Cloning, characterization and expression analysis of porcine microRNAs

Background: MicroRNAs (miRNAs) are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig.Results: By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined.Conclusion: Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans.Peer reviewedBiochemistry and Molecular BiologyAnimal Scienc

Genome-Wide DNA Methylation Maps in Follicular Lymphoma Cells Determined by Methylation-Enriched Bisulfite Sequencing

BACKGROUND: Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. METHODOLOGY/PRINCIPAL FINDINGS: We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19(+) B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19(+) B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19(+) B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. CONCLUSIONS/SIGNIFICANCE: This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples

Improved genome assembly and evidence-based global gene model set for the chordate Ciona intestinalis: new insight into intron and operon populations

An improved assembly of the Ciona intestinalis genome reveals that it contains non-canonical introns and that about 20% of Ciona genes reside in operons

Microarray analysis of femaleand larval-specific gene expression in the horn fly (Diptera: Muscidae)

The horn My, Haematobia irritans L., is an obligate blood-feeding parasite of cattle, and control of this pest is a continuing problem because the fly is becoming resistant to pesticides. Dominant conditional lethal gene systems are being studied as population control technologies against agricultural pests. One of the components of these systems is a female-specific gene promoter that drives expression of a lethality-inducing gene. To identify candidate genes to supply this promoter, microarrays were designed from a horn fly expressed sequence tag (EST) database and probed to identify female-specific and larval-specific gene expression. Analysis of dye swap experiments found 432 and 417 transcripts whose expression levels were higher or lower in adult female flies, respectively, compared with adult male flies. Additionally, 419 and 871 transcripts were identified whose expression levels were higher or lower in first-instar larvae compared with adult flies, respectively. Three transcripts were expressed more highly in adult females flies compared with adult males and also higher in the first-instar larval lifestage compared with adult flies. One of these transcripts, a putative nanos ortholog, has a high female-to-male expression ratio, a moderate expression level in first-instar larvae, and has been well characterized in Drosophila. melanogaster (Meigen). In conclusion, we used microarray technology, verified by reverse transcriptase-polymerase chain reaction and massively parallel pyrosequencing, to study life stageand sex-specific gene expression in the horn fly and identified three gene candidates for detailed evaluation as a gene promoter source for the development of a female-specific conditional lethality system

High-throughput sequence analysis of Ciona intestinalis SL trans-spliced mRNAs: Alternative expression modes and gene function correlates

Pre-mRNA 5′ spliced-leader (SL) trans-splicing occurs in some metazoan groups but not in others. Genome-wide characterization of the trans-spliced mRNA subpopulation has not yet been reported for any metazoan. We carried out a high-throughput analysis of the SL trans-spliced mRNA population of the ascidian tunicate Ciona intestinalis by 454 Life Sciences (Roche) pyrosequencing of SL-PCR-amplified random-primed reverse transcripts of tailbud embryo RNA. We obtained ∼250,000 high-quality reads corresponding to 8790 genes, ∼58% of the Ciona total gene number. The great depth of this data revealed new aspects of trans-splicing, including the existence of a significant class of “infrequently trans-spliced” genes, accounting for ∼28% of represented genes, that generate largely non-trans-spliced mRNAs, but also produce trans-spliced mRNAs, in part through alternative promoter use. Thus, the conventional qualitative dichotomy of trans-spliced versus non-trans-spliced genes should be supplanted by a more accurate quantitative view recognizing frequently and infrequently trans-spliced gene categories. Our data include reads representing ∼80% of Ciona frequently trans-spliced genes. Our analysis also revealed significant use of closely spaced alternative trans-splice acceptor sites which further underscores the mechanistic similarity of cis- and trans-splicing and indicates that the prevalence of ±3-nt alternative splicing events at tandem acceptor sites, NAGNAG, is driven by spliceosomal mechanisms, and not nonsense-mediated decay, or selection at the protein level. The breadth of gene representation data enabled us to find new correlations between trans-splicing status and gene function, namely the overrepresentation in the frequently trans-spliced gene class of genes associated with plasma/endomembrane system, Ca2+ homeostasis, and actin cytoskeleton

The Medicago genome provides insight into the evolution of rhizobial symbioses

Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation(1). Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species(2). Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing similar to 94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox