Characteristics of miRNA function during development

One of the most fascinating discoveries in the miRNA field was how deeply conserved some miRNAs are across long evolutionary distances. A small group of miRNAs, sometimes termed ‘ancient miRNAs’ have been discovered in both proteostome and deuterostome genomes indicating they may have been present in the last common bilaterian ancestor. This highly conserved nature of these miRNAs suggests that these genes have fundamental roles in animal biology. Yet, when individual miRNAs are removed from an animal, there are often very little phenotypic consequences. How to explain this disconnect?

The answer may lie in the manner in which miRNAs function. miRNAs are commonly seen performing two main regulatory functions. The first is ‘expression tuning’, where

level of target gene expression or by creating a stable ‘miRNA switch’ where only strong expression of a gene will lead to its correct function.

An excellent example of a miRNA tuning function was elucidated by Li et al, examining the role of miR-9a in the specification of external sensory cells in Drosophila. Peripheral sensory cells within Drosophila require the presence of the transcription factor Senseless (Sens) for their correct development. In non-sensory precursor cells,

Sens is repressed by activated Notch signalling. It was seen that miR-9a was required

in non-sensory precursor cells to also repress the expression of Sens through the inhibition of translation. Thus presence of miR-9a ensures that the expression of Sens is repressed within non-sensory precursor cells (Li et al., 2006).

Another study examining miR-9 function in vertebrates highlighted an interesting example of a miRNA behaving in a ‘switch-like’ function. The Notch signalling effector protein Hes1 is required for the continued proliferation of neural progenitors during development. For Hes1 to function, its expression must be cyclical. Bonev and colleagues observed that miR-9 was able to target Hes1 transcripts within these neural progenitors and that miR-9 transcription was also cyclical, however mature miR-9 levels were very stable. Therefore mature miR-9 expression levels constantly increase and concomitantly, their repression of Hes1 transcripts also increase. Eventually the repressive effect of miR-9 leads to limited Hes1 expression levels and the disruption of

Hes1 function. This causes the neural progenitors to abandon their proliferative state.

Here miR-9 expression acts as a switch, eventually reaching certain level of expression that is able to fully terminate Hes1 function (Bonev et al., 2012).

The second function is often termed ‘expression buffering’, where the miRNA acts to reduce any variation in target gene expression (Bartel and Chen, 2004; Herranz and Cohen, 2010; Hornstein and Shomron, 2006; Wu et al., 2009). An example of this miRNA function was uncovered by Li et al, in their analysis of miR-7 function during sensory cell development in Drosophila. The authors found that miR-7 was involved in the development of multiple types of sensory cells within the animal. However, expression of miR-7 target genes changed little when the miRNA was removed. This was until these animals were placed in fluctuating temperature conditions during their development cycle. This environmental perturbation led to irregular expression of miR-

7 targets indicating this miRNA functions by acting as a genetic buffer, stabilising gene

In both these functional roles, the effect on gene expression by miRNAs is relatively weak, therefore, when most miRNAs are removed from an animal, the resulting phenotype maybe subtle and difficult to detect. This would explain why very few miRNA mutants have easily observable phenotypes.

The relationship between the level of target gene expression and the level of mature miRNA expression can have important implications for target-miRNA interactions. Using a cell based reporter testing system, Mukherji et al., explored the dynamics of target-miRNA concentrations and the effect on target gene repression. A key finding was that the level of translational inhibition by miRNAs was related to the abundance of the target transcripts compared with miRNA abundance. Lowly expressed mRNAs were greatly repressed by large amounts of miRNA. Increased target expression led to a reduction in target gene repression – something comparable to a ‘fine-tuning’ role by the miRNA (Mukherji et al., 2011).

An important characteristic when considering miRNA function is the high degree of pleiotropic targeting by an individual miRNA. The miRNA targeting mechanism is a 6- 8nt ‘seed’ sequence. It is no surprise that many potential target genes will have a miRNA ‘seed’ site. Most estimates suggest each miRNA may have upwards of 100 targets within a cell at any given time.

Overall miRNAs provide subtle but very important regulatory behaviour within the cellular environment. Their ability to effect gene expression within the cytoplasm directly, gives them a fast acting regulatory activity that is perhaps not achievable through transcriptional control mechanisms alone. This allows miRNAs to act as intrinsic regulators of cell fate by maintaining the ‘status quo’ of gene expression within a cell, stabilising a particular cell phenotypic state. In some cases the miRNA profile within a cell can be used as a molecular marker for changing cell states. For example Neveu et al showed that similarities in miRNA profiles could be used to categorise pluripotent cell lines independent of their origin and that these profiles were indicative of p53 function within these cell lines (Neveu et al., 2010).