<Emphasis Type="Italic">Insilco</Emphasis> Prediction and Characterization of microRNAs from <Emphasis Type="Italic">Oncopeltus fasciatus</Emphasis> (Hemiptera: Lygaeidae) Genome

For studies on functional genomics, small RNAs, especially microRNAs (miRNAs), have emerged as a hot topic due to their importance in cellular and developmental processes. Identification of insect miRNAs largely depends on the availability of genomic sequences in the public domain. The large milkweed bug, Oncopeltus fasciatus (Dallas) is a hemimetabolous insect which has become a model hemipteran system for various molecular studies. In this study, we identified 96 candidate mature miRNAs from O. fasciatus genome using a blast search with the previously reported animal miRNAs. The secondary structure of predicted miRNA sequences was determined online using “mfold” web server and verified by calculating the minimal free energy index (MFEI). Six miRNAs let-7e, miR-133c, miR-219b, mir-466d, mir-669f, and mir-669l are reported for the first time in Insecta. Comparison of O. fasciatus mir-2 and mir-71 family clusters to those of diverse insect species showed that they are highly conserved. The phylogenetic analysis of miRNAs revealed the evolutionary relationship of conserved miRNAs of O. fasciatus with other insect species. Using a classical rule-based algorithm method, we predicted the possible targets of the new miRNAs. Our study not only identified the list of miRNAs in O. fasciatus but also provides a basic platform for developing novel pest management strategies based on artificial miRNAs.     

Computational identification of novel microRNA homologs in the chimpanzee genome

MicroRNAs are important negative regulators of gene expression in higher eukaryotes. The miRNA repertoire of the closest human animal relative, the chimpanzee (Pan troglodytes), is largely unknown. In this study, we focused on computational search of novel miRNA homologs in chimpanzee. We have searched and analyzed the chimp homologs of the human pre-miRNA and mature miRNA sequences. Based on a homology search of the chimpanzee genome with human miRNA precursor sequences as queries, we identified 639 chimp miRNA genes, including 529 novel chimp miRNAs. 91.8% of chimp mature miRNAs and 60.3% of precursors are 100% identical to their human orthologs. The pre-miRNA secondary structures, miRNA families, and clusters are also highly conserved. We also found certain sequence differences in pre-miRNAs and even mature miRNAs that occurred after the divergence of the two species. Some of these differences (especially in mature miRNAs) could have caused species-specific changes in the expression levels of their target genes which in turn could have resulted in phenotypic variation between human and chimp.     

Computational Approach for Identification of Anopheles gambiae miRNA Involved in Modulation of Host Immune Response

MicroRNAs (miRNAs) are small, noncoding RNAs that play key roles in regulating gene expression in animals, plants, and viruses, which involves in biological processes including development, cancer, immunity, and host–microorganism interactions. In this present study, we have used the computational approach to identify potent miRNAs involved in Anopheles gambiae immune response. Analysis of 217,261 A. gambiae ESTs and further study of RNA folding revealed six new miRNAs. The minimum free energy of the predicted miRNAs ranged from ?27.2 to ?62.63 kcal/mol with an average of ?49.38 kcal/mol. While its A + U % ranges from 50 to 65 % with an average value of 57.37 %. Phylogenetic analysis of the predicted miRNAs revealed that aga-miR-277 was evolutionary highly conserved with more similarity with other mosquito species. Observing further the target identification of the predicted miRNA, it was noticed that the aga-miR-2304 and aga-miR-2390 are involved in modulation of immune response by targeting the gene encoding suppressin and protein prophenoloxidase. Further detailed studies of these miRNAs will help in revealing its function in modulation of A. gambiae immune response with respect to its parasite.     

