There is a single cluster of smallRNA molecules that plays an important role in the regulation of a select cell type in the immune system. If this cluster of smallRNAs is over-expressed in mice, the animals develop a fatal over-activation of the immune system.{361}{362} On the other hand, mice that lack this cluster altogether die around the time of birth. In humans, the loss of one copy of this cluster leads to some cases of a rare condition called Feingold syndrome.{363} Patients with this disorder have variable symptoms, often including malformations of the skeleton, kidney problems, gut blockages and moderate learning disabilities.{364}
The consequences of disrupted expression of this cluster of just six smallRNAs seem puzzlingly diverse. But perhaps this isn’t so surprising, as researchers have calculated that this cluster alone may target over 1,000 protein-coding genes.{365}
The junk sequences that code for smallRNAs are often located within other junk regions, such as the genes producing the long non-coding RNAs.{366} There is a condition called human cartilage-hair hypoplasia, which was originally identified in an Amish community, where one in ten of the community is a carrier of the causative mutation. This is an incredibly high carrier frequency and almost certainly reflects the fact that this community was originally founded by just a small number of families. The affected children have defects in the formation of their skeletons, resulting in a short-limbed form of dwarfism, and light hair that is fine but sparse. The patients also tend to have a variable range of other defects.
The mutations that cause this condition lie in a long non-coding RNA gene. But this long gene encompasses two smallRNA genes, junk within junk, and many of the mutations affect the smaller moieties. The changes disrupt the structures of the smallRNAs so that they aren’t processed properly by the cutting enzyme represented by scissors in Figure 18.1. As a consequence, they aren’t expressed at their normal level. Between them, these two smallRNAs regulate over 900 protein-coding genes. These include genes known to be involved in skeletal and hair development, but also in a number of other systems. This is presumably why mutations that affect the levels and functions of these smallRNAs can also lead to problems in a range of organ systems in the affected children.{367}
Given how important smallRNAs are for fine-tuning of gene expression, it’s perhaps not surprising to learn that these junk molecules have a major role during development. This is the stage in life where apparently minor fluctuations in gene expression can have a significant impact (remember our Slinky falling down the stairs?).
A beautiful example of the importance of smallRNAs comes from reprogramming human tissue cells to become pluripotent stem cells, potentially capable of building any tissues we need. This is the technology that we first met in Chapter 12, and which is shown in Figure 12.1 (page 165). Although the original work for which the Nobel Prize was awarded so quickly was extraordinary, it had some limitations. Although the master regulator proteins could push the developmental Slinky back up a flight of stairs, they did so fairly inefficiently. Only a tiny percentage of cells were converted, and the process took many weeks. Five years after those ground-breaking findings, other researchers extended this work. They treated the adult cells with the same master regulators used in the original experiments. But they also added something else. They over-expressed a cluster of smallRNAs which had been shown to be highly expressed in normal embryonic stem cells. The scientists found that when they over-expressed these smallRNAs along with the original master regulators, adult cells changed back to pluripotent stem cells, as we would expect. But the percentage of cells that converted to stem cells was more than a hundred times greater than with just the master regulators alone. The process also happened much more quickly. Conversely, if they used the master regulators but knocked down the expression of the endogenous smallRNA cluster in the adult cells, the reprogramming efficiency dropped dramatically. This demonstrated that this particular cluster of smallRNAs does indeed play a critical role in helping to regulate the signalling networks that control cell identity.{368}{369}
Adult tissues also contain stem cells. These are able to create cells for their specific tissues, rather than multiple cell types. These are important for growth as we move from baby to adult, and also for repairing wear and tear. Some tissues retain a very active stem cell population even late into life. A classic example would be the bone marrow, which keeps producing the cells we need to fight infection and to patrol against potentially cancerous cells. One of the reasons the very elderly are particularly prone to infections and cancer is because their bone marrow stem cells eventually run out, leaving them with holes in their immune barricades.
There are data showing that stem cells and adult cells from human tissues express different patterns of smallRNAs. But expression data are always difficult to interpret, because of the cause-or-effect problem. Are the different patterns of smallRNAs driving the differences in cell activity and function, or are they simply a bystander consequence of the cellular changes? The fact that predicted sequence pairings between individual smallRNAs and the untranslated regions of at least half of all messenger RNA molecules have been preserved through evolution suggests a causal effect.{370} But to address this question more directly, scientists have frequently turned to our close cousin, the mouse.
Researchers have found ways of knocking out genes only in adult tissues, which has created a very powerful tool set for investigations. This handy technique means that mice develop in the usual way, so we don’t need to worry that symptoms are caused by pathways and networks going wrong during development. This approach has been used to work out what happens if the enzyme that is required to produce smallRNAs (the scissors in Figure 18.1) is inactivated in adult cells. This will interfere with production of all smallRNAs and so show us where they play an important role. It won’t, however, tell us exactly which smallRNAs are involved.
When scientists knocked out the scissors enzyme in all tissues of adult mice, they found defects in the bone marrow, but also in the spleen and the thymus. All three of these tissues produce cells required for fighting infection and were expected to have a large population of stem cells. This finding was consistent with the smallRNA systems having a role in stem cell control. The mice all died, but this was due to a massive deterioration of their intestinal tracts. This is also consistent with a role in stem cells. Our intestines are constantly losing cells that are sloughed off during the continuing activity of the digestive system. These cells have to be replaced every day so we would expect there to be a very active stem cell population.{371} However, it wasn’t clear exactly how the loss of the scissors enzyme resulted in dramatic damage to the intestines, although it may have been related to abnormalities in the way the mice processed fats in their diet.
These effects were very dramatic, but that doesn’t mean that these are the only tissues where smallRNAs play an important part. Because the mice died relatively quickly, this may have masked more subtle symptoms in other tissues. In order to investigate this, researchers can use a more discriminating version of the adult knockout technique. With this amended technology, they are able to inactivate the scissors gene in selected tissue types in adult mice.