Identification and characterization of microRNAs (miRNAs) and their transposable element origins in the saltwater crocodile, Crocodylus porosus.

MicroRNAs (miRNAs) are 18-24 nucleotide regulatory RNAs. They are involved in the regulation of genetic and biological pathways through post transcriptional gene silencing and/or translational repression. Data suggests a slow evolutionary rate for the saltwater crocodile (Crocodylus porosus) over the past several million years when compared to birds, the closest extant relatives of crocodilians. Understanding gene regulation in the saltwater crocodile in the context of relatively slow genomic change thus holds potential for the investigation of genomics, evolution, and adaptation. Utilizing eleven tissue types and sixteen small RNA libraries, we report 644 miRNAs in the saltwater crocodile with >78% of miRNAs being novel to crocodilians. We also identified potential targets for the miRNAs and analyzed the relationship of the miRNA repertoire to transposable elements (TEs). Results suggest an increased association of DNA transposons with miRNAs when compared to retrotransposons. This work reports the first comprehensive analysis of miRNAs in Crocodylus porosus and addresses the potential impacts of miRNAs in regulating the genome in the saltwater crocodile. In addition, the data suggests a supporting role of TEs as a source for miRNAs, adding to the increasing evidence that TEs play a significant role in the evolution of gene regulation.

Bioinformatics Analysis of miRNA Sequencing Data.

The bioinformatics analysis of miRNA is a complicated task with multiple operations and steps involved from processing of raw sequence data to finally identifying accurate microRNAs associated with the phenotypes of interest. A complete analysis process demands a high level of technical expertise in programming, statistics, and data management. The goal of this chapter is to reduce the burden of technical expertise and provide readers the opportunity to understand crucial steps involved in the analysis of miRNA sequencing data.In this chapter, we describe methods and tools employed in processing of miRNA reads, quality control, alignment, quantification, and differential expression analysis.

Dataset for miRNA expression analysis in the peripheral white blood cells of beef heifers at weaning.

Subfertility in beef heifers leads to a substantial economic loss for producers and beef industry. To overcome this problem, producers require an efficient system to discriminate beef heifers with varying reproductive potential as early as possible. MicroRNAs are short non-coding RNAs that post-transcriptionally regulate gene expression. Herein, we profiled the miRNAs in peripheral white blood cells (PWBC) of beef heifers at weaning to investigate the differences in the beef heifers with varying reproductive outcomes. Blood samples from Angus-Simmental crossbred heifers were collected at weaning. The blood was processed to extract the PWBC pellet and was stored at -80 °C until further processing. After the synchronization of estrus and breeding protocol (artificial insemination (AI) followed by natural bull service) and pregnancy diagnosis, the heifers were categorized as fertile (pregnant to AI) or subfertile (not pregnant to AI or bull exposure). Total RNA was extracted from PWBC collected at the time of weaning from the fertile and subfertile heifers. After quality assessment, the total RNA was used to prepare libraries. The quality-checked libraries (n = 14; 7 samples per fertile and subfertile group) were pooled and sequenced (single-end 50 bp) using a NextSeq 500 platform. The raw sequence reads were analyzed using a bioinformatics workflow utilizing FastQC and MultiQC for quality control, Cutadapt for adapter trimming, miRDeep2 for alignment, and DESeq2 for differential expression analysis. The raw and normalized miRNA counts were deposited and made publicly available on the gene expression omnibus database (GEO; GSE225854). This is the first dataset investigating the miRNA expression level in PWBC at weaning in beef heifers to predict the future reproductive outcome. The results from the data presented here are reported in the research article titled "miRNA expression profiles of peripheral white blood cells from beef heifers with varying reproductive potential" [1].

Bioinformatics analysis of miRNA and its associated genes to identify potential biomarkers of oral submucous fibrosis and oral malignancy.

