Homologous miRNAs Research Articles

Use of Flaviviral genetic fragments as a potential prevention strategy for HIV-1 Silencing.

Coinfection with certain members of the Flaviviridae, such as Dengue Virus (DV), West Nile Virus (WNV) Yellow Fever Virus (YFV) and most importantly, GBV-C have been documented to reduce HIV-1 viral load in vivo. Numerous studies strongly support the notion that persistent coinfection with non-pathogenic virus prolongs survival in HIV-1 infected individuals. Coinfected individuals show higher CD4+ cell counts, lower HIV-1 RNA viral loads and live three times longer than clinically matched HIV-1 monoinfected patients. We have previously shown that one of the major anti-HIV defenses conferred by GBV-C coinfection is the upregulation of intracellular miRNAs in CD4+ cells that share significant mutual homologies with GBV-C and HIV-1 (>80%) genomes. Genome-wide bioinformatics analyses were carried out to search for miRNA binding sites in mutual homologies between HIV and several members of the Flaviviridae. Several miRNAs shared significant mutual homology with HIV-1 genetic sequences and GBV-A, B, C, DV, WNV and YFV.These may be responsible for beneficial effects in HIV-1 infected individuals. Three highly mutual homologous miRNAs (i.e. miR-627-5, miR-369-5 and miR-548f), expressed in CD4+ cell lines, reduce HIV-1 replication by up to 90% whereas miRNAs with low mutual homologies (i.e. miR-34-1 and miR-508) impart only slight inhibition of HIV-1. We hypothesize that a recombinant GBV-C-based vector can be constructed which expresses several beneficial genetic motifs of the Flaviviridae without causing any side effects while stimulating a wide array of beneficial miRNAs that can more efficiently prevent HIV-1 infection.

Identification and Characterization of Novel miRNAs in Chlamydomonas reinhardtii by Computational Methods.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs with 18-24 nucleotides in length, which have important roles in posttranscriptional gene regulation. The resemblance of miRNA biogenesis in unicellular green algae and those in plants suggests probable evolutionary conserved pathways. This conservation provides a ground towards prediction of new homologs via computational biology. Here, conserved miRNA genes in Chlamydomonas reinhardtii and plants were examined through homology alignment. Previously known and unique plant miRNAs were BLASTed against expressed sequence tags (ESTs) and genomic survey sequences (GSSs) of C. reinhardtii. All candidate sequences with appropriate fold back structures were screened according to a series of miRNA filtering criteria. Homologous miRNAs (17), belonging to 9 miRNA gene families were predicted. Interestingly and for the first time, a miRNA family of genes was localized to chloroplast. Again and for the first time, here we report identification of C. reinhardtii miRNA orthologs in plants and animals. miRNA target genes were identified based on their sequence complementarities to the respective miRNAs using psRNATarget against C. reinhardtii, Unigene, and DFCI Gene Index (CHRGI). Totally, 152 potential target sites were identified. From the predicted miRNAs, 7 miRNAs had no target sequence in C. reinhardtii protein coding genes. Identifying miRNA and their target transcript(s) would be useful for other research concerned with the function and regulatory mechanisms of C. reinhardtii miRNAs and helps researchers to better understand the nature of its extensive metabolic flexibility and environmental compatibility to survive in distinct environmental niches and nutrient availability.

Japanese Macaque Rhadinovirus Encodes a Viral MicroRNA Mimic of the miR-17 Family.

