The Functions of MicroRNAs
In the past three years, several hundred novel genes encoding transcripts containing short double-stranded RNA hairpins, named microRNAs (miRNAs), were identified in plants and animals (Lee et al. 1993; Reinhart et al. 2000, 2002; Lagos-Quintana et al. 2001, 2002, 2003; Lau et al. 2001; Lee and Ambros 2001; Llave et al. 2002a; Mette et al. 2002; Mourelatos et al. 2002; Park et al. 2002; Ambros et al. 2003b; Aravin et al. 2003; Brennecke et al. 2003; Dostie et al. 2003; Grad et al. 2003; Houbaviy et al. 2003; Lai et al. 2003; Lim et al. 2003a, 2003b; Palatnik et al. 2003). More recently, miRNAs have also been identified in a large DNA virus, the Epstein Barr virus, and are likely to be found in other viruses (Pfeffer et al. 2004). The cellular functions of most animal miRNAs are unknown.
More than ten years after the discovery of the first miRNA gene, lin-4 (Chalfie et al. 1981; Lee et al. 1993), we know that miRNA genes constitute about 1%–2% of the known genes in eukaryotes. Investigation of miRNA expression combined with genetic and molecular studies in Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana have identified the biological functions of several miRNAs (recent review, Bartel 2004). In C. elegans, lin-4 and let-7 were first discovered as key regulators of developmental timing in early larval developmental transitions (Ambros 2000; Abrahante et al. 2003; Lin et al. 2003; Vella et al. 2004). More recently lsy-6 was shown to determine the left–right asymmetry of chemoreceptor expression (Johnston and Hobert 2003). In D. melanogaster, miR-14 has a role in apoptosis and fat metabolism (Xu et al. 2003) and the bantam miRNA targets the gene hid involved in apoptosis and growth control (Brennecke et al. 2003). In mouse, miR-181a modulates hematopoietic differentiation (Chen et al. 2004), and miR-196 directs the cleavage of Hox-B8 transcripts (Yekta et al. 2004).
miRNAs have specificity. In a range of organisms, miRNAs are differentially expressed in developmental stages, cell types, and tissues (Lee and Ambros 2001; Lagos-Quintana et al. 2002; Sempere et al. 2004). In particular, differential expression has been observed in mammalian organs (Lagos-Quintana et al. 2002; Krichevsky et al. 2003; Sempere et al. 2004) and embryonic stem cells (Houbaviy et al. 2003). Estimates in worm show that there are approximately 1,000 molecules of miRNA per cell, with some cells exceeding 50,000 molecules (Lim et al. 2003b). This dynamic range of regulation of miRNA expression underscores the regulatory functional importance of miRNAs.
The Mechanism of miRNA Action
How do miRNAs pair with their target messages? miRNAs cause the translational repression or cleavage of target messages (Doench and Sharp 2004). Some miRNAs may behave like small interfering RNAs (siRNAs) that direct mRNA cleavage between the nucleotide positions 10 and 11 in the siRNA:mRNA target duplex (Tuschl et al. 1999; Zamore et al. 2000; Elbashir et al. 2001; Hutvágner and Zamore 2002a; Llave et al. 2002b; Martinez et al. 2002; Bartel 2004; Yekta et al. 2004). It appears that the extent of base pairing between the small RNA and the mRNA determines the balance between cleavage and degradation (Hutvágner and Zamore 2002a). Recent examples of cleavage of target messages are, in mouse, mir-196 guiding cleavage of Hox-B8 transcripts (Yekta et al. 2004) and, in Epstein Barr virus, miR-BART2, a virus-encoded miRNA, guiding the cleavage of transcripts for virus DNA polymerase (gene BALF5) (Pfeffer et al. 2004). While cleavage of mRNA is a straightforward process, the details of the mechanism of translational repression are unknown.
The following rules for matches between miRNA and target messages have been deduced from a range of experiments. (1) Asymmetry: experimentally verified miRNA target sites indicate that the 5′ end of the miRNA tends to have more bases complementary to the target than its 3′ end. Loopouts in either the mRNA or the miRNA between positions 9 and 14 of the miRNA have been observed or deduced (Brennecke et al. 2003; Johnston and Hobert 2003; Lin et al. 2003; Vella et al. 2004). Recent experiments show some correlation between the level of translational repression and the free energy of binding of the first eight nucleotides in the 5′ region of the miRNA (Doench and Sharp 2004). However, confirmed miRNA:mRNA target pairs can have mismatches in this region (Moss et al. 1997; Johnston and Hobert 2003). (2) G:U wobbles: wobble base pairs are less common in the 5′ end of a miRNA:mRNA duplex, and recent work shows a disproportionate penalty of G:U pairing relative to standard thermodynamic considerations (Doench and Sharp 2004). (3) Cooperativity of binding: many miRNAs can bind to one gene (Reinhart et al. 2000; Ambros 2003; Vella et al. 2004), and the target sites may overlap to some degree (Doench and Sharp 2004).
