Distinct structural motifs are necessary for targeting and import of Tim17 in Trypanosoma brucei mitochondrion.

Nuclear-encoded mitochondrial proteins are correctly translocated to their proper sub-mitochondrial destination using location-specific mitochondrial targeting signals and via multi-protein import machineries (translocases) in the outer and inner mitochondrial membranes (TOM and TIMs, respectively). However, targeting signals of multi-pass Tims are less defined. Here, we report the characterization of the targeting signals of Trypanosoma brucei Tim17 (TbTim17), an essential component of the most divergent TIM complex. TbTim17 possesses a characteristic secondary structure including four predicted transmembrane (TM) domains in the center with hydrophilic N- and C-termini. After examining mitochondrial localization of various deletion and site-directed mutants of TbTim17 in T. brucei using subcellular fractionation and confocal microscopy, we located at least two internal targeting signals (ITS): (i) within TM1 (31-50 AAs) and (ii) TM4 + loop 3 (120-136 AAs). Both signals are required for proper targeting and integration of TbTim17 in the membrane. Furthermore, a positively charged residue (K122) is critical for mitochondrial localization of TbTim17. This is the first report of characterizing the ITS for a multipass inner membrane protein in a divergent eukaryote, like T. brucei.IMPORTANCEAfrican trypanosomiasis (AT) is a deadly disease in human and domestic animals, caused by the parasitic protozoan Trypanosoma brucei. Therefore, AT is not only a concern for human health but also for economic development in the vast area of sub-Saharan Africa. T. brucei possesses a single mitochondrion per cell that imports hundreds of nuclear-encoded mitochondrial proteins for its functions. T. brucei Tim17 (TbTim17), an essential component of the TbTIM17 complex, is a nuclear-encoded protein; thus, it is necessary to be imported from the cytosol to form the TbTIM17 complex. Here, we demonstrated that the internal targeting signals within the transmembrane 1 (TM1) and TM4 with loop 3, and residue K122 are required collectively for import and integration of TbTim17 in the T. brucei mitochondrion. This information could be utilized to block TbTim17 function and parasite growth.

Role of the translocase of the mitochondrial inner membrane in the import of tRNAs into mitochondria in Trypanosoma brucei.

Trypanosomatids are unicellular parasitic protozoa. Many of the species of this genera cause severe diseases in human, such as Leishmaniasis, African trypanosomiasis and Chagas disease. These parasites possess a single reticular mitochondrion with a concatenated structure of mitochondrial DNA known as kinetoplast or kDNA. kDNA encodes few essential mitochondrial proteins but no tRNAs. Therefore, trypanosomatid mitochondrion import a full set of nucleus-encoded tRNAs for mitochondrial translation. Recent advances indicated that mitochondrial protein translocases, particularly the subunits of the ATOM complex, are involved in the import of a tRNA in Trypanosoma brucei. However, the global picture and the role of the translocase components of the mitochondrial inner membrane (TbTims) are not well understood. Here we investigated the relative abundance of 16 different tRNAs in the cytosolic and mitochondrial fractions isolated from the six TbTims knockdown cell lines. We found that knockdown of TbTim17, one of the primary components of the TbTIM complex, reduced the abundance of all of these tRNAs into mitochondria and increased their abundance in the cytosol. Depletion of TbTim62, a TbTim17 associated proteins, also reduced the relative abundance of most of these tRNAs into mitochondria except for tRNAleu, tRNAmet, and tRNAglu. Whereas, knockdown of other TbTims, like TbTim50 and two small TbTims, TbTim10 and TbTim8/13, didn't have any effect on tRNA abundance either in the cytosol or mitochondria. Depletion of any of these TbTims showed minimal effect on the levels of total tRNAs in T. brucei. Absolute quantification of tRNA levels revealed that TbTim17 knockdown reduced the levels of different tRNAs in mitochondria from 3-6% to 0.8-1.4%, which is equivalent to ~70% reduction in average. Whereas, TbTim62 depletion showed somewhat selective effect. Overall, our results suggest that TbTim17 and TbTim62 are essential for tRNA import that further makes a connection between the tRNA and protein import into mitochondria in T. brucei.

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.

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.