Japanese macaque (JM) rhadinovirus (JMRV) is a novel, gamma-2 herpesvirus that was recently isolated from JM with inflammatory demyelinating encephalomyelitis (JME). JME is a spontaneous and chronic disease with clinical characteristics and immunohistopathology comparable to those of multiple sclerosis in humans. Little is known about the molecular biology of JMRV. Here, we sought to identify and characterize the small RNAs expressed during lytic JMRV infection using deep sequencing. Fifteen novel viral microRNAs (miRNAs) were identified in JMRV-infected fibroblasts, all of which were readily detectable by 24 h postinfection and accumulated to high levels by 72 h. Sequence comparisons to human Kaposi's sarcoma-associated herpesvirus (KSHV) miRNAs revealed several viral miRNA homologs. To functionally characterize JMRV miRNAs, we screened for their effects on nuclear factor kappa B (NF-κB) signaling in the presence of two proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). Multiple JMRV miRNAs suppressed cytokine-induced NF-κB activation. One of these miRNAs, miR-J8, has seed sequence homology to members of the cellular miR-17/20/106 and miR-373 families, which are key players in cell cycle regulation as well as inflammation. Using reporters, we show that miR-J8 can target 3' untranslated regions (UTRs) with miR-17-5p or miR-20a cognate sites. Our studies implicate JMRV miRNAs in the suppression of innate antiviral immune responses, which is an emerging feature of many viral miRNAs. Gammaherpesviruses are associated with multiple diseases linked to immunosuppression and inflammation, including AIDS-related cancers and autoimmune diseases. JMRV is a recently identified herpesvirus that has been linked to JME, an inflammatory demyelinating disease in Japanese macaques that mimics multiple sclerosis. There are few large-animal models for gammaherpesvirus-associated pathogenesis. Here, we provide the first experimental evidence of JMRV miRNAs in vitro and demonstrate that one of these viral miRNAs can mimic the activity of the cellular miR-17/20/106 family. Our work provides unique insight into the roles of viral miRNAs during rhadinovirus infection and provides an important step toward understanding viral miRNA function in a nonhuman primate model system.

Sensitive and specific detection of miRNA using an isothermal exponential amplification method using fluorescence-labeled LNA/DNA chimera primers.

MicroRNAs (miRNAs) are currently considered as potential biomarkers for various human diseases. In the present study, miRNA-triggered real-time fluorescent isothermal reaction with exponential amplification (ReFIRE) with or without Thermus aquaticus MutS (Taq MutS) was developed to analyze miRNAs using DNA polymerase, a nicking endonuclease, and fluorescently labeled primers. In the absence of Taq MutS, the ReFIRE system permitted the detection of 100ymol of targeted miRNA in 80min. However, this system enabled limited differentiation between homologous miRNA family members. Upon addition of Taq MutS to the ReFIRE system, non-specific amplification generated from the mishybridization between primers and primer dimers or primers and the template duplex was eliminated. The addition of Taq MutS enabled the ultrasensitive detection of as little as 10ymol of targeted miRNAs in 50min, which corresponds to less than 10 copies of miRNAs in a total volume of 20μl. Additionally, the assay exhibited a dynamic range of up to 12 orders of magnitude. The ReFIRE system also showed high specificity, enabling differentiation between homologous miRNA family members exhibiting only single-base differences. The sensitivity, specificity, and dynamic range associated with this system were greater than most currently available miRNA isothermal amplification assays. Moreover, when target-specific primers were labeled with different fluorescent reporters, multiplex analysis was easily performed in a single tube, permitting accurate normalization of miRNA expression. This simple, fast, ultrasensitive, highly specific, and easy-to-multiplex method could significantly contribute to research investigations pertaining to the biological roles of miRNA, as well as clinical diagnosis of various diseases that involve miRNA disruptions. Graphical Abstract The principle of ReFIRE system.

Ultrasensitive, colorimetric detection of microRNAs based on isothermal exponential amplification reaction-assisted gold nanoparticle amplification

MicroRNAs (miRNAs) play important roles in a wide range of biological processes, and their aberrant expressions are linked to a large number of human diseases and disorders. In this work, we developed a colorimetric method for rapid, ultrasensitive miRNA detection via isothermal exponential amplification reaction (EXPAR)-assisted gold nanoparticle (AuNP) amplification. The sensing probe designed with a tandem phosphorothioate modification in the backbone of the polyadenines at the 5′ terminus was employed to directly assemble onto the surface of AuNP with high adsorption affinity. The recognition domain at the 3′ terminus of the sensing probe hybridizes with target miRNAs to trigger EXPAR with exponential signal amplification. With the amplification reaction with the action of DNA polymerase, the sensing probe gradually detaches from the AuNP, resulting in the aggregation of bare AuNPs in the high-salt reaction environment due to lack of DNA protection. The presence of AuNP aggregation is conveniently measured by UV–vis spectroscopy. Our proposed method could provide a linear detection range from 50fM to 10nM with a detection limit of ∼46fM within 60min, and also discriminate a single-nucleotide difference between homologous miRNAs.

