Muhammad Din

Identification of MicroRNAs and their targets in Helianthus

MicroRNAs (miRNAs) are small non-coding RNAs about 20–24 nucleotides long, playing regulatory role. The conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach using bioinformatic tools. A systematic search approach was used for inter-species homologs of miRNA precursors (pre-miRNAs), from known Helianthus expressed sequence tags (ESTs). The study resulted in 61 novel miRNAs belonging to 34 families from Helianthus ESTs. The 28 miRNA families; mir 159,160, 164, 170, 390, 393, 413, 415, 419, 426, 446, 530, 822, 842, 846, 1310, 1888, 2086, 2657, 2667, 2678, 2659, 2911, 2938, 3440, 3521, 3623, and 3630 are reporting for the first time in Helianthus. In the 61 new miRNAs, 20 are from H.tuberosus, 17 miRNAs belong to H.annus, 8 are from H.ciliaris, 5 to H.exilis, 4 is from H.argophyllous, H.petiolaris each and 3 are from H.paradoxus. All the pre-miRNAs form stable minimum free energy (mfe) stem-loop structure as their orthologues form and the mature miRNAs reside in the stem portion of the stem-loop structures. Their targets consist of growth and development related, transcription factors, signalling pathway kinases, stress resistant proteins and transport related proteins.

Profiling microRNAs and their targets in an important fleshy fruit: Tomato (Solanum lycopersicum)

Tomato (Solanum lycopersicum) is an important and the most useful plant based diet. It is widely used for its antioxidant property. Presently, only two digits, tomato microRNAs (miRNAs) are reported in miRBase: a miRNA database. This study is aimed to profile and characterize more miRNAs and their targets in tomato. A comprehensive comparative genomic approach is applied and a total of 109 new miRNAs belonging to 106 families are identified and characterized from the tomato expressed sequence tags (ESTs). All these potential miRNAs are profiled for the first time in tomato. The profiled miRNAs are also observed with stable stem-loop structures (Precursor-miRNAs), whose length ranges from 45 to 329 nucleotides (nt) with an average of 125 nt. The mature miRNAs are found in the stem of pre-miRNAs and their length ranges from 19 to 24 nt with an average of 21 nt. Furthermore, twelve miRNAs are randomly selected and experimentally validated through RT-PCR. A total of 406 putative targets are also predicted for the newly 109 tomato miRNAs. These targets are involved in structural protein, metabolism, transcription factor, growth & development, stress related, signaling pathways, storage proteins and other vital processes. Some important proteins like; 9-cisepoxycarotenoid dioxygenase (NCED), transcription factor MYB, ATP-binding cassette transporters, terpen synthase, 14-3-3 and TIR-NBS proteins are also predicted as putative targets for tomato miRNAs. These findings improve a baseline data of miRNAs and their targets in tomato. This baseline data can be utilized to fine tune this important fleshy fruit for nutritional & antioxidant properties and also under biotic & abiotic stresses.

Structural and functional based identification of the bean (Phaseolus) microRNAs and their targets from expressed sequence tags

MicroRNAs (miRNAs) are small, 18–26 nucleotides long, non-coding RNAs that play role in post-transcriptional gene regulation. Many of these are evolutionarily conserved. This suggests a powerful approach to predict new miRNAs in other species. In this research, structural and functional approaches were combined to make computational prediction of potential miRNAs and their targets in Bean (Phaseolus). Total 55 novel miRNAs were detected from 38 miRNAs families in Bean (Phaseolus). These families are; miR156, 160, 164, 168, 170, 171, 172, 319, 393, 396, 397, 398, 408, 414, 438, 444, 535, 1310, 1424, 1426, 1848, 1860, 1863, 2055, 2091, 2093, 2094, 2102, 2103, 2105, 2864, 2866, 2925, 2926, 4221, 4245, 4246 and 4250. In the 55 putative miRNAs; 28 miRNAs belong to Phaseolus acutifolius, 23 to Phaseolus vulgaris, 4 to Phaseolus coccineus. All the mature miRNAs reside in the stem portion of the hairpin structures. Total 146 potential protein targets were predicted for these miRNAs.

Profiling and characterization of eggplant (Solanum melongena L.) microRNAs and their targets

MicroRNAs (miRNAs) are small, non-protein coding and functional RNAs. They play negative regulatory role in gene regulation. They are endogenous in nature and are ~21 nucleotides in length. They are reported in many plant species, but still missing and need to be identified in other important plants like; eggplant. The comparative genomic methodology due to their conserved nature is a reasonable approach for the novel miRNAs discovery. In this research, total 76 novel miRNAs from 67 families were identified in the important vegetable eggplant (Solanum melongena L.). All precursor miRNAs form stable minimum free energy secondary structures and the mature miRNAs reside in the stem regions. Furthermore, eight miRNAs were randomly selected and experimentally validated through RT-PCR. A total of 345 putative targets were also identified for the novel 76 eggplant miRNAs. Their targets are involved in regulation, metabolism, transcription factors, growth and development and other physiological processes. These findings provide a baseline to unravel the miRNAs role in eggplant and utilize them for the improvement of the plant biology.

