences.

Genotyping by re-sequencing has become a standard approach to estimate single nucleotide polymorphism (SNP) diversity, haplotype structure and the biodiversity and has been defined as an efficient approach to address geographical population genomics of several model species. To access core SNPs and insertion/deletion polymorphisms (indels), and to infer the phyletic patterns of speciation, most such approaches map short reads to the reference genome. Variant calling is important to establish patterns of genome-wide association studies (GWAS) for quantitative trait loci (QTLs), and to determine the population and haplotype structure based on SNPs, thus allowing content-dependent trait and evolutionary analysis. Several tools have been developed to investigate such polymorphisms as well as more complex genomic rearrangements such as copy number variations, presence/absence variations and large deletions. The programs available for this purpose have different strengths (e.g. accuracy, sensitivity and specificity) and weaknesses (e.g. low computation speed, complex installation procedure and absence of a user-friendly interface). Here we introduce Altools, a software package that is easy to install and use, which allows the precise detection of polymorphisms and structural variations.

Altools: a user friendly NGS data analyser.

The Arabidopsis Tóxicos en Levadura (ATL) protein family is a class of E3 ubiquitin ligases with a characteristic RING-H2 Zn-finger structure that mediates diverse physiological processes and stress responses in plants. We carried out a genome-wide survey of grapevine (Vitis vinifera L.) ATL genes and retrieved 96 sequences containing the canonical ATL RING-H2 domain. We analysed their genomic organisation, gene structure and evolution, protein domains and phylogenetic relationships. Clustering revealed several clades, as already reported in Arabidopsis thaliana and rice (Oryza sativa), with an expanded subgroup of grapevine-specific genes. Most of the grapevine ATL genes lacked introns and were scattered among the 19 chromosomes, with a high level of duplication retention. Expression profiling revealed that some ATL genes are expressed specifically during early or late development and may participate in the juvenile to mature plant transition, whereas others may play a role in pathogen and/or abiotic stress responses, making them key candidates for further functional analysis. Our data offer the first genome-wide overview and annotation of the grapevine ATL family, and provide a basis for investigating the roles of specific family members in grapevine physiology and stress responses, as well as potential biotechnological applications.

Genome-wide characterisation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members.

Mistranslation errors compromise fitness by wasting resources on nonfunctional proteins. In order to reduce the cost of mistranslations, natural selection chooses the most accurately translated codons at sites that are particularly important for protein structure and function. We investigated the determinants underlying selection for translational accuracy in several species of plants belonging to three clades: Brassicaceae, Fabidae, and Poaceae. Although signatures of translational selection were found in genes from a wide range of species, the underlying factors varied in nature and intensity. Indeed, the degree of synonymous codon bias at evolutionarily conserved sites varied among plant clades while remaining uniform within each clade. This is unlikely to solely reflect the diversity of tRNA pools because there is little correlation between synonymous codon bias and tRNA abundance, so other factors must affect codon choice and translational accuracy in plant genes. Accordingly, synonymous codon choice at a given site was affected not only by the selection pressure at that site, but also its participation in protein domains or mRNA secondary structures. Although these effects were detected in all the species we analyzed, their impact on translation accuracy was distinct in evolutionarily distant plant clades. The domain effect was found to enhance translational accuracy in dicot and monocot genes with a high GC content, but to oppose the selection of more accurate codons in monocot genes with a low GC content.

The Evolutionary Basis of Translational Accuracy in Plants.

The genetic diversity among grapevine (Vitis vinifera L.) cultivars that underlies differences in agronomic performance and wine quality reflects the accumulation of single nucleotide polymorphisms (SNPs) and small indels as well as larger genomic variations. A combination of high throughput sequencing and mapping against the grapevine reference genome allows the creation of comprehensive sequence variation maps. We used next generation sequencing and bioinformatics to generate an inventory of SNPs and small indels in four widely cultivated Sardinian grape cultivars (Bovale sardo, Cannonau, Carignano and Vermentino). More than 3,200,000 SNPs were identified with high statistical confidence. Some of the SNPs caused the appearance of premature stop codons and thus identified putative pseudogenes. The analysis of SNP distribution along chromosomes led to the identification of large genomic regions with uninterrupted series of homozygous SNPs. We used a digital comparative genomic hybridization approach to identify 6526 genomic regions with significant differences in copy number among the four cultivars compared to the reference sequence, including 81 regions shared between all four cultivars and 4953 specific to single cultivars (representing 1.2 and 75.9% of total copy number variation, respectively). Reads mapping at a distance that was not compatible with the insert size were used to identify a dataset of putative large deletions with cultivar Cannonau revealing the highest number. The analysis of genes mapping to these regions provided a list of candidates that may explain some of the phenotypic differences among the Bovale sardo, Cannonau, Carignano and Vermentino cultivars.