Sensitive electrochemical determination of miRNAs based on a sandwich assay onto magnetic microcarriers and hybridization chain reaction amplification

A novel electrochemical approach for determination of miRNAs involving a sandwich hybridization assay onto streptavidin-magnetic beads (Strep-MBs), hybridization chain reaction (HCR) amplification and amperometric detection at disposable screen-printed carbon electrodes is reported. Using miRNA-21 as the target analyte, a dynamic linear range from 0.2 to 5.0nM with a 60pM (1.5fmol in 25μL) detection limit was obtained. The achieved sensitivity is 24-fold higher than a non-HCR amplification approach involving conventional sandwich type assay onto MBs. Moreover, the whole assay time lasted 1h 45min which is remarkably shorter than other reported methodologies. The methodology exhibited full selectivity against other non-complementary miRNAs as well as an acceptable discrimination between homologous miRNA family members. The applicability of this novel approach was demonstrated by determining mature miRNA-21 in total RNA (RNAt) extracted from tumor cells and human tissues.

Sequence data mining in search of hookworm (Necator americanus) microRNAs

The new world hookworm, Necator americanus is a soil-transmitted nematode responsible for Necatoriasis (a type of helminthiasis) in hosts such as humans, dogs, and cats. N. americanus genome and transcriptome has been sequenced and a draft assembly analysis has been published highlighting protein coding genes and possible drug target proteins. Hookworm microRNA identification, annotations and their public release is yet to be attempted. The same is evident from lack of hookworm miRNA information in related popular public nucleotide sequence repositories such as miRBase, GenBank, WormBase etc. Therefore, in the present study we addressed these issues using EST and assembled transcript sequence information of hookworm. Using computational approaches, we identified three miRNAs precursor sequences and their mature forms. We also identified their potential targets from hookworm ESTs and transcripts, and from human transcriptome. Overall, the results indicate presence of nematode specific miRNA homologs in N. americanus and shades light on their putative targets in worm itself and the human host.

Computational prediction of micrornas and their target genes in rainbow trout (Oncorhynchus mykiss)

MicroRNAs (miRNA) are non-coding small RNA molecules consisting of 20–24 nucleotides (nt), which regulate gene expression negatively at the post-transcriptional levels depending on the extent of complementation between miRNA and mRNA in a variety of eukaryotic organisms. They have attracted increasing attention recently. To date, a total of 1637 mature miRNAs from fishes have been deposited in the miRBase database (Release 21) and they are just a small portion of the miRNAs described in fish species. Especially in rainbow trout (Oncorhynchus mykiss) miRNAs, it is far from the numbers of miRNAs found in other fishes and many more miRNA remain to be discovered. In present study, we used known animal miRNAs to systematically search miRNA homologs in available expressed sequence tags (EST), genomic survey sequences (GSS), and transcript sequence assemblies (TSA) of O. mykiss based on the conservation of miRNA sequences. Following the filtering with a combination of stringent criteria, 39 miRNAs belonging to 25 independent families were identified, of which 14 are novel identified miRNAs firstly in O. mykiss. The randomly selected 13 miRNAs were verified by stem-loop RT-PCR indicating that the prediction method that we used to identify the miRNAs was effective. Furthermore, total of 94 target genes were predicted and their probable functions were illustrated. Their target genes are involved in transcription factors, development, metabolism, signaling, immunity, and cell division. The current study has provided an update on O. mykiss miRNAs and their targets, which will be helpful for future research on miRNA-mediated gene regulation in this important fish species.