Given the overlap between the siRNA and miRNA pathways, it is reasonable to assume that rules of regulation in the siRNA pathway will partly apply to miRNA target recognition (Hutvágner and Zamore 2002b; Boutet et al. 2003; Doench et al. 2003). Lately, detailed characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the3′ terminus, lack of inverted repeats, and strand base preferences (positions 3, 10, 13, and 19) (Jackson et al. 2003; Reynolds et al. 2004). These observations may provide clues for better quantitative description of miRNA:mRNA interaction. Regions adjacent or near to the target site can be important for miRNA specificity. In lin-41, a 27-nucleotide (nt) intervening sequence between two consecutive let-7 sites is necessary for its regulation (Vella et al. 2004). Because of lack of conservation of this 27-nt intervening sequence in C. briggsae, incorporation of a corresponding rule is premature.
Maturation of miRNAs and Assembly in RNA-Induced Silencing Complex
miRNAs are transcribed as longer precursors, termed pre-miRNAs (Lee et al. 2002), sometimes in clusters and frequently in introns (25% of human miRNAs; Table S1). Upon transcription, miRNAs undergo nuclear cleavage by the RNase III endonuclease Drosha, producing the 60–70-nt stem-loop precursor miRNA (pre-miRNA) with a 5′ phosphate and a 2-nt 3′ overhang (Lee et al. 2003). The pre-miRNA is subsequently transported across the nuclear membrane, dependent on the protein exportin 5 (Lund et al. 2003; Yi et al. 2003). Dicer cleaves the pre-miRNA in the cytoplasm about two helical turns away from the ends of the pre-miRNA stem loop, producing double-stranded RNA. A helicase unwinds the cleaved double-stranded RNA in a strand-specific direction (Khvorova et al. 2003; Schwarz et al. 2003).
One of the unwound strands is subsequently incorporated into a ribonuclear particle (RNP) complex, RNA-induced silencing complex (RISC) (Hutvágner and Zamore 2002a; Martinez et al. 2002). Every RISC contains a member of the Argonaute protein family, which tightly binds the RNA in the complex (Hammond et al. 2001; Hutvágner and Zamore 2002a; Martinez et al. 2002; Mourelatos et al. 2002). There are at least eight members of the Argonaute family in mammals (Sasaki et al. 2003), and only a small subset has been functionally characterized. The Argonautes and Dicer bind single-stranded RNA via their PAZ domains (Lingel et al. 2003; Sasaki et al. 2003; Song et al. 2003; Yan et al. 2003), and the known structures of the PAZ domains may have implications for prediction of miRNA targets (Lingel et al. 2003; Song et al. 2003; Yan et al. 2003).
Association of mRNAs and miRNAs with Fragile X Mental Retardation Protein
Among the prime candidates for miRNA control are the genes that are posttranscriptionally regulated. The mRNA-binding protein fragile X mental retardation protein (FMRP) is involved in the regulation of local protein synthesis (Antar and Bassell 2003) and binds 4% of mRNAs expressed in the rat brain, as tested in vitro (Brown et al. 2001). The loss of function of FMRP causes fragile X syndrome, the most prevalent form of mental retardation (one in every 2,000 children). Over the past three years a number of different groups have identified in vivo mRNA cargoes of FMRP. The Warren and Darnell laboratories have identified ligands by co-immunoprecipitation followed by microarray analysis, complemented by extraction of polyribosomal fractions (Brown et al. 2001). They discovered that FMRP and one of its three RNA-binding domains specifically binds to G-rich quartet motifs (Brown et al. 2001; Darnell et al. 2001; Denman 2003; Miyashiro et al. 2003). Three more studies found that mRNAs containing U-rich motifs bind recombinant FMRP in vitro and associate with FMRP-containing mRNPs in vivo (Chen et al. 2003; Denman 2003). Lastly, antibody-positioned RNA amplification as a primary screen followed by traditional methods identified over 80 FMRP-regulated mRNAs, with a combination of G-quartet and U-rich motifs in their mRNA sequences (Miyashiro et al. 2003).
Independently, FMRP has been shown to be associated with RISC components and miRNAs (Jin et al. 2004). The Drosophila homolog of FMRP (FXR) and the Vasa intronic gene were identified as components of RISC (Caudy et al. 2002). More recent studies have proved that mammalian FMRP interacts with miRNAs and with the components of the miRNA pathways including Dicer and the mammalian orthologs of Argonaute (AGO) 1 (Ishizuka et al. 2002; Jin et al. 2004). Given the association of FMRP with Argonaute-containing complexes, we propose and investigate the hypothesis that the cargoes carried by FMRP are also miRNA targets, and we derive hypotheses of specific pairing interactions.
Here, we predict miRNA targets in five vertebrate genomes as a way of facilitating experiments and exploring a number of open questions. What proportion of all genes is regulated by miRNAs? How many genes are regulated by each miRNA? Are specific cellular processes targeted by specific miRNAs or by miRNAs in general? What is the extent of cooperativity in miRNA:mRNA binding?