Computational identification of 48 potato microRNAs and their targets

MicroRNAs (miRNAs) are a new family of small RNA molecules known in animals and plants, whose conservation among species suggests that they bear conserved biological functions. So far, little is known about miRNA in Solanum tuberosum species. Using previously known miRNAs from Arabidopsis, rice and other plant species against expressed sequence tags (ESTs), genomic survey sequence (GSS) and nucleotide databases, we identified 48 potential miRNAs in S. tuberosum. These potato miRNAs may regulate 186 potential targets, which are involved in floral, leaf, root, and stem development, signal transduction, metabolism pathways, and stress responses. To validate the prediction of miRNAs in potato, we performed a RT-PCR analysis and found that potato miRNAs have diverse expression patterns during development.     

Molecular evolution and structural variations in nuclear encoded chloroplast localized heat shock protein 26 (sHSP26) from genetically diverse wheat species

Heat shock proteins are an important class of molecular chaperones known to impart tolerance under high temperature stress. sHSP26, a member of small heat shock protein subfamily is specifically involved in protecting plant’s photosynthetic machinery. The present study aimed at identifying and characterizing sequence and structural variations in sHSP26 from genetically diverse progenitor and non-progenitor species of wheat. In silico analysis identified three paralogous copies of TaHSP26 to reside on short arm of chromosome 4A while one homeologue each was localized on long arm of chromosome 4B and 4D of cultivated bread wheat. Wild DD-genome donor Aegilops tauschii carried an additional sHSP26 gene (AET4Gv20569400) which was absent in the cultivated DD genome of bread wheat. In vitro amplification of this novel gene in wild accessions of Ae. tauschii and synthetic hexaploid wheat but not in cultivated bread wheat validated this finding. Further, significant length polymorphism could be identified in exon1 from diverse sHSP26 sequences. Multiple sequence alignment of procured sequences revealed numerous sSNPs and nsSNPs. D3A, P125 L, Q242 K were designated as homeolog specific- while A49 G as non-progenitor specific amino acid replacements. A 9-bp indel in TmHSP26-1(GA) translated into a deletion of SPM amino acid segment in chloroplast specific conserved consensus region III. High degree of divergence in nucleotide sequence between cultivated and wild species appeared in the form of higher ω values (Ka/Ks >1) indicating positive selection during the course of evolution. Phylogenetic analysis elucidated ancestral relationships between wheat sHSP26 proteins and orthologous proteins across plant kingdom. Overall, data mining approach may be employed as an effective pre-breeding strategy to identify and mobilize novel stress responsive genes and distinct allelic variants from wider germplasm collections of wheat to enhance climate resilience of present day elite wheat cultivars.     

In Silico Mining of Conserved miRNAs of Indian Catfish <Emphasis Type="Italic">Clarias batrachus</Emphasis> (Linnaeus, 1758) from the Contigs,ESTs, and BAC End Sequences

MicroRNAs are small non-coding RNAs which play significant role in RNA interference. The present work deals with mining of the conserved miRNA and their target genes from the contigs, ESTs, and BAC end sequences of commercially important catfish, Clarias batrachus, from India. A total of 138, 1 and 1 conserved pre-miRNA sequences, were mined from the contigs, ESTs, and BAC end sequences, respectively. The analysis of families of the conserved pre-miRNA revealed conservation of the fish-specific family mir-430 and other important families, such as mir-455, let-7, mir-133, and mir-137. The mir-455 is involved in hypoxia signaling, let-7 family represents potential anti-tumor molecules involved in human cancer therapy, whereas mir-133 and mir-137 have high therapeutic potentials. Using an alternate computational in silico approach, mining of mature miRNAs resulted in identification of 210 mature miRNAs from contigs, 1 from EST, and 2 each from forward as well as reverse BAC end sequences. Target prediction of these putative miRNAs resulted in the identification of 66,758 and 18,747 target genes in C. batrachus and Danio rerio, respectively. Functional annotation of these miRNAs indicated their involvement in diverse biological functions. The findings of the present study can serve as a valuable resource for further functional genomics studies in C. batrachus.     