MiRNAS from shrimp penaeus monodon and its possible role in innate immunity

MicroRNAs (miRNAs) are short noncoding RNAs of the RNA interference pathways that regulate gene expression through partial complementary base pairing to target mRNAs. Here, miRNAs that are expressed in the white spot syndrome virus (WSSV)-infected Penaeus monodon hemocyte, were identified by next generation sequencing. Forty-six miRNA homologs were identified. Of those, the expression of 16 miRNAs in response to WSSV infection was analyzed by stem-loop real-time PCR. Eleven out of sixteen miRNAs were differently expressed upon WSSV infection. Two miRNAs, pmo-miR-315 and pmo-miR-750, were highly responsive upon WSSV infection. From the miRNA target prediction, miRNAs were targeted at 5UTR, ORF, and 3UTR of several immune-related genes involved in apoptosis, antimicrobial peptides, prophenoloxidase system, proteinase and proteinase inhibitor. To characterize the miRNA function, the highly conserved miRNA homolog, pmo-bantam, was further analyzed. A highly conserved miRNA, pmo-bantam was predicted to target the 3'UTR of an immune gene, Kunitztype serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was confirmed by luciferase reporter assay. Also, correlation of pmo-bantam and KuSPI expression was revealed in lymphoid organ of WSSV-infected shrimp suggesting that the regulatory role of pmo-bantam in shrimp lymphoid organ. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response.

Shrimp miRNAs regulate innate immune response against white spot syndrome virus infection

MicroRNAs are short noncoding RNAs of RNA interference pathways that regulate gene expression through partial complementary base-pairing to target mRNAs. In this study, miRNAs that are expressed in white spot syndrome virus (WSSV)-infected Penaeus monodon, were identified using next generation sequencing. Forty-six miRNA homologs were identified from WSSV-infected shrimp hemocyte. Stem-loop real-time RT-PCR analysis showed that 11 out of 16 selected miRNAs were differentially expressed upon WSSV infection. Of those, pmo-miR-315 and pmo-miR-750 were highly responsive miRNAs. miRNA target prediction revealed that the miRNAs were targeted at 5′UTR, ORF, and 3′UTR of several immune-related genes such as genes encoding antimicrobial peptides, signaling transduction proteins, heat shock proteins, oxidative stress proteins, proteinases or proteinase inhibitors, proteins in blood clotting system, apoptosis-related proteins, proteins in prophenoloxidase system, pattern recognition proteins and other immune molecules. The highly conserved miRNA homolog, pmo-bantam, was characterized for its function in shrimp. The pmo-bantam was predicted to target the 3′UTR of Kunitz-type serine protease inhibitor (KuSPI). Binding of pmo-bantam to the target sequence of KuSPI gene was analyzed by luciferase reporter assay. Correlation of pmo-bantam and KuSPI expression was observed in lymphoid organ of WSSV-infected shrimp. These results implied that miRNAs might play roles as immune gene regulators in shrimp antiviral response.

Synteny and comparative analysis of miRNA retention, conservation, and structure across Brassicaceae reveals lineage- and sub-genome-specific changes.

The recent availability of genome sequences together with syntenic block information for Brassicaceae offers an opportunity to study microRNA (miRNA) evolution across this family. We employed a synteny-based comparative genomics strategy to unambiguously identify miRNA homologs from the genome sequence of members of Brassicaceae. Such an analysis of miRNA across Brassicaceae allowed us to classify miRNAs as conserved, lineage-, karyotype- and sub-genome-specific. The differential loss of miRNA from sub-genomes in polyploid genomes of Brassica rapa and Brassica oleracea shows that miRNA also follows the rules of gene fractionation as observed in the case of protein-coding genes. The study of mature and miR* region of precursors revealed instances of in-dels and SNPs which reflect the evolutionary history of the genomes. High level of conservation in miR* regions in some cases points to their functional relevance which needs to be further investigated. We further show that sequence and length variability in precursor sequences can affect the free energy and foldback structure of miRNA which may ultimately affect their biogenesis and expression in the biological system.

Identification and Expression Profiles of microRNA in Dolphin.

