Computational protocol: Role of bioinformatics in establishing microRNAs as modulators of abiotic stress responses: the new revolution

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[…] The detection and validation of miRs by molecular cloning was supported by systematic approaches using computational techniques (Bonnet et al., ). These approaches also complemented the experimental methods by identifying difficult to clone miR families such as miR395 and miR399 (Jones-Rhoades and Bartel, ; Adai et al., ) which were difficult to detect by experimental approaches due to their low expression levels. Computational predictions strategies have been quite useful in miR identification in various plant species such as Arabidopsis (Wang et al., ; Adai et al., ; Li et al., ), rice (Li et al., ; Zhang et al., ), maize (Zhang et al., , ), tomato (Yin et al., ; Zhang et al., ), foxtail millet (Khan et al., ), soybean (Zhang et al., ), Brassica napus (Xie et al., ), apple (Gleave et al., ), grape (Carra et al., ), and some other plants (Zhang et al., ; Sunkar and Jagadeeswaran, ).It had been verified that a majority of known miRs are evolutionarily conserved and are expected to have homologs or orthologs in other species. So search criteria allowed up-to three sequence mismatches while looking for conserved miRs in heterologous species. Using this approach 85 conserved sequences which were showing perfect match to miRs reported in miRBase (Release 19) were predicted from Morus notabilis tissues (Jia et al., ). Whereas in another study 35 miR families were identified in heat stressed Brassica napus by allowing two mismatches with A. thaliana miRs (Yu et al., ). Thus, the conserved sequence of plant miRs and other structural features were used for developing suitable strategies and rules for identifying and annotating (Discussed in Section The Influence of Bioinformatics Approaches on microRNA Nomenclature and Annotation) new miR genes (Lagos-Quintana et al., ; Reinhart et al., ; Floyd and Bowman, ; Wang et al., ; Adai et al., ; Zhang et al., ; Lukasik et al., ). One of the early comprehensive computational analysis by Jones-Rhoades and Bartel () systematically identified plant miRs and their regulatory targets that are conserved between Arabidopsis and rice. Using MIRcheck algorithm they predicted that the miRs could target mRNAs like superoxide dismutases (SOD), laccases, and ATP sulfurylases that are involved in plant stress responses. Such studies lead to identification of involvement of Ath-miR398 in the ROS pathway by targeting sites on Cu/Zn-SOD (Jones-Rhoades and Bartel, ; Sunkar and Zhu, ; Lu et al., ; Sunkar et al., ) A similar approach was used in miRFinder computational pipeline, to identify 91 conserved plant miRs in rice and Arabidopsis (Bonnet et al., ).Another strategy was based on the property of miRs to bind with perfect complementarity to their target transcripts (Laufs et al., ). In plant species where the target sequence was available the conserved miRs could be easily predicted by using 20 mer genomic segments with not more than two mismatches as in silico probes. This target-guided strategy was adopted to identify 16 families of drought stress-associated miRs from Physcomitrella patens (Wan et al., ).The computational predictions also utilized the criteria for conservation of miR sequence and key secondary structure features of pre-miRs like their characteristic fold-back structure, thermodynamic stability etc. to predict new miRs (Berezikov et al., ). Seventy-nine putative miRs were identified in wheat using traditional computational strategy, out of which 9 were validated by northern blot experiments (Jin et al., ). Subsequently bioinformatics tools like miRAlign were developed based on the requirement of structural similarity and sequence conservation between new candidates and experimentally identified miRs (Wang et al., ). Though numerous miR profiles were generated by the computational algorithms, this was not found to be appropriate for species with less annotated genomes (Chen and Xiong, ).The non-availability of complete genome annotation was overcome by employing the Expressed Sequence Tags (EST) database. These represented the true gene expression entities so they emerged as better indicators of dynamic expressions of the miR. A detailed study by identified 123 miRs from stress-induced ESTs of 60 plant species (Zhang et al., ). This study confirmed that irrespective of evolutionary divergence miRs are highly conserved in plant kingdom and miR genes may exist as orthologs or homologs in different species within the same kingdom (Weber, ; Zhang et al., ). The EST database was also used to confirm some novel miRs identified earlier by computational strategies in citrus (Song et al., ) and peach (Zhang et al., ). In a recent study ESTs of abiotic stress treated libraries of Triticum aestivum were used to identify novel miRs in drought, cold, and salt stressed cDNA libraries by searching all mature sequences deposited in the miRBase (Release 19) (Pandey et al., ). […]

Pipeline specifications

Software tools MIRcheck, MiRFinder, MiRAlign
Databases miRBase
Application Genome annotation