Prediction of novel putative miRNAs and their targets in buffalo

Frontiers in genetics, 2017

The domestication of cattle from the now-extinct wild aurochs (Bos primigenius) involved selection for physiological and behavioral traits, with underlying genetic factors that remain largely unknown. Non-coding microRNAs have emerged as key regulators of the spatio-temporal expression of target genes controlling mammalian growth and development, including in livestock species. During the domestication process, selection of mutational changes in miRNAs and/or miRNA binding sites could have provided a mechanism to generate some of the traits that differentiate domesticated cattle from wild aurochs. To investigate this, we analyzed the open reading frame DNA sequence of 19,994 orthologous protein-coding gene pairs from extant Bos taurus genomes and a single extinct B. primigenius genome. We identified miRNA binding site polymorphisms in the 3' UTRs of 1,620 of these orthologous genes. These 1,620 genes with altered miRNA binding sites between the B. taurus and B. primigenius linea...

Molecular Ecology Resources, 2014

An intriguing question in biology is how the evolution of gene regulation is shaped by natural selection in natural populations. Among the many known regulatory mechanisms, regulation of gene expression by microRNAs (miR-NAs) is of critical importance. However, our understanding of their evolution in natural populations is limited. Studying the role of miRNAs in three-spined stickleback, an important natural model for speciation research, may provide new insights into adaptive polymorphisms. However, lack of annotation of miRNA genes in its genome is a bottleneck. To fill this research gap, we used the genome of three-spined stickleback to predict miRNAs and their targets. We predicted 1486 mature miRNAs using the homology-based miRNA prediction approach. We then performed functional annotation and enrichment analysis of these targets, which identified over-represented motifs. Further, a database resource (GAmiRdb) has been developed for dynamically searching miRNAs and their targets exclusively in three-spined stickleback. Finally, the database was used in two case studies focusing on freshwater adaptation in natural populations. In the first study, we found 44 genomic regions overlapping with predicted miRNA targets. In the second study, we identified two SNPs altering the MRE seed site of sperm-specific glyceraldehyde-3-phosphate gene. These findings highlight the importance of the GAmiRdb knowledge base in understanding adaptive evolution.

Computational Biology and Chemistry, 2009

MicroRNAs (miRNAs) are a class of endogenous, noncoding, short RNAs directly involved in regulating gene expression at the posttranscriptional level. High conservation of miRNAs in plant provides the foundation for identification of new miRNAs in other plant species through homology alignment. Here, previous known plant miRNAs were BLASTed against the Expressed Sequence Tag (EST) and Genomic Survey Sequence (GSS) databases of Vigna unguiculata, and according to a series of filtering criteria, a total of 47 miRNAs belonging to 13 miRNA families were identified, and 30 potential target genes of them were subsequently predicted, most of which seemed to encode transcription factors or enzymes participating in regulation of development, growth, metabolism, and other physiological processes. Overall, our findings lay the foundation for further researches of miRNAs function in Vigna unguiculata. High protein and carbohydrate contents make it not only important to the human diet, but also suitable as high protein feed and fodder to livestock. With its greater tolerance to heat, drought, and low soil fertility, Vigna unguiculata is a particularly valuable component of low-input farming systems of resource-poor farmers. Also, it is able to enhance soil fertility through nitrogen fixation . Until recently limited progress has been made in basic gene discovery and gene regulation in Vigna unguiculata, and only two new miRNAs of it have been reported . Despite the limited genome resources of Vigna unguiculata, publishing of EST and GSS databases in GenBank has provided the chance to get more genetic information. In this study, new miRNAs were mined for the purpose of understanding their roles in regulating growth, development, metabolism, and other physiological processes in Vigna unguiculata.

2015

microRNAs represent a class of noncoding small RNAs of approximately 20-23 nt length, which are evolutionarily conserved and play a vital role in various biological processes by either degrading or repressing mRNA translation. The Felis catus (cat) genome sequence has been published, and just revealed the number of miRNAs in the genome-without mention of any further details on these miRNAs. This paper discusses an in silico comparative approach using all known sequences of vertebrate pre-miRNA as query sequence, and report 405 putative miRNAs from cat genome. We determine the identity values of pre-miRNAs and mature miRNAs besides statistical sequence characteristics. Interestingly, among 405 miRNAs-90, 53 and 50 showed 100% identity to cattle, human and dog, respectively. Further, we have validated 6 miRNAs, whose identity are ,85% with the query sequence and validated them using MiPred algorithm. We also identify 25 miRNA clusters in cat based on their homologs in other vertebrates. Most importantly, based on identities among pre-miRNA, mature miRNA, miRNA families and clusters, we observe that miRNAs from cat are more identical to cattle, than humans. Our results, therefore may add a new dimension to the studies related to the evolution of cat.