Recently, microRNAs (miRNAs) are focused on the role of biomarker because they are stable in serum and plasma, and some of them express in the specific organs and increase with the organ injury. Thus miRNAs may be very useful as biomarkers for monitoring the health and condition of dolphins and for detecting disorders in aquariums. Here, a small RNA library was made from dolphin lung, liver and spleen, and miRNA expression patterns were then determined for 15 different tissues. We identified 62 conserved miRNA homologs in the dolphin small RNA library and found high expression miRNAs in specific tissues: miR-125b and miR-221 were highly expressed in brain, miR-23b in heart, miR-199a and miR-223 in lung, and miR-122-5p in liver. Some of these tissue-enriched miRNAs may be useful as specific and sensitive diagnostic blood biomarkers for organ injury in dolphins.

Characterization and Profiling of Liver microRNAs by RNA-sequencing in Cattle Divergently Selected for Residual Feed Intake.

MicroRNAs (miRNAs) are short non-coding RNAs that post-transcriptionally regulate expression of mRNAs in many biological pathways. Liver plays an important role in the feed efficiency of animals and high and low efficient cattle demonstrated different gene expression profiles by microarray. Here we report comprehensive miRNAs profiles by next-gen deep sequencing in Angus cattle divergently selected for residual feed intake (RFI) and identify miRNAs related to feed efficiency in beef cattle. Two microRNA libraries were constructed from pooled RNA extracted from livers of low and high RFI cattle, and sequenced by Illumina genome analyser. In total, 23,628,103 high quality short sequence reads were obtained and more than half of these reads were matched to the bovine genome (UMD 3.1). We identified 305 known bovine miRNAs. Bta-miR-143, bta-miR-30, bta-miR-122, bta-miR-378, and bta-let-7 were the top five most abundant miRNAs families expressed in liver, representing more than 63% of expressed miRNAs. We also identified 52 homologous miRNAs and 10 novel putative bovine-specific miRNAs, based on precursor sequence and the secondary structure and utilizing the miRBase (v. 21). We compared the miRNAs profile between high and low RFI animals and ranked the most differentially expressed bovine known miRNAs. Bovine miR-143 was the most abundant miRNA in the bovine liver and comprised 20% of total expressed mapped miRNAs. The most highly expressed miRNA in liver of mice and humans, miR-122, was the third most abundant in our cattle liver samples. We also identified 10 putative novel bovine-specific miRNA candidates. Differentially expressed miRNAs between high and low RFI cattle were identified with 18 miRNAs being up-regulated and 7 other miRNAs down-regulated in low RFI cattle. Our study has identified comprehensive miRNAs expressed in bovine liver. Some of the expressed miRNAs are novel in cattle. The differentially expressed miRNAs between high and low RFI give some insights into liver miRNAs regulating physiological pathways underlying variation in this measure of feed efficiency in bovines.

