Lorne 2012: Modelling microRNAs

MicroRNAs (miRNAs) work by modifying gene expression at the post-translational level across a range of species, from plants to worms to humans.

Discovered only in 1993, it is now clear that miRNAs, while not coding for proteins, have a tremendous impact on the gamut of mammalian functions, including cell growth and proliferation, development, apoptosis, immune function and metabolism.

These tiny stretches of ribonucleic acid (only 21-23 bases) bind to complementary sequences on target messenger RNA transcripts to usually shelve the transcriptional information (gene silencing) and thus effectively act as negative regulators of gene expression.

As may be expected, aberrant expression of miRNA has also been named and shamed in a range of disease processes, fueling a whole new field of research aimed at finding miRNA-based targets for diagnostics and therapies.

One of the most respected names in the field of Drosophila genetics is Dr Stephen Cohen from the Institute of Molecular and Cell Biology (IMCB) in Singapore, who in February will head to the sunny but cooler climes of Lorne for the 2012 Lorne Genome conference.

Cohen’s lab uses the fruit fly as a genetic model for the study of cancer, metabolic disease and neurodegeneration and in recent years has focussed on understanding the biological functions of miRNAs in this context.

All animal genomes have miRNA genes, explains Cohen. In fact, up to five per cent of all genes are thought to be miRNAs.

“Latest estimates put the regulatory capacity of miRNAs at 100s of potential targets for each individual miRNA, so they are similar to transcription factors in that respect. miRNAs could potentially regulate 50 per cent or more of the protein-coding genes in animal genomes based on the predicted miRNA target sites in those genes.

“Over several years we and others have found that miRNAs are key regulators of a range of biological processes relevant to development and disease, including cell growth, death and metabolism,” says Cohen.

“In disease, the miRNA is sometimes misexpressed, leading to inappropriate downregulation of genes that might be important for normal function. Other times, miRNA expression is lost and, because of that absence, the levels of some genes go up and that may be detrimental.”

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Old tools, new questions

Primarily using a forward genetic approach, Cohen is using Drosophila as a model system to study the regulatory potential of miRNA genes by knocking them out in the fly genome and then studying the nature of the defects in the mutants that lack the specific miRNA function.

“The idea is that learning what can go wrong in the fly mutants might help us find new links to disease.” Flies are ideal for this type of approach as they have an “unparalleled flexibility for genetic manipulation and, at the cellular level there are many similarities between flies and mammals.”

Indeed, Cohen’s team has now completed a large-scale project to knock out all miRNA genes in the fly genome, and apparently that was the easy part. “We are now focussing on a systematic functional analysis of all those genes and I will talk about some of our findings at Lorne.”

Cohen plans to discuss a couple of results, hot off the lab bench, wherein identifying targets of miRNAs in flies has turned up new candidate molecules for diseases, and he will give specific examples relating to cancer and to neurodegenerative disease. Unfortunately, because this work is yet unpublished, he could only reveal that these candidate targets look to be very promising.

“Anecdotally, I can tell you that we are finding relatively few mutations that are incompatible with survival of the animals, although relatively many mutations that affect viability in one way or another,” says Cohen.

“It is not really surprising that if you mutate a gene you make the animal sick. But in some instances the fly viability is affected in surprising ways such that it makes the flies healthier, including a handful of mutations that make them live longer than normal. Of course, understanding how that happens will be very interesting indeed.”

Cohen also suggested that a large number of the Drosphila miRNAs are expressed in the brain, sometimes representing all or a significant fraction of their total expression. Then, when they looked further at the fly mutants with this brain-centric miRNA expression, a very high proportion showed evidence of impaired nervous system function.

“What we are hoping to find from these flies is evidence of genes whose misregulation in the brain can be linked to defects of a specific sort. For example, we have some mutations that cause too much activity in a particular type of neuron that leads to neurodegeneration because that part of the nervous system is overactivated.

“So that is the kind of thing that we are seeing in our analysis and the sort of direction this work could take as we move into collaborations with the clinical research community.”

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New targets

Cohen hopes his team’s efforts will eventually generate completely new targets for the miRNA-linked disease functions they identify, things that could not have been revealed by previous strategies.

“The potential success of this strategy is much higher using the fly because you can feasibly screen the entire genome for function using forward genetics. That is just not possible with the mammalian genome because such screens usually have to be phenotype driven, and it is difficult to pick up modest effects that may be important.

“With flies, you can also directly test the hundreds of potential candidates for those that are functionally important in the defect you are looking at. You can basically address so many more questions with fly genetics.

“For example, if a particular gene is upregulated as a consequence of knocking out the miRNA, can I then limit that effect in some way such that the disease condition, the defect, goes away or at least is made better? We have all the tools to address these questions, and by doing so we assign function.”

Cohen’s ultimate goal is to identify genes that are linked to a cancer or other disease phenotypes in the fly that he can extrapolate to the human condition.

“In a good scenario, these findings might reveal new biological markers for use in the diagnosis and prognosis testing of clinical cancer. In a slightly better world, we might be able to use that to guide therapeutic choice. And in an even better world, we would find a new drug target.

“In our cancer-related work, the prospects of rapid translation to the clinic are not all that bad for some instances. However, the nervous system picture is much more complex and more likely to be a longer and slower process,” says Cohen.

Unlike in cancer where the amount of disease genetic and genomic data available is increasing all the time, the depth of information about neurodegenerative diseases and their causes is much more scarce and translation work in this space will almost certainly involve moving into mouse models at some stage.

Having said that, one of Cohen’s team’s hopes is to largely bypass other animal models such as the mouse, if possible. To this end, they have started to work with clinicians in Singapore who work on neurodegenerative disease.

“We are generating hypotheses based on our findings in flies for them to look at in their patient material. In turn, they have hypotheses coming based on the patient data and samples that we can, in turn, investigate by generating Drosophila genetic models to see if we can recapitulate what they think is causing disease. So the possibility is there to make those direct and meaningful connections.”

Very recently Cohen is also on the way to forging collaborations with clinical researchers in the cancer area. “Based on what we are finding in these very weeks, we will be seeking out further relationships on the cancer side, and we believe we have something that they will find very interesting.”