ExRNA

Background: MicroRNAs play key roles in host-pathogen-interactions and disease pathogenesis. Our aim was to characterize the differentially expressed miRNAs in the blood cells of diseased (Brucellosis-positive, Johne's diseasepositive) and healthy-water buffaloes. The pooled small-RNA samples of each group were sequenced on Ion Torrent Personal Genome Machine (PGM) sequencer and the data were analyzed for differential expression. Results: Here we identified 274 known miRNAs with bovine homologs and 36 novel mature-star miRNAs from the sequnces of small RNA libraries. Overall 195 miRNAs were common to all the three groups. Certain miRNAs such as bta-miR-21-5p, −26a, −29a/b, −30d − 103, − 140, − 150, − 191, − 374, − 1434-5p,-1260b, − 2484 and let-7 members were abundantly expressed in diseased groups. Bta-miR-1434-5p, − 188, −200c were up-regulated (> 1.5 folds) while bta-miR-27a-5p, −34b and-2285x were down-regulated (> 100 folds) in Brucellosis group. In Johne's Disease group, only 3 miRNAs (bta-miR-1434-5p, − 2340 and − 2484) were up-regulated (> 1.5 folds). The functional classification of miRNA target genes into gene ontology (GO) terms indicated their involvement in innate immunity and cellular process of disease pathogenesis. Expression profile of four differentially expressed miRNAs (bta-miR-9-5p, − 677, − 331-3p and − 2440) and eight predicted target-genes were validated through reverse transcriptase qPCR. Conclusion: This study provides a valuable frame of reference for elucidation of regulatory roles of miRNAs associated with disease pathogenesis in water buffaloes as well as identification of miRNA biomarkers for disease diagnosis and treatment.

MicroRNAs (miRNAs) are endogenous, small, noncoding RNAs of 18-25 nucleotides in length that negatively regulate their complementary messenger-RNAs at the post transcriptional level. Presently there are three approaches for miRNA identification. Two approaches i.e. bioinformatic and small RNA library construction and sequencing are examples of reverse genetics approach. In these two approaches researchers try to find miRNAs in the known genotypes (sequences). A third approach called the classical approach is example of forward genetics in which researchers determine the unknown genotype (miRNA sequence) of a known phenotype. The miRNAs have shown both evolutionarily converged nature (conserved miRNAs) from species to species within the same kingdom and species specific expression (species specific miRNAs). Majority of the miRNA genes are observed as conserved miRNAs as orthologs. This conserved nature of majority of miRNAs becomes an important logical tool for bioinformatics discovery of miRNAs in other species. Identification of miRNAs by using bioinformatic tools is now a commonplace and of the most widely used methods and it has facilitated the prediction of new miRNAs in both plant and animal systems. This is largely used due to its low cost and high efficiency. In 2001 the 1 st release of miRBases (an online repository of miRNAs) contained only 218 miRNA entries and now its latest release 20 (released in June 2013) contains 24521 entries. The bioinformatics hunting of miRNAs has also a reasonable contribution in identification of new miRNAs. Around 25% of entries in miRBase (across all species) are predicted computationally. Likewise in Plant MicroRNA Database (PMRD) within the total 8433 miRNAs, 1297 of them are experimentally demonstrated and 7136 of them are from computational predictions . This review is aimed at describing the bioinformatic resources used in miRNA identification. Furthermore, the limitations of these resources are also described.

Scientific Reports, 2013

BMC Genomics, 2010

Background: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution. Results: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells. The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.

2013

Background: MicroRNAs (miRNAs) are a family of~22 nucleotide small RNA molecules that regulate gene expression by fully or partially binding to their complementary sequences. Recently, a large number of miRNAs and their expression patterns have been identified in various species. However, to date no miRNAs have been reported to modulate muscle development in beef cattle. Results: Total RNAs from the Chinese Qinchuan bovine longissimus thoracis at fetal and adult stages were used to construct small RNA libraries for Solexa SBS technology sequencing. A total of 15,454,182 clean reads were obtained from the fetal bovine library and 13,558,164 clean reads from the adult bovine library. In total, 521 miRNAs including 104 novel miRNA candidates were identified. Furthermore, the nucleotide bias, base edit and family of the known miRNAs were also analyzed. Based on stem-loop qPCR, 25 high-read miRNAs were detected, and the results showed that bta-miRNA-206, miRNA-1, miRNA-133, miRNAn12, and miRNAn17 were highly expressed in muscle-related tissue or organs, suggesting that these miRNAs may play a role in the development of bovine muscle tissues. Conclusions: This study confirmed the authenticity of 417 known miRNAs, discovered 104 novel miRNAs in bos taurus, and identified five muscle-specific miRNAs. The identification of novel miRNAs significantly expanded the repertoire of bovine miRNAs and could contribute to further studies on the muscle development of cattle.

PLoS ONE, 2011

MicroRNAs (miRNAs) are small non-coding RNA molecules that are processed from large 'hairpin' precursors and function as post-transcriptional regulators of target genes. Although many individual miRNAs have recently been extensively studied, there has been very little research on miRNA transcriptomes in teleost fishes. By using high throughput sequencing technology, we have identified 167 and 166 conserved miRNAs (belonging to 108 families) in bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix), respectively. We compared the expression patterns of conserved miRNAs by means of hierarchical clustering analysis and log2 ratio. Results indicated that there is not a strong correlation between sequence conservation and expression conservation, most of these miRNAs have similar expression patterns. However, high expression differences were also identified for several individual miRNAs. Several miRNA* sequences were also found in our dataset and some of them may have regulatory functions. Two computational strategies were used to identify novel miRNAs from un-annotated data in the two carps. A first strategy based on zebrafish genome, identified 8 and 22 novel miRNAs in bighead carp and silver carp, respectively. We postulate that these miRNAs should also exist in the zebrafish, but the methodologies used have not allowed for their detection. In the second strategy we obtained several carp-specific miRNAs, 31 in bighead carp and 32 in silver carp, which showed low expression. Gain and loss of family members were observed in several miRNA families, which suggests that duplication of animal miRNA genes may occur through evolutionary processes which are similar to the protein-coding genes.