In silico identification of conserved microRNAs and their target transcripts from expressed sequence tags of three earthworm species

MicroRNAs are a recently identified class of small regulatory RNAs that target more than 30% protein-coding genes. Elevating evidence shows that miRNAs play a critical role in many biological processes, including developmental timing, tissue differentiation, and response to chemical exposure. In this study, we applied a computational approach to analyze expressed sequence tags, and identified 32 miRNAs belonging to 22 miRNA families, in three earthworm species Eisenia fetida, Eisenia andrei, and Lumbricus rubellus. These newly identified earthworm miRNAs possess a difference of 2-4 nucleotides from their homologous counterparts in Caenorhabditis elegans. They also share similar features with other known animal miRNAs, for instance, the nucleotide U being dominant in both mature and pre-miRNA sequences, particularly in the first position of mature miRNA sequences at the 5' end. The newly identified earthworm miRNAs putatively regulate mRNA genes that are involved in many important biological processes and pathways related to development, growth, locomotion, and reproduction as well as response to stresses, particularly oxidative stress. Future efforts will focus on experimental validation of their presence and target mRNA genes to further elucidate their biological functions in earthworms.     

Phylogenetic implications and secondary structure analyses of Vigna mungo (L.) Hepper genotypes based on nrDNA ITS2 sequences

The internal transcribed spacers are highly preferred nuclear markers for the phylogenetic assessment of most eukaryotes, including plants. More recently, ITS2 has shown to possess equivalent phylogenetic significance as the entire ITS region. Vigna mungo L. Hepper is comparatively less explored from the molecular aspects as compared to the other species of the Vigna genus. The study presents the intra-individual characterization of 24 distinct genotypes Vigna mungo L. Hepper, using morphological as well as nrDNA ITS2 sequences and secondary structural data. The morphological characterization has been carried out using nine important agro-morphological traits. The molecular phylogeny of the sequence data, using the maximum parsimony and neighbor joining methods, shows the significant distinction based on the haplotypic variations amongst blackgram genotypes. The ITS2 secondary structures predicted using the homology modeling approach were compatible with the eukaryote-universal ITS2 secondary structure. The sequence-structure phylogeny reconstructed using the profile neighbour joining approach, also showed the presence of haplotypic variations in form of clusters on the phylogenetic tree. Further, the high GC content in the sequence data and highly negative ΔG values of the folded secondary structures ruled out the possibilities of the presence of any pseudogenes in the data set. Our analysis recommends the use of ITS2 sequence and secondary structure data at the intraspecific levels of plant taxonomical classification. Moreover, this study for the very first time reports the combined use morphological, and molecular data (using ITS2 sequence and secondary structural information) for the characterization of plants at the varietal level of taxonomical classification.     

Computational Identification of MicroRNAs and Their Targets in Perennial Ryegrass (Lolium perenne)

MicroRNAs (miRNAs) are small non-coding RNA molecules of 22 nucleotides in length that have been characterized as regulators of messenger RNA (mRNA) regulating a number of developmental processes in plants and animals by silencing genes using multiple mechanisms. miRNAs have been extensively studied in various plant species; however, few information are available about miRNAs in perennial ryegrass, animal feed, and industrial raw materials. In this study, the 12 potential perennial ryegrass miRNAs were identified for the first time by computational approach. Using the newly identified miRNA sequences, the perennial ryegrass mRNA database was further used for BLAST search and detected 33 potential targets of miRNAs. Prediction of potential miRNA target genes revealed their functions involved in various important plant biological processes. Our result should be useful for further investigation into the biological functions of miRNAs in perennial ryegrass. The selected miRNAs representing four families were verified by RT-PCR experiment, indicating that the prediction method that we used to identify the miRNAs was effective.     