BACKGROUND: MicroRNAs are a group of non-coding RNA that controls the gene expression. The interaction between miRNA and mRNA is thought to be dynamic. Oral cancer "The cancer of mouth" is quite prevailing in developing countries. miRNA has been found associated with oral cancer targeting tumor growth, cell proliferation, metastasis, invasion. The significant association of miRNA with genes could be used as a remarkable tool for diagnosis as well as prognostic analysis of oral cancer. AIM: The aim of the present study is to evaluate common upregulated and downregulated miRNAs in oral submucous fibrosis (OSMF) and oral malignancy (OM) patients that can be used as diagnostic biomarkers, and to find out their interactions with target genes to establish associated networks in cancer pathways. METHODS AND RESULTS: Using miRDeep2 and DESeq analysis, the upregulated and downregulated miRNA in OSMF (Oral Submucous Fibrosis) and OM (Oral Malignancies) samples were compared to GEO (Gene Expression Omnibus) control dataset. There were 50 common downregulated miRNAs and 13 common upregulated miRNAs in OSMF and OM samples. miRNet analysis of common upregulated miRNA and common downregulated miRNA identified 1295 and 5954 genes, respectively connected with cancer pathways. From analysis of Hub genes, HRAS, STAT3, TP53, MYC, PTEN, CTNNB1, CCND1, JUN, VEGFA, KRAS were found associated with downregulated miRNA and VEGFA, TP53, MDM2, PTEN, MYC, ERBB2, CDKN1A, HSP90AA1, CCND1, AKTI were found associated with upregulated miRNA. The gene enrichment analysis of these hub genes were associated with cell communication, metabolic process, cell proliferation, and cellular component organization. Hub Genes linked with upregulated miRNA had an enrichment ratio of 11.828, whereas hub genes linked with downregulated miRNA had an enrichment ratio of 45.912. CONCLUSION: We identified common deregulated miRNAs between OSMF and OM patients, which were further analyzed to find out associations with the genes correlated to cancer pathways. The hub genes identified in this study were found to have a significant impact on tumor growth and carcinogenesis. Also, the enrichment of these genes has revealed that the genes are associated with cellular communication, metabolic processes and various biological regulation. These deregulated miRNAs can be used to make a panel of biomarkers to diagnose oral cancer from blood even before its onset.

Isolation and Analysis of MicroRNAs from Extracellular Vesicles of the Parasitic Model Nematodes Nippostrongylus brasiliensis and Trichuris muris.

The identification, detection, and use of small RNA species have rapidly gained interest-especially to study parasite-host interactions. Parasite-to-host communication is contributed by small secreted extracellular vesicle (EV)-derived nucleic acid species. In particular, microRNAs (miRNAs) and small interfering RNAs can regulate the host response by targeting cells at both transcriptional and posttranscriptional levels. Here, modified protocols for density gradient purification of EVs from nematodes and the subsequent extraction of EV-derived small RNAs using commercially available reagents and kits are provided with a special focus on basic background information. Further, considerations for Next-Generation Sequencing using the Illumina NextSeq500 sequencing technology (kit-based library preparation, small RNA sequencing, and miRNA sequence analysis pipelines using the miRDeep2 package) are introduced.

Insect and plant-derived miRNAs in greenbug (Schizaphis graminum) and yellow sugarcane aphid (Sipha flava) revealed by deep sequencing.

Schizaphis graminum (green bug; GB) and Sipha flava (yellow sugarcane aphid; YSA) are two cereal aphid species with broad host ranges capable of establishing on sorghum (Sorghum bicolor) and several switchgrass (Panicum virgatum) cultivars. Switchgrass and sorghum are staple renewable bioenergy crops that are vulnerable to damage by aphids, therefore, identifying novel targets to control aphids has the potential to drastically improve yields and reduce losses in these bioenergy crops. Despite the wealth of genomic and transcriptomic information available from a closely related model aphid species, the pea aphid (Acyrthosiphon pisum), similar genomic information, including the identification of small RNAs, is still limited for GB and YSA. Deep sequencing of miRNAs expressed in GB and YSA was conducted and 72 and 56 miRNA candidates (including 14 and eight novel) were identified, respectively. Of the identified miRNAs, 45 were commonly expressed in both aphid species. Further, plant derived miRNAs were also detected in both aphid samples, including 13 (eight known and five novel) sorghum miRNAs and three (novel) barley miRNAs. In addition, potential aphid gene targets for the host plant-derived miRNAs were predicted. The establishment of miRNA repertoires in these two aphid species and the detection of plant-derived miRNA in aphids will ultimately lead to a better understanding of the role of miRNAs in regulating gene expression networks in these two aphids and the potential roles of plant miRNAs in mediating plant-insect interactions.