Computational analysis of microRNA targets in Caenorhabditis elegans Yuka Watanabe a,b , Nozomu Yachie a,c , Koji Numata a,c , Rintaro Saito a , Akio Kanai a, ⁎ , Masaru Tomita a,b a Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, 997-0017, Japan b Department of Environmental Information, Keio University, Fujisawa, 252-8520, Japan c Bioinformatics Program, Graduate School of Media and Governance, Keio University, Fujisawa, 252-8520, Japan Received 28 March 2005; received in revised form 4 August 2005; accepted 27 September 2005 Available online 13 December 2005 Abstract MicroRNAs (miRNAs) are endogenous ∼ 22-nucleotide (nt) non-coding RNAs that post-transcriptionally regulate the expression of target genes via hybridization to target mRNA. Using known pairs of miRNA and target mRNA in Caenorhabditis elegans, we first performed computational analysis for specific hybridization patterns between these two RNAs. We counted the numbers of perfectly complementary dinucleotide sequences and calculated the free energy within complementary base pairs of each dinucleotide, observed by sliding a 2-nt window along all nucleotides of the miRNA-mRNA duplex. We confirmed not only strong base pairing within the 5′ region of miRNAs (nts 1–8) in C. elegans, but also the required mismatch within the central region (nt 9 or nt 10), and we found weak binding within the 3′ region (nts 13–14). We also predicted 687 possible miRNA target transcripts, many of which are thought to be involved in C. elegans development, by combining the abovementioned hybridization tendency with the following analyses: (1) prediction of the miRNA-mRNA duplex with free-energy minimization; (2) identification of the complementary pattern within the miRNA–mRNA duplex; (3) conservation of target sites between C. elegans and C. briggsae, a related soil nematode; and (4) extraction of mRNA candidates with multiple target sites. Rigorous tests using shuffled miRNA controls supported these predictions. Our results suggest that miRNAs recognize their target mRNAs by their hybridization pattern and that many target mRNAs may be regulated through a combination of several specific miRNA target sites in C. elegans. © 2005 Elsevier B.V. All rights reserved. Keywords: Bioinformatics; Nematode; C. briggsae; MicroRNA targets; MiRNA–mRNA duplex 1. Introduction Noncoding RNA (ncRNA) genes produce various kinds of functional RNA molecules. The short sequences known as microRNAs (miRNAs), a family of ncRNAs, imperfectly hybridize to target mRNAs and regulate their messages (Bar- tel, 2004; He and Hannon, 2004). Although the first miRNA was identified not long ago, experimental and computational approaches have now found more than 1000 different miR- NAs. Many endogenously encoded miRNAs have been cloned from a number of species (Lau et al., 2001; Lee and Ambros, 2001; Reinhart et al., 2002; Ambros et al., 2003; Lim et al., 2003), and a combination of computational approaches has yielded even more (Ambros et al., 2003; Lai et al., 2003; Lim et al., 2003). MiRNA sequences are archived and accessible at the Rfam miRNA registry website (Griffiths- Jones, 2004). The database contains a total of 1420 miRNAs from 13 species, including Homo sapiens, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana, and even the Epstein-Barr virus. However, experimental studies have touched on only a handful of functions (Lee et al., 1993; Wightman et al., 1993; Moss et al., 1997; Reinhart et al., 2000; Brennecke et al., 2003; Johnston and Hobert, 2003) of miRNAs such as lin-4 (Lee et al., 1993) and let-7 (Reinhart et al., 2000), well known as the founding members of the miRNA family and having central roles as key regulators of developmental timing through cell fate decisions. It was found recently that some miRNAs regulate a large number of mes- sages (Lim et al., 2005), supporting the proposition that miRNAs work as key participants in gene regulatory networks (Ke et al., 2003; John et al., 2004). Gene 365 (2006) 2 – 10 www.elsevier.com/locate/gene Abbreviations: miRNA, microRNA; C. elegans, Caenorhabditis elegans; C. briggsae, Caenorhabditis briggsae; UTR, untranslated region(s). ⁎ Corresponding author. Tel.: +81 235 29 0524; fax: +81 235 29 0525. E-mail address: akio@sfc.keio.ac.jp (A. Kanai). 0378-1119/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.09.035