148 FOLLICULAR FLUID microRNA SEQUENCES AS BIOMARKERS OF COMPETENT OOCYTES IN CATTLE

Oocyte developmental competence is correlated with antral follicle count through ill-defined mechanisms. Oocytes from ovaries with fewer than 10 mid-antral follicles of 2 to 6 mm in diameter (low group) show reduced competence compared with those from ovaries with more than 10 follicles (high group). To unravel mechanisms underlying this phenomenon, this work explored the role of follicular fluid microRNAs (miRNAs, short non-coding RNAs regulating gene expression at the post-transcriptional level). A total of 3 pools of 300 µL of follicular fluid (FF) were collected from mid-antral follicles of low (L) and high (H) groups, respectively. Following miRNA extraction and library preparation, deep sequencing was carried out on Illumina HisEqn 2000 (Illumina Inc., San Diego, CA, USA). Differentially expressed miRNAs were identified with R package edgeR (http://bioconductor.org/packages/release/bioc/html/edgeR.html). Target genes of differentially expressed miRNAs were predicted with DIANA miRPath using homologous human miRNA and gene union options (P < 0.01). Gene ontology (GO) analysis was carried out by Cytoscape (http://www.cytoscape.org/) using a bovine database. In total, 1279 miRNAs were identified in FF: 805 ± 139 in L and 862 ± 36 in H (P > 0.05). We found that 27 miRNAs were differentially expressed (false discovery rate ≤0.001): 17 were up-regulated in L and 10 in H. Up-regulated miRNAs in L group were predicted to target 121 genes, 39 of which are specific for ovarian function (e.g. BCL2, FOXO3, KIT, TP53, and PTK2). The GO analysis indicated that these genes were kinases, anti-apoptotic and oncogenic factors, and enriched stress-activated MAPK cascade mediated by oxygen reactive species, G1/S transition checkpoint, cellular response to interleukin-1, and negative regulation of cellular adhesion. Overexpressed miRNAs in H group were predicted to target 92 genes, 22 of which (e.g. MAPK, APC, JNK, PKA) are involved in folliculogenesis. These genes were represented by kinases, apoptotic and cytoskeleton remodelling factors, and enriched very important ovarian processes such as cell cycle, ephrin receptor, smoothened and phosphatidylinositol 3-kinase activity GO processes. Only 7 target genes were common between the 2 groups, 2 of which were important in ovarian functionality (CDKN1A and ITGA5). Interestingly, overexpressed miRNAs in both groups regulate several genes involved in processes apparently not related to folliculogenesis. Finally, regulation can be exerted also by low levels of specific miRNAs such as miR-320, which was reduced in L group and is known to be associated with premature ovarian senescence in women and decreased developmental potential of mouse oocytes. Our results indicate that the different oocyte quality is associated with a different miRNA blueprint, which may alter the expression of several genes relevant for intra- and extra-ovarian processes. Further studies will be necessary to determine if FF miRNAs can act outside the ovary and if their levels can be detected in the bloodstream thereby becoming possible noninvasive, real-time markers to determine oocyte quality in living animals. This study was supported by FP7-KBBE-2012-FECUND-312097.

Survival and Diversity of Human Homologous Dietary MicroRNAs in Conventionally Cooked Top Sirloin and Dried Bovine Tissue Extracts

Dietary microRNAs (miRNAs), notably those found in milk, are currently being investigated for their potential to elicit biological effects via canonical binding to human messenger RNA targets once ingested. Besides milk, beef and other bovine tissue-derived ingredients could also be a relevant source of potentially bioactive dietary miRNAs. In this study, we characterized the human homologous miRNA profiles in food-grade, bovine-sourced sirloin, heart and adrenal tissue (raw, cooked, and pasteurized, freeze-dried extracts) via deep-sequencing and quantitative reverse transcription PCR (RT-qPCR). A total of 198 human homologous miRNAs were detected at 10 or more normalized reads in all replicates (n = 3) of at least one preparation method. Tissue origin rather than preparation method was the major differentiating factor of miRNA profiles, and adrenal-based miRNA profiles were the most distinct. The ten most prevalent miRNAs in each tissue represented 71–93% of the total normalized counts for all annotated miRNAs. In cooked sirloin, the most abundant miRNAs were miR-10b-5p, (48.8% of total annotated miRNA reads) along with the muscle-specific miR-1 (24.1%) and miR-206 (4.8%). In dried heart extracts, miR-1 (17.0%), miR-100-5p (16.1%) and miR-99a-5p (11.0%) gave the highest normalized read counts. In dried adrenal extracts, miR-10b-5p (71.2%) was the most prominent followed by miR-143-3p (7.1%) and 146b-5p (3.7%). Sequencing results for five detected and two undetected miRNAs were successfully validated by RT-qPCR. We conclude that edible, bovine tissues contain unique profiles of human homologous dietary miRNAs that survive heat-based preparation methods.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

High throughput sequencing of small RNA component of leaves and inflorescence revealed conserved and novel miRNAs as well as phasiRNA loci in chickpea

Among legumes, chickpea (Cicer arietinum L.) is the second most important crop after soybean. MicroRNAs (miRNAs) play important roles by regulating target gene expression important for plant development and tolerance to stress conditions. Additionally, recently discovered phased siRNAs (phasiRNAs), a new class of small RNAs, are abundantly produced in legumes. Nevertheless, little is known about these regulatory molecules in chickpea. The small RNA population was sequenced from leaves and flowers of chickpea to identify conserved and novel miRNAs as well as phasiRNAs/phasiRNA loci. Bioinformatics analysis revealed 157 miRNA loci for the 96 highly conserved and known miRNA homologs belonging to 38 miRNA families in chickpea. Furthermore, 20 novel miRNAs belonging to 17 miRNA families were identified. Sequence analysis revealed approximately 60 phasiRNA loci. Potential target genes likely to be regulated by these miRNAs were predicted and some were confirmed by modified 5′ RACE assay. Predicted targets are mostly transcription factors that might be important for developmental processes, and others include superoxide dismutases, plantacyanin, laccases and F-box proteins that could participate in stress responses and protein degradation. Overall, this study provides an inventory of miRNA–target gene interactions for chickpea, useful for the comparative analysis of small RNAs among legumes.