In-silico identification and characterization of organic and inorganic chemical stress responding genes in yeast (Saccharomyces cerevisiae).

To study the life processes of all eukaryotes, yeast (Saccharomyces cerevisiae) is a significant model organism. It is also one of the best models to study the responses of genes at transcriptional level. In a living organism, gene expression is changed by chemical stresses. The genes that give response to chemical stresses will provide good source for the strategies in engineering and formulating mechanisms which are chemical stress resistant in the eukaryotic organisms. The data available through microarray under the chemical stresses like lithium chloride, lactic acid, weak organic acids and tomatidine were studied by using computational tools. Out of 9335 yeast genes, 388 chemical stress responding genes were identified and characterized under different chemical stresses. Some of these are: Enolases 1 and 2, heat shock protein-82, Yeast Elongation Factor 3, Beta Glucanase Protein, Histone H2A1 and Histone H2A2 Proteins, Benign Prostatic Hyperplasia, ras GTPase activating protein, Establishes Silent Chromatin protein, Mei5 Protein, Nondisjunction Protein and Specific Mitogen Activated Protein Kinase. Characterization of these genes was also made on the basis of their molecular functions, biological processes and cellular components.

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.

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.

The MicroRNAs and their targets in the channel catfish (Ictalurus punctatus).

MicroRNAs (miRNAs) are small, non-coding and negative regulatory RNAs about 22 nucleotides. They are mostly conserved among the organisms and this conservation makes them a good source for the identification of novel miRNAs by computational genomic homology. The miRNA repertoire of the major aquaculture species, channel catfish (Ictalurus punctatus), is unknown. This study is focused on computational search for novel miRNA homologs and their targets along with their characterization in channel catfish. Total 60 novel precursor miRNAs having 73 mature sequences belong to 45 families in channel catfish were identified and characterized. They belong to the miRNA families; ipu-let-7, miR-7, 10, 16, 24, 29, 32, 93, 99, 101, 105, 126, 127, 133, 135, 141, 142, 143, 144 145, 148, 150, 152, 153, 203, 210, 214, 221, 223, 293, 429, 430, 466, 682, 731, 737, 1388, 1594, 1642, 1701, 1782, 1814, 2145, 2182 and 3074 are reported for the first time in channel catfish. All the 73 mature miRNAs are observed in the stem portion of the stable minimum free energy stem-loop structures. Total 341 proteins targeted by the novel channel catfish miRNAs were also identified. They are involved in immune-related (32 %), signaling (15 %), transcription factors (15 %), metabolism (12 %), transportation (8 %), growth & development (5 %), structural (5 %) and others (8 %) proteins.

Identification and characterization of the microRNAs and their targets in Salmo salar.

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about 18 to 26 nucleotides long. Their conserved nature among the various organisms makes them a good source of new miRNAs discovery by comparative genomics approach. The study resulted in novel 75 precursor miRNAs containing 102 mature sequences belonging to 46 families in an important aquatic environmental monitoring fish (Salmo salar). All the miRNA families (let-7, mir-1, 7, 9, 21, 22, 92, 96, 122, 126, 128, 129, 132, 133, 142, 144, 147, 148, 196, 202, 212, 223, 375, 429, 430, 449, 451, 457, 466, 682, 700, 1388, 1594, 1600, 1607, 1616, 1642, 1681, 1701, 1720, 1772, 1782, 1787, 1814, 2189 and 3540) are found for the first time in S. salar. All 75 miRNA precursors form stable minimum free energy stem loop and the mature miRNAs reside in the stem portion of the stem loop structure. Their target proteins are involved in transcription factors (28%), metabolism (23%), signaling (18%), transportation (9%), immunity (8%), stress related activity (5%), cancer and tumor related activity (5%), growth and development (3%), and cell division (1%).

The novel 172 sheep (Ovis aries) microRNAs and their targets.

MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs about ≈22 nucleotides in length. The comparative genomics approach due to their conserved nature is a good source for the novel miRNAs discovery. In this study, total 172 novel miRNAs from 140 precursor sequences belonging to 114 families were identified in sheep (Ovis aries), the most important livestock animal. All the miRNA families (oar-mir-95, 129, 130, 186, 214, 219, 223, 324, 339, 423, 450, 499, 544, 562, 568, 584, 669, 671, 763, 935, 1281, 1282, 1306, 1552, 1584, 1587, 1603, 1607, 1706, 1711, 1718, 1732, 1738, 1761, 1771, 1777, 1778, 1780, 1807, 1843, 1895, 1930, 2127, 2139, 2182, 2284, 2287, 2295, 2296, 2310, 2311, 2314, 2315, 2316, 2320, 2359, 2378, 2381, 2382, 2395, 2400, 2404, 2410, 2412, 2423, 2426, 2435, 2470, 2477, 2482, 2487, 2881, 2883, 2885, 2888, 2889, 2896, 2901, 2904, 2917, 2964, 3063, 3064, 3074, 3080, 3432, 3529, 3533, 3613, 3649, 3654, 3658, 3661, 3662, 3940, 3960, 4273, 4426, 4447, 4459, 4468, 4493, 4507, 4647, 4680, 4785, 4788, 4800, 5102, 5105, 5109, 5115, 5125 and 5132) are found for the first time in Sheep. All 140 miRNA precursors form stable minimum free energy stem loop and the mature miRNAs reside in the stem portion of the stem loop structure. Their putative targets are involved in transcription factors (26%), signaling (19%), metabolism (18%), transportation (10%), immunity (9%), cancer and tumor related (5%), growth and development (5%), stress related (4%), and structural proteins (3%).

Identification of biotic and abiotic stress up-regulated ESTs in Gossypium arboreum.

Asiatic desi cotton (Gossypium arboreum) shows great potential against biotic and abiotic stresses. The stress resistant nature makes it a best source for the identification of biotic and abiotic stress resistant genes. As in many plants same set of genes show responding behavior against the various abiotic and biotic stresses. Thus in the present study the ESTs from the G. arboreum drought stressed leaves were subjected to find the up-regulated ESTs in abiotic and biotic stresses through homology and in-silico analysis. A cDNA library has been constructed from the drought stressed G. arboreum plant. 778 clones were randomly picked and sequenced. All these sequences were subjected to in-silico identification of biotic and abiotic up-regulated ESTs. Total 39 abiotic and biotic up-regulated ESTs were identified. The results were further validated by real-time PCR; by randomly selection of ten ESTs. These findings will help to develop stress resistant crop varieties for better yield and growth performance under stresses.

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.

[Isolation and functional analysis of cotton universal stress protein promoter in response to phytohormones and abiotic stresses].

The 949 bp promoter fragment upstream from the translation initiation site of the GUSP gene encoding a universal stress protein was isolated from the genomic DNA of Gossypium arboream. Some putative cis-acting elements involved in stress responses including E-box, ABRE, DPBF-box, and MYB-core elements were found in the promoter region. In an Agrobacterium-mediated transient expression assay, strong activation of the GUSP full promoter region occurred in tobacco leaves following dehydration, abscisic acid, salt, heavy metal, gibberellic acid and dark treatments. Deletion analysis of the promoter revealed that the dehydration, abscisic acid and salt responses were affected by the deletion between -208 and -949 bp and showed 2-4-fold induction. However, in response to dark, gibberellic acid and heavy metals the induction was only 2-fold. This is an important study as no report of this universal stress protein promoter is available in literature.

Functional analysis of cotton small heat shock protein promoter region in response to abiotic stresses in tobacco using Agrobacterium-mediated transient assay.

The cotton (Gossypium arboreum) stress-related gene GHSP26 responds to dehydration. To elucidate its stress tolerant mechanism at the transcriptional level, we isolated and characterized the promoter region (PGHSP26, -2,831 bp) flanking the 5' GHSP26 coding region from the genomic DNA. A series of PGHSP26 deletion derivatives was created for the identification of the upstream region of the gene required for the promoter activity. Each deletion construct was analyzed by agrobacterium mediated transient transformation in tobacco leaves after treatment with abscissic acid (ABA), heavy metals and dehydration. Promoter fragments of 716 bp or longer showed two-fold or greater induction after each treatment. These findings further our understanding of the regulation of GHSP26 expression and provide a new drought-inducible promoter system in transgenic plants.