Figure 10.2 ncRNAs were thought to repress expression of target genes. If this hypothesis were correct, then decreasing the expression of a specific ncRNA should result in more expression of the target gene, as the repression diminishes. This is shown in the middle panel. However, it is now becoming clear that a large number of ncRNAs actually drive up expression of their target genes. This has been shown by cases in which experimentally decreasing the expression of an ncRNA has the effect shown in the right hand side of this figure.
Twelve ncRNAs were tested, and in seven cases the scientists found the result shown in the right-hand panel of Figure 10.2. This was contrary to expectations, because it suggests that about 50 per cent of long ncRNAs may actually increase expression of neighbouring genes, not decrease it[137].
Rather pithily, the authors of the paper stated, ‘The precise mechanism by which our ncRNAs function to enhance gene expression is not known.’ It’s a statement that is very hard to argue with. It has considerable merit as it makes clear that we currently have no idea how this is happening. Ramin Shiekhattar’s work does demonstrate rather convincingly that there is a lot we don’t understand about long ncRNAs, and that we should be wary of creating new dogma too quickly.
We should also be wary of assuming that size is everything and that big is best. The long ncRNAs clearly have major importance in cell function, but there is another equally importance class of ncRNAs that also has a significant impact in the cell. The ncRNAs in this class are short (usually 20–24 bases in length), and they target mRNA molecules, not DNA. This was first shown in our favourite worm, C. elegans.
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.
This can make it very difficult to unravel exactly what a miRNA is doing in a cell, as the effects will vary depending on the cell type and the other genes (protein-coding and non-protein-coding) that the cell is expressing at any one time. That can be important experimentally, but also has significant consequences for normal health and disease. In conditions where there are an abnormal number of chromosomes, for example, it won’t just be protein-coding genes that change in number. There will also be abnormal production of ncRNAs (large and small). Because miRNAs in particular can regulate lots of other genes, the effects of disrupting miRNA copy numbers may be very extensive.
The fact that 98 per cent of the human genome does not code for protein suggests that there has been a huge evolutionary investment in the development of complicated ncRNA-mediated regulatory processes. Some authors have even gone so far as to speculate that ncRNAs are the genetic features that have underpinned the development of Homo sapiens’ greatest distinguishing feature – our higher thought processes[141].