An in-silico approach to study the possible interactions of miRNA between human and SARS-CoV2

BackgroundThe progressive SARS-CoV2 outbreaks worldwide have evoked global investigation. Despite the numerousin-silico approaches, the virus-host relationship remains a serious concern. MicroRNAs are the small non-coding RNAs that help in regulating gene profiling. The current study utilized miRNA prediction tools along with the PANTHER classification system to demonstrate association and sequence similarities shared between miRNAs of SARS-CoV2 and human host.MethodAn in-silico approach was carried out using Vmir analyzer to predict miRNAs from SARS-CoV2 viral genomes. Predicted miRNAs from SARS-CoV2 viral genomes were used for effective hybridization sequence identification along the nucleotide similarities with human miRNAs from miRbase database. Further, it was proceeded to analyze the gene ontology using miRDB with PANTHER classification.ResultBased on the prediction and analysis, we have identified 22 potential miRNAs from five genomes of SARS-CoV2 linked with 12 human miRNAs. Analysis of human miRNAs hsa-mir-1267, hsa-mir-1-3p, hsa-mir-5683 were found shared between all the five viral SARS-CoV2 miRNAs. Further, PANTHER classification analyzed the gene-ontology being carried by these associations showed that 44 genes were involved in biological functions that includes genes specific for signaling pathway, immune complex generation, enzyme binding with effective role in the virus-host relationship.ConclusionOur analysis concludes that the genes identified in this study can be effective in analyzing the virus-host interaction. It also provides a new direction to understand viral pathogenesis with a probable new way to link, that can be used to understand and relate the miRNAs of the virus to the host conditions.     

MicroRNA sequence motifs reveal asymmetry between the stem arms

The processing of micro RNAs (miRNAs) from their stemloop precursor have revealed asymmetry in the processing of the mature and its star sequence. Furthermore, the miRNA processing system between organism differ. To assess this at the sequence level we have investigated mature miRNAs in their genomic contexts. We have compared profiles of mature miRNAs within their genomic context of the 5' and 3' stemloop precursor arms and we find asymmetry between mature sequences of the 5' and 3' stemloop precursor arms. The main observation is that vertebrate organisms have a characteristic motif on the 5' arm which is in contrast to the 3' arm motif which mainly show the conserved U at the position of the mature start. Also the vertebrate 5' arm motif show a semi-conserved G 13 nucleotides upstream from the first position. We compared the 5' and 3' arm profiles using the average log likelihood ratio (ALLR) score, as defined by Wang and Stormo (2003) [Wang T., Stormo, G.D., 2003. Combining phylogenetic data with co-regulated genes to identify regulatory motifs. Bioinformatics 2369-2380.] and computing a p-value we find that the two profiles differs significantly in their 3' end where the 5' arm motif (in contrast to the 3' arm motif) has a semi-conserved GU rich region. Similar findings are also obtained for other organisms, such as fly, worm and plants. The observed similarities and differences between closely and distantly related organisms are discussed and related to current knowledge of miRNA processing.     

A computational model for non-conserved mature miRNAs from the rice genome

Several computational approaches employ the high complementarity of plant miRNAs to target mRNAs as a filter to recognize miRNA. Numerous non-conserved miRNAs are known with more recent evolutionary origin as a result of target gene duplication events. We present here a computational model with knowledge inputs from reported non-conserved mature miRNAs of Oryza sativa (rice). Sequence- and structure-based approaches were used to retrieve miRNA features based on rice Argonaute protein and develop a multiple linear regression (MLR) model (r² = 0.996, q²_cv = 0.989) which scored mature miRNAs as predicted by the MaturePred program. The model was validated by scoring test set (q² = 0.990) and computationally predicted mature miRNAs as external test set (q²_test = 0.895). This strategy successfully enhanced the confidence of retrieving most probable non-conserved miRNAs from the rice genome. We anticipate that this computational model would recognize unknown non-conserved miRNA candidates and nurture the current mechanistic understanding of miRNA sorting to unveil the role of non-conserved miRNAs in gene silencing.