microRNA sequencing

microRNA sequencing

MicroRNA 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. There are mounting evidence demonstrating important role of micro-RNA 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) . Plant microRNA have also been reported to act by repressing translation or by inducing methylation of DNA. Repression of the target transcript by miRNA may occur through translational inhibition, accelerated exonucleolytic mRNA decay or slicing within miRNA-mRNA base pairing). RNA polymerase II transcription units yield a primary miRNA transcript called a pri-miRNA. The pri-miRNA typically forms an imperfect fold-back structure, which is processed into a stem loop precursor . This precursor molecule is than cleaved by Dicer-like 1 protein resulting in a miRNA: miRNA* complex which after transport 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 .

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.  Several investigators have  reported identification of novel as well as conserved miRNA using computational approach in recent past . 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  as it generates millions of bases per run . 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.