Genetic redundancy and pleiotropism have limited the discovery of functions associated with miRNAs and other regulatory mechanisms. To overcome this, we performed an enhancer screen for developmental defects caused by compromising both global miRISC function and individual genes in Caenorhabditis elegans. Among 126 interactors with miRNAs, we surprisingly found the CED-3 caspase that has only been well studied for its role in promoting apoptosis, mostly through protein activation. We provide evidence for a non-apoptotic function of CED-3 caspase that regulates multiple developmental events through proteolytic inactivation. Specifically, LIN-14, LIN-28, and DISL-2 proteins are known miRNA targets, key regulators of developmental timing, and/or stem cell pluripotency factors involved in miRNA processing. We show CED-3 cleaves these proteins in vitro. We also show CED-3 down-regulates LIN-28 in vivo, possibly rendering it more susceptible to proteasomal degradation. This mechanism may critically contribute to the robustness of gene expression dynamics governing proper developmental control.
For an organism to develop from a single cell into a collection of many different, specialized cells, different genes must be switched on or off at particular times. However, some of these genes involved in development are ‘redundant’ and carry out the same or similar tasks. This acts like a backup system, so if one of the genes is unable to complete a task, the others can compensate and the organism will still develop correctly.
To produce a protein from a gene, the DNA sequence that makes up the gene is used as a template to create another molecule called messenger RNA. Genes can also be ‘silenced’—prevented from making proteins—by small molecules called microRNAs, which bind to messenger RNA molecules and mark them for destruction. MicroRNA molecules therefore play an important role in controlling development. However, as many microRNA molecules often work together, and as many genes are redundant, it can be difficult to discover the effects of specific microRNAs. It is also difficult to discover whether any other mechanisms work alongside the microRNAs to control development.
Weaver, Zabinsky et al. used mutant forms of the nematode worm Caenorhabditis elegans, in which microRNA gene regulation did not work correctly, to investigate the mechanisms that work alongside microRNAs to control development. Genes in these worms were silenced; those silenced genes that caused additional developmental defects were considered likely to work ‘redundantly’ in the same role as a microRNA molecule. This revealed over one hundred genes that were previously unknown to work with microRNA molecules.
Weaver, Zabinsky et al. focused on one of these genes, called ced-3. The CED-3 protein produced from this gene is known to execute programmed cell death, a carefully controlled process also known as apoptosis, but was not known to have other developmental functions. However, the worms with mutant forms of the ced-3 gene already have problems performing apoptosis but are otherwise relatively normal, so Weaver, Zabinsky et al. reasoned that the CED-3 protein must also have another role in development.
Further investigation revealed that ced-3 mutations most severely disrupt development when they are combined with mutations in one particular family of microRNAs. These microRNAs are particularly important for controlling both when cells specialize into a particular type of cell, and the timing of when certain stages of development happen. Experiments using purified proteins showed that CED-3 breaks down three proteins that are produced from genes controlled by this family of microRNA molecules, and one of these proteins was also broken down by CED-3 in experiments with mutant worms. Weaver, Zabinsky et al. therefore propose that CED-3 is part of a semi-redundant system that ensures the proteins are produced at the right level and at the right time even if the microRNAs insufficiently regulate them. This finding demonstrated both a specific role and specific targets for the CED-3 protein during development, entirely distinct from its role in apoptosis.
Although Weaver, Zabinsky et al. have identified a large number of genes that work alongside microRNAs to control development, these are only the genes that cause obvious developmental defects in healthy worms. Further experiments using similar techniques performed on worms under stress may reveal yet more such genes.