The Epigenetics Revolution

As we have already discussed, C. elegans is a very useful model system because we know exactly how every cell should normally develop. The timing and sequence of the different stages is very tightly regulated. One of the key regulators is a protein called LIN-14. The LIN-14 gene is highly expressed (a lot of LIN-14 protein is produced) during the very early embryo stages, but is down-regulated as the worms move from larval stage 1 to larval stage 2. If the LIN-14 gene is mutated the worm gets the timing of the different stages wrong. If LIN-14 protein stays on for too long the worm starts to repeat early developmental stages. If LIN-14 protein is lost too early the worm moves into later larval stages prematurely. Either way, the worm gets very messed up, and normal adult structures don’t develop.

In 1993 two labs working independently showed how LIN-14 expression was controlled[138][139]. Unexpectedly, the key event was binding of a small ncRNA to the LIN-14 mRNA molecule. This is shown in Figure 10.3. It is an example of post-transcriptional gene silencing, where an mRNA is produced but is prevented from generating a protein. This is a very different way of controlling gene expression from that used by the long ncRNAs.

^{Figure 10.3 Schematic to demonstrate how expression of microRNAs at specific developmental stages can radically alter expression of a target gene.}

The importance of this work is that it laid the foundation for a whole new model for the regulation of gene expression. Small ncRNAs are now known to be a mechanism used by organisms throughout the plant and animal kingdoms to control gene expression. There are various different types of small ncRNAs, but we’ll concentrate mainly on the microRNAs (miRNAs).

At least 1,000 different miRNAs have been identified in mammalian cells. miRNAs are about 21 nucleotides (bases) in length (sometimes slightly smaller or longer) and most of them seem to act as post-transcriptional regulators of gene expression. They don’t stop production of an mRNA, instead they regulate how that mRNA behaves. Typically, they do this by binding to the 3′ untranslated region (3′ UTR) of an mRNA molecule. This region is shown in Figure 10.3. It’s present in the mature mRNA, but it doesn’t code for any amino acids.

When genomic DNA is copied to make mRNA, the original transcript tends to be very long because it contains both exons (which code for amino acids) and introns (which do not). As we saw in Chapter 3, introns are removed during splicing to create an mRNA which codes for protein. But the Chapter 3 description passed over something. There are stretches of RNA at the beginning (known as 5′ UTR) and the end (3′ UTR) which don’t code for amino acids, but don’t get spliced out like introns either. Instead, these non-coding regions are retained on the mature mRNA and act as regulatory sequences. One of the functions of the 3′ UTR in particular is to bind regulatory molecules, including miRNAs.

How does a miRNA bind to an mRNA and what happens when it does? The miRNA and the 3′ UTR of the mRNA only interact if they recognise each other. This uses base-pairing, quite similar to that in double stranded DNA. G can bind C, A can bind U (in RNA, T is replaced by U). Although miRNAs are usually 21 bases in length, they don’t have to match the mRNA over the entire 21 nucleotides. The key region is positions 2 to 8 on the miRNA.

Sometimes the match from 2 to 8 is not perfect, but it’s still close enough for the two molecules to pair up. In these cases, binding of the miRNA prevents translation of the mRNA into protein (this is what happened in the case shown in Figure 10.3). If, however, the match is perfect, the binding of miRNA to mRNA triggers destruction of the mRNA, by enzymes that attach to the miRNA[140]. It’s not yet clear if positions 9 to 21 on the miRNAs also influence in a less direct way how these small molecules are targeted, or what the consequences of their targeting are. One thing we do know, however, is that a single miRNA can regulate more than one mRNA molecule. We saw in Chapter 3 how one gene could encode lots of different protein molecules, by altering the way in which messenger RNA is spliced. A single miRNA can influence many of these differently spliced versions simultaneously. Alternatively, a single miRNA can also influence quite unrelated proteins that are encoded by different genes but have similar 3′ UTR sequences.