What's that gene for, again?

I'm always skeptical when you hear prepositions such as "gene X has function Y," as often there are very complicated mechanisms nestled between the "gene" and the "function/phenotype." If you've been following me over the past year (yes, I've been blogging for a year already, time flies!), we've learned that between-gene interactions (epistasis), and changes in gene expression (epigenetics) can completely change the picture.

Recent reviews on the use of RNA interference have given me additional reasons to be skeptical.

Gene function in vivo has been studied through a procedure called "gene knockdown," which uses RNA interference (RNAi) to "tune down" the expression of the gene. RNAi has also been used in to mimic human genetic diseases that would otherwise have no somatic equivalent in the animal world, in particular in studies aimed at discovering novel drug targets. By introducing synthetic RNA into the cell, researchers can effectively silence target genes and thus identify their functions within specific cellular processes.

It certainly is a brilliant tool, but there are several issues one needs to keep in mind when using RNAi. The target specificity, for example, is not always perfect, and several off-target effects (down-regulation of genes different from the target ones) have been documented. When this happens, you can no longer be sure of what genes, if not all, caused the observed change in phenotype. Ideally, in order to minimize off-target effects, one should repeat the experiment with different types of RNAi targeting the same gene. Rescuing the loss of function by re-inserting the mRNA (or making it "immune" to the RNAi) would also provide further evidence. However, this is very hard to realize in practice.

It gets more complicated.

Many genes regulate cellular fitness. The change observed change in phenotype, rather than reflect the knockdown gene, could instead be a direct consequence of lower cell proliferation. In addition, we tend to simplify things thinking that the relationship between gene and phenotype is linear, or that the effect from different genes is additive, when in fact such simple mathematical frameworks often don't capture the reality of the biological world. Interactions and non-linearity are difficult to model. Experiments that target multiple genes rank the results in terms of dose-responses, though such results are often contaminated by false positives and knockdown efficiencies.

All this not to say that this is the end of RNAi experiments, rather, that additional thought has to be given when interpreting the results. We still have a long way to go before we can fully encompass the complexity of our genome, and we are taking one baby step at the time.

William G. Kaelin Jr. (2012). Use and Abuse of RNAi to Study Mammalian Gene Function Science, 337 (6093), 421-422 DOI: 10.1126/science.1225787