Tiny Yet Powerful

microRNA (miRNA) are one of a group of small, noncoding, regulatory RNA; approximately 22 nucleotides (nt) in length, they regulate posttranscriptional gene expression by repressing or breaking down messenger RNA (mRNA). They occur in plants, invertebrates, and vertebrates, including humans. In a commentary in Cell last year, Gary Ruvkun, Bruce Wightman, and Ilho Ha described the technical roadblocks they and colleagues have faced along a 20-some year journey of identifying miRNA and determining their function. Overtime, the discovery and development of molecular techniques have revealed increasing numbers of miRNA families and are allowing researchers to identify their target genes, as well as the mechanism by which they are processed from larger precursor transcripts to their tiny, functional selves.

Small Worms, Abundant Information

The existence of a detailed analysis of Caenorhabditis elegans cell lineage and developmental genetics, along with a collection of mutants, including some that caused developmental timing defects, played a role in identifying the first miRNA. Victor Ambros was working on lin-4, a negative regulator of developmental timing, in Robert Horvitz’ laboratory, while Gary Ruvkun was working on lin-14, a heterochronic gene, which temporally regulated the fate of cells during larval development. lin-4 encoded an RNA but did not appear to have an open reading frame. lin-14 looked like a protein-coding sequence (in fact, it encodes a novel transcription factor) with a long 3′ untranslated region. They eventually discovered that lin-4 RNA was complementary to a region in the lin-14 3′ region, which suggested that lin-4 might be binding to lin-14. lin-4, the first known miRNA, progressively represses the translation of lin-14 mRNA, the first miRNA target. Subsequently, Frank Slack, Associate Professor, Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT, joined Ruvkun's lab and worked on let-7, which was the second miRNA and which, like lin-4, is involved in developmental timing. Although in the same pathway as lin-4 and lin-14, conservation of the let-7 sequence in other organisms, including fruit flies and other invertebrates and humans, suggested that this system had wide-reaching importance beyond C. elegans.

In animals, there may be sequence differences of seven to eight or more nucleotides between an miRNA and its targets, making target prediction difficult. In addition, a single miRNA could have several to hundreds of different targets. Slack says there may be 120 to 500 miRNA in C. elegans, “depending on who you believe. A lot is known about let-7, and there are validated targets in C. elegans.” Slack suggests that looking for conserved regions can allow one to design rules to identify new targets using a bioinformatics approach, “although not every miRNA works the same.” The rules can be validated in C. elegans before the human or other homologues are examined. C. elegans shares 60% of genes with humans, and there are at least 30 to 40 C. elegans miRNA conserved in other organisms that have been around since the last common ancestor of C. elegans and the mouse.

Looking at Trees

miRNA in plants differs from those in other classes of organisms in key ways. Vincent Chiang, Professor, Department of Forestry, North Carolina State University, Raleigh, points out that although in animal systems one may detect both the precursor transcript and the mature miRNA, in plants, for reasons that are unknown, the precursor is transient, and only the mature 21-to 24-nt-long miRNA is detectable. Also, in plants, miRNA have almost perfect complementarity to their targets, so it is easier to predict targets through a computational approach. Chang notes “hybridization methods (e.g., microarrays and Northern blots) detect collective expression of an miRNA family, but you can't tell which family member contributes the most to expression.” He and colleagues have developed a real-time PCR technique that detects mature miRNA in plants. “In principle, it is simple: add a poly(A) tail to make small sequences longer, then treat the longer molecule as usual in a quantitative PCR assay. Our technique varies the annealing temperature of the PCR, so it can distinguish miRNA differing by only one nucleotide in the sequence. It is very useful to determine quantitative expression of individual miRNA and can differentiate the most expressed family member. I think this is a very important step in the linear path from discovery to functional analysis,” he concludes.