Research article Identification of micro-RNAs in cotton Muhammad Younas Khan Barozai a,b, * , Muhammad Irfan b , Rizwan Yousaf b , Imran Ali b , Uzma Qaisar b , Asma Maqbool b , Muzna Zahoor b , Bushra Rashid b , Tayyab Hussnain b , Sheikh Riazuddin b a Botany Department, University of Baluchistan, Quetta, Pakistan b Centre of Excellence in Molecular Biology, 87 West Canal Bank Road, Thoker Niaz Baig, Lahore, Pakistan Received 28 April 2008 Available online 28 May 2008 Abstract The plant genome has conserved small non-coding microRNAs (miRNAs) genes about 20e24 nucleotides long. They play a vital role in the gene regulation at various stages of plant life. Their conserved nature among the various organisms not only suggests their early evolution in eukaryotes but also makes them a good source of new miRNA discovery by homology search using bioinformatics tools. A systematic search approach was used for interspecies orthologues of miRNA precursors, from known sequences of Gossypium in GenBank. The study resulted in 22 miRNAs belonging to 13 families. We found 7 miRNA families (miR160, 164, 827, 829, 836, 845 and 865) for the first time in cotton. All 22 miRNA precursors form stable minimum free energy (mfe) stem loop structure as their orthologues form in Arabidopsis and the mature miRNAs reside in the stem portion of the stem loop structure. Fifteen miRNAs belong to the world’s most commercial fiber producing upland cotton (Gossypium hirsutum), five are from Gossypium raimondii and one each is from Gossypium herbaceum and Gossypium arboreum. Their targets consist of transcription factors, cell division regulating proteins and virus response gene. The discovery of 22 miRNAs will be helpful in future for detection of precise function of each miRNA at a particular stage in life cycle of cotton. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Cotton; Micro RNAs; Post-transcriptional gene silencing; Homology search 1. Introduction Micro RNA genes that encode miRNAs reside in a specific genomic region. The non-coding RNA such as miRNA, transfer RNA (tRNA) and others constitute 3% out of the total 5% of the functional genome [1]. miRNAs are endogenous, non-coding, small RNAs about 20e24 nucleotides long [2] and are conserved in plants and animals [3,4]. They have a cru- cial role in post-transcriptional gene regulation [5,6]. Mature miRNAs are produced by a chain of reaction with the help of enzymes [7]. Primary transcripts of mature miRNAs (pri- miRNAs) fold into a stable stem loop structure forming miRNA precursor (pre-miRNA). The loop of pre-miRNA is cleaved, producing a short double-stranded RNA (dsRNA); a single strand of the dsRNA acts as mature miRNA [8]. The processing occurs in nucleus and is processed by a special RNaseIII-like endonuclease, Drosha and Dicer in animals [9] and Dicer-like enzyme (DCL) in plants [10], that also predom- inantly incorporate the mature miRNA into the RNA induced silencing complex (RISC) [8]. The RISC complex negatively regulates gene expression either by inhibiting translation Abbreviations: ath, Arabidopsis thaliana; BLAST, basic local alignment search tool; ch_ratio, core hairpin ratio; DCL, dicer-like enzyme; dsRNA, dou- ble-stranded RNA; ESTs, expressed sequence tags; ghr, Gossypium hirsutum; mRNA, messenger RNA; MIR, micro-RNA; miRNAs, microRNAs; mfe, min- imum free energy; pre-miRNAs, miRNAs precursor; NCBI, National Center for Biotechnology Information; osa, Oryza sativa; Ptc, Populus trichocarpa; Ptn, position of terminal nucleotide; Pfn, position of the first nucleotide; pri- miRNAs, primary transcripts of mature miRNAs; rRNA, ribosomal RNA; RISC, RNA induced silencing complex; tRNA, transfer RNA; UTRs, untrans- lated regions. * Corresponding author. Centre of Excellence in Molecular Biology, 87 West Canal Bank Road, Thoker Niaz Baig, Lahore, Pakistan. Tel.: þ92 (0)33 781 7319. E-mail address: barozaikhan@gmail.com (M.Y. Khan Barozai). 0981-9428/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2008.05.009 Available online at www.sciencedirect.com Plant Physiology and Biochemistry 46 (2008) 739e751 www.elsevier.com/locate/plaphy