Small but mighty timekeepers

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A tiny RNA molecule ensures that the larvae of a roundworm develop into adults. The discovery of this RNA in many other animal groups implies that this way of keeping developmental time may be universal.

How is the timing of major developmental transitions in animals genetically controlled? How, for instance, does a larva know when to transform into an adult? One possible answer was suggested by the discovery of 'heterochronic' mutations in a small, intensely studied roundworm, the nematode Caenorhabditis elegans. These mutations, in a series of genes, either advance or retard the schedule of many developmental events as the worm progresses through its larval stages1.

Some of these 'timekeeping' genes — such as lin-4, which acts between larval stages 1 and 2, and let-7, which is involved in the transition from larval stage 4 to adult — work in a rather quirky way. Most genes encode long messenger RNAs, which are translated into protein (Fig. 1a). But genes like lin-4 and let-7 instead encode small RNAs that inhibit the expression of a quite different gene, probably by stopping the translation of the mRNA it encodes (Fig. 1b ). It is the inhibition of the target genes that sets the genetic cascade controlling a particular developmental transition. These genes, by acting on yet another set of genes, are the major regulators of the transition in question2,3. But ultimately, the precise timing of these events is achieved by regulating the amount, and the time of synthesis, of the small RNAs2,3.

Figure 1: Keeping time with small RNAs.
figure1

a, Conventionally, genes are transcribed into messenger RNAs, which are then translated into proteins. It is the proteins that do the 'work' specified by the gene sequences. b, A few genes, such as the nematode let-7 gene (left), are instead transcribed in response to upstream signals (not shown) and processed to produce small RNAs — the let-7 small RNA is a mere 21 nucleotides in length. Here it is the small RNA that does the work: it pairs up with the untranslated nucleotide sequence at the 3′ untranslated end of a target messenger RNA (the in-41 RNA in the case of let-7), and probably prevents it from being translated into protein. This relieves the inhibition on other genes, setting off a genetic cascade (not shown) that steers the organism through a major developmental transition. This mechanism of gene inhibition by small RNAs is probably not the same as 'RNA silencing', which also involves small RNAs but results in the degradation of the target RNAs. Pasquinelli et al.5 have found the let-7 small RNA in all major groups of bilaterally symmetrical animals, a result that hints that this mechanism of developmental timekeeping is also conserved.

From bacteria to mammals, there are precedents to the use of RNA for controlling gene expression4. Unlike most groups of animals, however, nematodes develop through a strictly defined pattern of cell divisions to yield precise cell lineages. So, as neat as this particular mechanism seems, it might simply have been a developmental timing device specific to nematodes.

But on page 86 of this issue5, Pasquinelli and colleagues show that one of the nematode small RNAs, that encoded by the let-7 gene, is amazingly conserved across all the major groups of bilaterally symmetrical animals — from flies to vertebrates, and from worms and molluscs to echinoderm larvae. Moreover, let-7 is expressed during the development of these different animals at times — such as just before metamorphosis in the fruitfly Drosophila melanogaster — that suggest it is involved in controlling a major developmental transition. Finally, because the genomes of some of the animals tested have been completely sequenced, or almost so, the authors were able to identify let-7's target gene in both Drosophila and vertebrates. This result hints that the whole genetic cascade has been conserved during evolution.

The degree of conservation of the let-7 small RNA is quite remarkable5. In its mature form, the C. elegans let-7 RNA comprises just 21 nucleotides. Drosophila has one exact match, and humans have three, as well as another small RNA in which 20 of the 21 nucleotides are identical. This renders these small RNAs among the most highly evolutionarily conserved molecules — similar levels of conservation are found only in stretches of ribosomal RNA or small nucleolar RNAs. The degree of conservation hints that there are considerable constraints on the nucleotide sequence of the let-7 small RNA. One such constraint could be the fact that other RNAs (such as the lin-41 mRNA in Drosophila, vertebrates5 and C. elegans) and proteins interact with let-7. This in a sense 'freezes' the evolution of the RNA–protein complex, because any change in one of the partners would require compensatory changes in the other. Indeed, the target sequence on the interacting mRNA is also highly conserved. The conservation of the size of let-7 is in itself not too surprising, however. This RNA is as small as it could be — it has barely enough nucleotides to establish a thermodynamically stable pairing with its target mRNA.

The remarkable conservation of this timing device raises a host of questions. For example, how might a system involving such a tight interaction between two complementary RNA sequences have come into existence? This 'invention' probably occurred in the last common ancestor of bilaterally symmetrical animals, as the other descendants of this ancestor — sponges and cnidarians (such as jellyfish) — are apparently without it. It probably started as a gene duplication, which somehow became subverted for use in controlling translation.

Another mystery is how the binding of a small RNA at the untranslated end of the target mRNA affects that mRNA's translation. Also, increased expression of the let-7 gene sets off the genetic cascade, but how is let-7 itself regulated? And which are the 'effector' genes that actually produce the developmental transition? But perhaps the most intriguing question is whether the whole regulatory cascade — including the effector genes — is the same across animal groups.

In the late 1980s, biologists were baffled by the discovery of the extraordinary conservation of 'developmental genes' across very distant animal groups. Organisms as distantly related as Drosophila (an arthropod) and mice and humans (vertebrates) were found to share a gene array known as the Hox complex. One of the functions of this complex is to specify the destiny of different parts of the body along the head-to-tail axis of these animals. Since then, the list of similar gene networks that are conserved across animal phylogeny has been steadily increasing. The implication is that the genes were already present in the last common ancestor of all the animals that share them today — it is unlikely that identical genes would be 'invented' independently in different lineages.

But the key question is whether the processes in which these genes are involved are put to the same use in different animals. This would indicate that the processes were also present in the last common ancestor. Alternatively, the genes might have been 'co-opted' for use in quite different functions in the varying animal lineages. The same question applies to the let-7 small RNA and its genetic cascade. With time, the regulators and effectors of let-7 in several phyla will no doubt be identified. Then we will be able to answer the question of whether this timekeeping mechanism is truly universal.

References

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    Ambros, V. Curr. Opin. Genet. Dev. 10, 428–433 (2000).

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    Reinhart, B. J. et al. Nature 403, 901–906 (2000).

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    Slack, F. J. et al. Mol. Cell 5, 659–669 (2000).

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    Eddy, S. R. Curr. Opin. Genet. Dev. 9, 695–699 (1999).

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    Pasquinelli, A. E. et al. Nature 408, 86–89 (2000).

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Correspondence to André Adoutte.

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