In-silico based identification and functional analyses of miRNAs and their targets in Cowpea ( Vigna unguiculata L.)

Cowpea (Vigna unguiculata L.) is an important leguminous plant and a good diet due to presence of carbohydrate and high protein contents. Currently, only few cowpea microRNAs (miRNAs) are reported. This study is intended to identify and functionally analyze new miRNAs and their targets in cowpea. An in-silico based homology search approach was applied and a total of 46 new miRNAs belonging to 45 families were identified and functionally annotated from the cowpea expressed sequence tags (ESTs). All these potential miRNAs are reported here for the first time in cowpea. The 46 new miRNAs were also observed with stable hairpin structures with minimum free energy, ranging from −10 to −132 kcal mol−1 with an average of −40 kcal mol−1. The length of new cowpea miRNAs are ranged from 18 to 26 nt with an average of 21 nt. The cowpea miRNA-vun-mir4414, is found as pre-miRNA cluster for the first time in cowpea. Furthermore, a set of 138 protein targets were also identified for these newly identified 46 cowpea miRNAs. These targets have significant role in various biological processes, like metabolism, transcription regulation as transcription factor, cell transport, signal transduction, growth & development and structural proteins. These findings are the significant basis to utilize and manage this important leguminous plant-cowpea for better nutritional properties and tolerance for biotic and abiotic stresses.

Initial screening of plant most conserved MicroRNAs targeting infectious viruses: HBV and HCV

MicroRNAs (miRNAs) belong to small RNA world are involve in posttranslational gene regulation. They are small in size (18–26 nt), non-coding and endogenously produced by cell. Recently, some evidence showing cross gene regulation of plants miRNAs targeting animal genes is attracting the researchers to do research in this interesting area of regulatory RNAs. This research is aimed to initially screen the role of plant most conserved miRNAs against the hepatitis B and C viruses causing an infectious diseases that affect the human liver. Combinations of various bioinformatics tools were applied to predict the cross-kingdom interactions of plant miRNAs targeting HBV and HCV. For this purpose six most conserved plant miRNAs miR156/157, 166, 169, 172 390 and 399 were subjected to find targets in the reported nucleic acid sequences of HBV and HCV in GenBank. This initial screening is resulted a total of 12 HBC and HCV genes as potential targets by plant conserved miRNAs. These findings provide a baseline data of plant based miRNAs and their targets in HBV and HCV. This initial baseline data would be helpful to elucidate in detail the link between plant miRNAs against animal and human infectious viruses such as; HBV and HCV.

Identification and annotation of newly conserved microRNAs and their targets in wheat (Triticum aestivum L.)

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs produce by cell endogenously. They are 18–26 nucleotides in length and play important roles at the post-transcriptional stage of gene regulation. Evolutionarily, miRNAs are conserved and their conservation plays an important role in the prediction of new miRNAs in different plants. Wheat (Triticum aestivum L.) is an important diet and consumed as second major crop in the world. This significant cereal crop was focused here through comparative genomics-based approach to identify new conserved miRNAs and their targeted genes. This resulted into a total of 212 new conserved precursor miRNAs (pre-miRNAs) belonging to 185 miRNA families. These newly profiled wheat’s miRNAs are also annotated for stem-loop secondary structures, length distribution, organ of expression, sense/antisense orientation and characterization from their expressed sequence tags (ESTs). Moreover, fifteen miRNAs along with housekeeping gene were randomly selected and subjected to RT-PCR expressional validation. A total of 32927 targets are also predicted and annotated for these newly profiled wheat miRNAs. These targets are found to involve in 50 gene ontology (GO) enrichment terms and significant processes. Some of the significant targets are RNA-dependent DNA replication (GO:0006278), RNA binding (GO:0003723), nucleic acid binding (GO:0003676), DNA-directed RNA polymerase activity (GO:0003899), magnesium ion transmembrane transporter activity (GO:0015095), antiporter activity (GO:0015297), solute:hydrogen antiporter activity (GO:0015299), protein kinase activity (GO:0004672), ATP binding (GO:0005524), regulation of Rab GTPase activity (GO:0032313) Rab GTPase activator activity (GO:0005097), regulation of signal transduction (GO:0009966) and phosphoprotein phosphatase inhibitor activity (GO:0004864). These findings will be helpful to manage this economically important grain plant for desirable traits through miRNAs regulation.