Discriminative identification of miRNA let-7 family members with high specificity and sensitivity using rolling circle amplification.

Rolling circle amplification (RCA) is a new method based on virus DNA reproduction, which has been widely used in the field of miRNA detection. However, discrimination of highly homologous miRNAs is a bottleneck in the research of miRNA. In this study, the RCA process was creatively used to conduct the discrimination of miRNAs. Results showed that T4 RNA ligase 2 could reach the highest circularization efficiency during the RCA process with higher specificity. By using RCA technology, a member of highly homologous miRNAs, let-7, could be discriminated at the amount of 2.5 fmol. This sensitivity could not be achieved by using traditional reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. In addition, detection of miRNAs by using RCA could reach the amount limit of fmol with a good linearity. Optimal RCA technology used in this study is better than RT-qPCR in discriminating highly homologous family miRNAs. Results from this study can promote the applications of RCA in clinical diagnosis, environment protection, health care, disease inspection and prevention, and national security.

In silico screening of alleged miRNAs associated with cell competition: an emerging cellular event in cancer

Cell competition is identified as a crucial phenomenon for cancer and organ development. There is a possibility that microRNAs (miRNAs) may play an important role in the regulation of expression of genes involved in cell competition. In silico screening of miRNAs is an effort to abridge, economize and expedite the experimental approaches to identification of potential miRNAs involved in cell competition, as no study has reported involvement of miRNAs in cell competition to date. In this study, we used multiple screening steps as follows: (i) selection of cell competition related genes of Drosophila through a literature survey; (ii) homology study of selected cell competition related genes; (iii) identification of miRNAs that target conserved cell competition-related genes through prediction tools; (iv) sequence conservation analysis of identified miRNAs with human genome; (v) identification of conserved cell competition miRNAs using their expression profiles and exploration of roles of their homologous human miRNAs. This study led to the identification of nine potential cell competition miRNAs in the Drosophila genome. Importantly, eighteen human homologs of these nine potential Drosophila miRNAs are well reported for their involvement in different types of cancers. This confirms their probable involvement in cell competition as well, because cell competition is well justified for its involvement in cancer initiation and maintenance.

A genome-wide screen for non-template nucleotides and isomiR repertoires in miRNAs indicates dynamic and versatile microRNAome.

miRNA variants (termed isomiRs) have been reported as potential functional molecules that may affect miRNA stability or target selection. The aims of the present study were to comprehensively survey and characterize non-template nucleotides (NTNs) and isomiR repertoires in miRNAs. Over 50 % of the NTNs were located in the 3' ends (also termed 3' additions), followed by the 5' ends and adjacent positions to 5' and 3' ends. The similar distributions of NTNs and isomiR repertoires might be detected between homologous or clustered miRNAs. miRNA might be stably expressed based on the typical analysis, but its isomiRs might be strongly up- or down-regulated. IsomiRs with novel seed sequences were mainly derived from "seed shifting" events in 5' isomiRs, NTNs in 5' ends or in seed sequences. IsomiRs from a miRNA locus or homologous miRNA loci maybe have the same seed sequences, but they would have various enrichment levels and 3' ends. Interestingly, isomiR species with novel seed sequences via NTNs in the seed region were always stably expressed. These novel seed sequences could lead to novel functional roles through driving the potential novel target mRNAs. Integrated predicted target mRNAs and further microarray validation showed that these isomiRs have versatile biological roles. Collectively, multiple isomiR products and miRNA maturation processes provide opportunities to perform versatile roles in the regulatory network, which further enriches and complicates the regulation of biological processes.