A few years ago, the terms ‘switch target’, ‘tuning target’ and ‘neutral target’ were coined to describe possible outcomes of miRNA-mediated repression.21
A switch target was defined as one whose protein output is reduced to functionally inconsequential levels by the miRNA. A tuning target was defined as one for which miRNA-mediated regulation acts to to ensure functional, but not undesirably high levels of the gene product. A neutral target was defined as one whose regulation by a miRNA is entirely incidental, such that any change in target levels brought about by loss of the miRNA has no biological consequence.
In some cases of miRNA control of developmental regulators, the elimination of the target is essential for a given biological process. However, it is instructive to note that the “switch” category, as defined above, does not necessarily imply that a miRNA-target relationship is of substantial importance.2
Since the level of transcription of many targets is already low in cognate miRNA-expressing cells,20,22
target outputs might essentially be inconsequential even without the involvement of a miRNA. Such “de-noising” roles in clearing away low levels of unwanted transcripts might be quite broad for miRNAs. For such targets we might expect their loss of miRNA-mediated regulation to be of minimal consequence.
Conversely, one should not assume that mild amounts of target regulation are correspondingly of little consequence. In some cases, a “thresholding” function of miRNAs has been posited, which sets a minimum level of target activity that must be exceeded in order to get the ball rolling on some larger chain of events.2,23
For such cases of finely balanced target activity, the loss of miRNA-mediated regulation might be quantitatively subtle, but nonetheless of great phenotypic significance if a pathway is inappropriately tripped.
These considerations make clear that it is not a trivial matter to infer substantial biological functions of miRNAs strictly from perusal of target predictions.24
Phenotype-driven gain-of-function screening is one expedient approach to identify specific activities of miRNAs. In cultured cells or in whole animals, many miRNAs produce spectacular phenotypes due to the ectopic repression of one or more targets.25–29
Such studies can lend insight into potent miRNA-target relationships, and strongly support the notion that miRNA deregulation can directly induce disease and cancer. Nevertheless, the activities observed in such experiments are not necessarily germane to the normal function of the miRNA. For instance, if a miRNA is misexpressed entirely outside of its normal domain of expression, it might encounter and repress completely inappropriate targets.
As is the case for protein-encoding genes, then, loss-of-function analysis is needed to divine the endogenous function of miRNA genes. Antisense inhibitors of miRNAs are currently in broad usage for their technical convenience. However, the extent to which they might induce off-target effects is currently uncertain, and the phenotypes attributed to the inhibition of several miRNAs30
were not observed following the analysis of corresponding deletion mutants.31–33
Therefore, the unambiguous determination of miRNA requirements requires genuine loss-of-function alleles. A substantial number of miRNA deletions have now been isolated in worms, flies and mice (summarized in ), and we review some of the lessons learned from their analysis.
Summary of miRNA deletion mutants in worms, flies and mice