Sequence Polymorphisms and Structural Variations among Four Grapevine (Vitis vinifera L.) Cultivars Representing Sardinian Agriculture.

Pseudogenes are gene copies that have lost the capability to encode a functional protein. Based on their structure, pseudogenes are classified in two types. Processed pseudogenes arise by a process of retrotranscription from a spliced mRNA and subsequent integration into the genome. Nonprocessed (or duplicated) pseudogenes are generated by genomic duplication and subsequent mutations that disable their functionality so that they cannot longer encode a functional protein. Differently from processed pseudogenes, duplicated pseudogenes are expected to conserve the exon-intron structure of their functional paralogs.Here, we describe a computational pipeline for identifying pseudogenes of both types in B. distachyon chromosomes. Our pipeline (1) identifies pseudogenes based on tBLASTn searches of B. distachyon proteins against the noncoding genomic complement of the same species, (2) identifies the most homologous pseudogenes functionally paralogous as the pseudogene paternal locus, (3) uses the intron-exon structure of paternal genes to distinguish between pseudogene types.The pipeline is presented in its composing steps and tested on the Brachypodium distachyon Bd1 scaffold.

Identification of Pseudogenes in Brachypodium distachyon Chromosomes.

Genome overview of eight Candida boidinii strains isolated from human activities and wild environments.

A molecular approach was applied to the study of the carotenoid biosynthetic pathway of Rhodotorula mucilaginosa. At first, functional annotation of the genome of R. mucilaginosa C2.5t1 was carried out and gene ontology categories were assigned to 4033 predicted proteins. Then, a set of genes involved in different steps of carotenogenesis was identified and those coding for phytoene desaturase, phytoene synthase/lycopene cyclase and carotenoid dioxygenase (CAR genes) proved to be clustered within a region of ~10 kb. Quantitative PCR of the genes involved in carotenoid biosynthesis showed that genes coding for 3-hydroxy-3-methylglutharyl-CoA reductase and mevalonate kinase are induced during exponential phase while no clear trend of induction was observed for phytoene synthase/lycopene cyclase and phytoene dehydrogenase encoding genes. Thus, in R. mucilaginosa the induction of genes involved in the early steps of carotenoid biosynthesis is transient and accompanies the onset of carotenoid production, while that of CAR genes does not correlate with the amount of carotenoids produced. The transcript levels of genes coding for carotenoid dioxygenase, superoxide dismutase and catalase A increased during the accumulation of carotenoids, thus suggesting the activation of a mechanism aimed at the protection of cell structures from oxidative stress during carotenoid biosynthesis. The data presented herein, besides being suitable for the elucidation of the mechanisms that underlie carotenoid biosynthesis, will contribute to boosting the biotechnological potential of this yeast by improving the outcome of further research efforts aimed at also exploring other features of interest.

Article	Year
Mutational biases and selective forces shaping the structure of Arabidopsis genes. PloS one\| PubMed ID: 19633720	2009
Spatial analyses of mono, di and trinucleotide trends in plant genes. PloS one\| PubMed ID: 21829660	2011
The relation of codon bias to tissue-specific gene expression in Arabidopsis thaliana. Genetics\| PubMed ID: 22865738	2012
The signatures of selection for translational accuracy in plant genes. Genome biology and evolution\| PubMed ID: 23695187	2013
gff2sequence, a new user friendly tool for the generation of genomic sequences. BioData mining\| PubMed ID: 24020993	2013
Seforta, an integrated tool for detecting the signature of selection in coding sequences. BMC research notes\| PubMed ID: 24739143	2014
Altools: a user friendly NGS data analyser. Biology direct\| PubMed ID: 26883204	2016
Genome-wide characterisation and expression profile of the grapevine ATL ubiquitin ligase family reveal biotic and abiotic stress-responsive and development-related members. Scientific reports\| PubMed ID: 27910910	2016
The Evolutionary Basis of Translational Accuracy in Plants. G3 (Bethesda, Md.)\| PubMed ID: 28533334	2017
Sequence Polymorphisms and Structural Variations among Four Grapevine (Vitis vinifera L.) Cultivars Representing Sardinian Agriculture. Frontiers in plant science\| PubMed ID: 28775732	2017
Identification of Pseudogenes in Brachypodium distachyon Chromosomes. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29039010	2018
Genome overview of eight Candida boidinii strains isolated from human activities and wild environments. Standards in genomic sciences\| PubMed ID: 29213357	2017
CAR gene cluster and transcript levels of carotenogenic genes in Rhodotorula mucilaginosa. Microbiology (Reading, England)\| PubMed ID: 29219805	2017

Salvatore Camiolo

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