MicroRNAs (miRNAs) are derived from endogenous genes that are initially transcribed as longer RNA transcripts (Lee et al. 1993) (Wightman et al. 1993) (Reinhart et al. 2000) (Lagos-Quintana et al. 2001) (Lau et al. 2001) (Lee and Ambros 2001) (Mourelatos et al. 2002) (reviewed in (Nelson et al. 2003), (Bartel 2004)). In mammals, the primary miRNA transcripts (pri-miRNAs) are processed by the nuclease Drosha (Lee et al. 2003) into ˜70 nt precursor miRNAs (pre-miRNAs) that are exported by exportin-5 to the cytoplasm (Yi et al. 2003; Lund et al. 2004) (Bohnsack et al. 2004). The Dicer nuclease excises the mature miRNAs from pre-miRNAs (Hutvagner et al. 2001) (Ketting et al. 2001) (Knight and Bass 2001) (Grishok et at. 2001). miRNAs are bound to proteins that belong to the Argonaute family and, in humans, may also assemble with other proteins, including the Gemin3 and Gemin4 proteins, to form micro-Ribonucleoprotein complexes (miRNPs) (Mourelatos et al. 2002) (Nelson et al. 2004). Dicer also processes another class of ˜22 nt RNAs termed short interfering RNAs (siRNAs) (Elbashir et al. 200)) (Hamilton and Baulcombe 1999) from double stranded RNAs (Bernstein et al. 2001). Analogous to miRNAs, siRNAs are bound to Argonaute proteins (Hammond et al. 2001) (Martinez et al. 2002) and may also assemble with additional proteins to form RNA-induced Silencing Complexes (RISCs) (Hammond et al. 2001). siRNAs and miRNAs (and RISCs and miRNPs) are functionally equivalent and the main difference between the two classes of small RNAs are the fact that miRNAs are derived from endogenous genes (Ambros et al. 2003a).
Many miRNAs and siRNAs function by base pairing with miRNA-recognition elements (MREs) found in their mRNA targets and direct either target RNA endonucleolytic cleavage (Elbashir et al. 2001) (Hutvagner and Zamore 2002) or translational repression (Olsen and Ambros 1999; Seggerson et al. 2002; Zeng et al. 2002; Doench et al. 2003). The manner by which a miRNA or siRNA base pairs with its mRNA target correlates with its function: if the complementarity between a miRNA and its target is extensive, the RNA target is cleaved (Hutvagner and Zamore 2002) (Rhoades et al. 2002) (Llave et al. 2002) (Tang et al. 2003) (Xie et al. 2003); if the complementarity is partial, the stability of the target mRNA in not affected but its translation is repressed (Olsen and Ambros 1999; Seggerson et al. 2002; Zeng et al. 2002; Doench et al. 2003). However, how general this correlation is and the factors and mechanisms that determine the function of any given miRNA are unknown.
In plants, the computational identification of miRNA targets was facilitated by the extensive complementarity between plant miRNAs and their mRNA targets (Llave et al. 2002). (Rhoades et al. 2002). Plant miRNA targets have been verified experimentally (Llave et al. 2002) (Xie et al. 2003) (Kasschau et al. 2003) (Palatnik et al. 2003) (Aukerman and Sakai 2003) (Chen 2004) reviewed in (Bartel and Bartel 2003). Two mouse miRNAs (miR-127 and miR-136) show perfect antisense complementarity with the coding region of a retrotransposon-like gene (Rtl1) (Seitz et al. 2003). However, most animal miRNAs are thought to recognize their mRNA targets via partial antisense complementarity (Lee et al. 1993) (Wightman et al. 1993) (Moss et al. 1997) (Reinhart et al. 2000) (Olsen and Ambros 1999; Zeng et al. 2002; Doench et al. 2003). Because of this partial complementarity, simple homology-based searches have failed to uncover targets for miRNAs in organisms other than plants (Bartel and Bartel 2003) (Ambros et al. 2003b). Animal miRNA targets were initially identified in genetic screens. In particular, genetic dissection of the heterochronic gene pathway in C. elegans identified the lin-14 and lin-28 mRNAs as targets for the lin-4 miRNA (Lee et al. 1993) (Wightman et al. 1993) (Moss et al. 1997), and the lin-41 mRNA as a target for the let-7 miRNA (Reinhart et al. 2000). In Drosophila, the bantam miRNA regulates the pro-apoptotic gene hid (Brennecke et al. 2003). Importantly, these and other studies demonstrated that MRE sequences are necessary and sufficient to confer miRNA-dependent gene expression regulation in MRE-bearing target mRNAs (Moss et al. 1997) (Reinhart et al. 2000) (Zeng et a). 2002) (Doench et al. 2003) (Vella et al. 2004). Putative targets for other miRNAs have been proposed (Lai 2002; Abrahante J E 2003; Lin S Y 2003; Xu et al. 2003), but these are predominantly based on visual inspection of putative mRNA targets for partial complementarity with miRNAs and lack experimental verification of specific miRNA:MRE interactions.
Very recently, carefully designed bioinformatic approaches have been used to predict mRNA targets for Drosophila (Stark et al. 2003) (Enright et al. 2003) and mammalian miRNAs (Lewis et al. 2003). In particular, Bartel, Burge and colleagues have presented a robust bioinformatics strategy that allows prediction of conserved, mammalian miRNA targets along with accurate estimates of false positive rates (at 31% for miRNA targets identified in human mouse and rat and 22% for targets identified in mammals and in pufferfish) and experimental validation of 11 (out of 15 tested) predicted targets (Lewis et al. 2003). Most of the targets identified by Lewis et al. contain multiple MREs for the same miRNA or are regulated by more that one miRNA. The targets reported for Drosophila miRNAs also contain, for the most part, multiple MREs (Stark et al. 2003) (Enright et al. 2003). However, the rules guiding single miRNA:MRE (target mRNA) interactions have not been investigated and as a result predictions of miRNA targets containing single MREs are lacking.
There remains a need for an algorithm providing rules that guide single miRNA:MRE (target mRNA) recognition. There remains a need for methods, systems and computer programs which use the rules to identify MREs.