Abstract
microRNAs are important regulators of gene expression. In the retina, the mir-183/96/182 cluster is of particular interest due to its robust expression and studies in which loss of the cluster caused photoreceptor degeneration. However, it is unclear which of the three miRNAs in the cluster are ultimately required in photoreceptors, whether each may have independent, contributory roles, or whether a single miRNA from the cluster compensates for the loss of another. These are important questions that will not only help us to understand the role of these particular miRNAs in the retina, but will deepen our understanding of how clustered microRNAs evolve and operate. To that end, we have developed a complete panel of single, double, and triple mir-183/96/182 mutant zebrafish. While the retinas of all mutant animals were normal, the triple mutants exhibited acute hair cell degeneration which corresponded with impaired swimming and death at a young age. By measuring the penetrance of this phenotype in each mutant line, we determine which of the three miRNAs in the cluster are necessary and/or sufficient to ensure normal hair cell development and function.
Highlights
MiRNA primary transcripts are generated principally by RNA Polymerase II1 and form hairpin structures that are recognized and excised into pre-miRNAs by the “microprocessor,” a complex of proteins including the ribonuclease Drosha[2] and the RNA binding protein Dgcr[83]
MiR-182 is co-expressed along with miR-183 and miR-96 on a single primary transcript[11] (Fig. 1a,b). This type of microRNA clustering is caused primarily by gene duplication events which can lead to redundancies[12], meaning that organisms are often unaffected by the loss of individual miRNAs from the cluster[13,14]
A gene trap that inactivated the entire cluster resulted in progressive photoreceptor degeneration and increased susceptibility to light damage[18], and targeted deletion of mir-183/96 had a similar effect[19]
Summary
MiRNA primary transcripts are generated principally by RNA Polymerase II1 and form hairpin structures that are recognized and excised into pre-miRNAs by the “microprocessor,” a complex of proteins including the ribonuclease Drosha[2] and the RNA binding protein Dgcr[83]. Combined re-expression of miR-182 and closely-related miR-183 was sufficient to rescue cone degeneration in C-DGCR8-KO mice[9] Taken together, these studies indicate that is miRNA-dependent gene regulation of critical importance in the retina, but that several of these miRNAs make a compelling case for immediate investigation. Loss of only miR-182 had a similar effect on light damage sensitivity, caused a decrease in ERG amplitude, and resulted in downregulation of several photoreceptor-specific genes[21,22] This cluster is highly expressed in hair cells[11,23], and mutations in mir-96 have been linked to hearing disorders in humans[24,25], as well as in the diminuendo mouse[26]. Additional studies in zebrafish have implicated the mir-183/96/182 cluster in the development of these organs[31]
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