Figure 1 Enlarge Image Figure1: This pre-miRNA hairpin will be processed to release a mature miRNA (shaded region).

Gene regulation by microRNAs

A trove of small RNAs

A surprise of recent years was the revelation that small RNAs preside over a previously hidden universe of negative gene regulation. Of particular interest was the discovery that eukaryotic cells process RNAs with double-stranded character into 21-22 nucleotide regulatory RNAs. In the RNA interference (RNAi) pathway, exogenous double-stranded RNA is converted into 21-22 nt small interfering (siRNAs), which guide the cleavage and destruction of perfectly complementary transcripts. In a related pathway, endogenous transcripts containing hairpin structures are processed into microRNAs (miRNAs) (Figure 1), which negatively regulate host transcripts by cleavage or by translational inhibition. Although only recognized as a gene family since late 2001, miRNAs proved to constitute one of the most abundant gene classes in plants and animals.

Control of development and physiology by microRNAs

Figure 2 Enlarge Image Figure 2. (Left) The 3’ UTR of the Notch target gene E(spl)m contains two miR-4 binding sites, as defined by 7 nt of Watson-Crick pairing to the 5’ end of the miRNA. (Right) A GFP reporter fused to the E(spl)ma 3’ UTR (in green) is strongly repressed in miR-4 expressing cells (in red).

Many targets of plant miRNAs were easily identified as transcripts with near-perfect or even perfect complementarity to miRNAs. In contrast, the known regulatory targets of animal miRNAs generally exhibit only limited complementarity to miRNAs, and have been correspondingly more difficult to identify. Nevertheless, genetic studies of the first few animal miRNAs have uncovered dramatic mutant phenotypes and biological activities for a few miRNAs. For example, the founding miRNAs lin-4 and let-7 control developmental transitions in worms, the worm miRNAs lsy-6 and miR-273 regulate neuronal cell fates, the fly miRNAs bantam and miR-14 collectively regulate apoptosis, growth and fat metabolism. Therefore, it is widely presumed that miRNAs as a class will prove to play diverse and important roles in developmental patterning and adult physiology.

Genomewide studies of miRNAs in Drosophila

In previous studies, we established insights into miRNA gene identification and miRNA target discovery. First, we developed computational methods to recognize miRNA genes as conserved hairpin structures that display appropriate patterns of nucleotide divergence, and used this to identify most of the miRNAs of Drosophila. We also demonstrated that two large classes of Notch target genes constitute one of the largest known sets of biologically relevant miRNA targets (Figure 2). This work helped define the critical determinant of miRNA:target interaction, namely by conserved ~7 nucleotide 3' UTR sequence motifs with Watson-Crick basepairing to the 5' end of the miRNA.

Figure 3 Enlarge Image Figure 3. Each mutant wing misexpresses a different miRNA, that alters wing morphology in a distinct manner.

We are continuing our work with miRNAs by exhaustively cataloguing the miRNAs of Drosophila and other insects using both computational and experimental methods. More than a genome annotation effort, we will also use this knowledge to investigate the evolution of miRNA genes. However, the primary focus will be to understand the biological functions of miRNAs. To this end, we have also created a library of transgenic flies that permits the targeted misexpression of most known Drosophila miRNA genes. These induce a wide variety of specific phenotypes (Figure 3), which may give clues as to their normal functions. We are also conducting genetic screens to identify other loci that are generally important for miRNA biogenesis and function, or alternatively, that are required specifically for the activity of individual miRNAs.