microRNA sequencing

MicroRNas initially discovered in C.elegans , are newly identified class of non-protein-coding small (~20nt) RNA that widely exist in animals, plants and in some viruses. Large number of microRNA  have been report in various plants species (Llave et al., 2002b; Park et al., 2002; Reinhart et al.,2002; Palatnik et al., 2003; Bonnet et al., 2004; Floyd and Bowman, 2004; Jones-Rhoades and Bartel, 2004; Sunkar and Zhu, 2004; Wang et al., 2004a, 2004b; Adai et al., 2005; Bedell et al., 2005). There are mounting evidence demonstrating important role of microRNA in various plant biological process, including tissue identity, developmental timing and response to environmental stress . In contrast to animal, the preferred mechanism of action of microRNA in plants is cleavage of target mRNAs by the RNA-induced silencing complex (RISC), guided by the miRNANA (Bartel 2004). Plant microRNA have also been reported to act by repressing translation or by inducing methylation of DNA (Bao et al 2004). Repression of the target transcript by miRNANAs may occur through translational inhibition, accelerated exonucleolytic mRNA decay or slicing within miRNANA-mRNA base pairing (Eulalio et al 2008). Most characterized eukaryotic MIRNA genes are RNA polymerase II transcription units that yield a primary miRNANA transcript called a pri-miRNANA (Lee et al 2004). The pri-miRNANA typically forms an imperfect fold-back structure, which is processed into a stem loop precursor (Kim 2005). This precursor molecule is than cleaved by Dicer-like 1 protein resulting in a miRNA: miRNA* complex which after tansport to cytoplasm separates into the miRNA and miRNA* unit. One strand (miRNA) serve as guide for the  RNA-induced silencing complex  (RISC), which cleave the RNA of target genes at the paired region (Llave et al 2002).

Until recently, most experimental miRNA isolation studies involved cloning and capillary sequencing. Though, the concatemerization of sRNA clones, followed by cloning and cDNA isolation from bacteria before sequencing makes this approach laborious and costly (Barakat et al 2007).  However, lot of researcher used this approach to identify miRNANA in different plant species. Shanfa Lu et al 2005, reported novel and mechanical stress-reponsive microRNA in Populus trichocarpa using such an approach. Similarly using the cloning approach, Sunkar and Zhu (2004) identified a significant number of miRNA from Arabidopsis grown under abiotic stress conditions. However, fact that most of the miRNANA identified using this approach are highly expressed or tissure or development specific, shifted identification efforts towards computational analysis which have resulted in a considerable larger number of identified Arabidopsis miRNA (Bonnet et al 2004, Wang er al 2004, Adai et al 2005). Several investigators have  reported identification of novel as well as conserved miRNA using computational approach in recent past (Zhang et al 2005;2006; 2006b; 2007; 2008; Pan et al 2007; Wang et al 2005; Sunkar and Jagadeeswaran 2008). However this method is limited by the number of nucleotide sequences available in the database. Recently introduced high through put sequencing technology provided better alternative (Abdelali et al 2007) as it generates millions of bases per run and has been used successfully for sequencing the genomes of bacteria (Goldberg et al 2006), chloroplast (Moore et al 2006) and mitochondria( Poinar et al 2006) as well as for transcriptiome analysis (Weber et al 2007). Recently Zhao et al 2010, utilized deep sequencing to identified novel and conserved microRNA in peanut, earlier Rajagopalan et al 2006, Fahlgren et al 2007 and Barakat et al 2007 used ultrahigh throughput sequencing technology for small RNA sequencing in Arabidopsis and basal eudicot Eschscholzia californica.  In these studies, the number of microRNA identified in Arabidopsis doubled the number previously discovered in total of over 30 studies using capillary sequencing. The greater efficiency to detect variants that are expressed at low levels derives from the much deeper coverage of the sRNA and avoidance of cloning, makes deep sequencing technology favorable approach for microRNA discovery, specially in species where genome is not completely sequenced yet.