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James Birchler on Apoptosis and RNAi Inhibition in Drosophila

Name: James Birchler
Position: Professor of biological sciences, University of Missouri
Background: Assistant professor, organismic and evolutionary biology, Harvard University —1985-1991
- Postdoc, University of California, Berkeley — 1981-1995
- Postdoc, Oak Ridge National Laboratory — 1977-1981
- PhD, genetics, Indiana University at Bloomington — 1977
- BS, botany and zoology, Eastern Illinois University — 1972

At the University of Missouri, James Birchler researches regulatory gene balance, dosage compensation, gene silencing, and centromere activity, among other things.
Recently, he began a National Science Foundation-funded project to investigate his unpublished finding that RNAi is inhibited in cells adjacent to ones undergoing apoptosis in Drosophila.
Last week, RNAi News spoke with Birchler about his efforts.
Could you give some background on the inhibition of RNAi by apoptosis?
We were using a construct that creates RNAi for the white eye color gene in Drosophila … [using] mutants that have cell death as part of their mutant effect in the eye. [This] … was the classical Bar eye mutation [in which] the anterior part of the eye undergoes cell death [and] therefore it looks like a kidney shape instead of the normal round eye.
Next to the place where the cell death has occurred, we saw that instead of the white eye color gene being silenced as it ought to be with the RNAi construct present, there were red sectors, which is the normal effect of the white eye color gene. This suggested that there was a signal from the dying cells that was interfering with RNAi.
There were a variety of other mutations that caused cell death, as well, that produced a related kind of phenomenon. Then, we started intentionally using constructs that would induce cell death in the eye. Those, too, would reverse the effect of RNAi in the eye.
What exactly were these constructs? Was the RNAi inhibition something related to them?
These were constructs that, when over-expressed, would cause cell death. … Whenever the white eye color gene and the RNAi construct are present, you induce cell death by over-expressing these cell-death constructs, and in adjacent sectors one finds the reversal of the RNAi effect.
Was this just in the eye that you’ve seen this effect, or have you seen it in other parts of the fly?
The eye is the best place to investigate cell death. It’s also possible to look in the wing, but in the mature wing the cells have died.
You can do without the eye without killing the fly, whereas if you cause cell death in other tissues, the fly is dead and so your experiment really can’t work.
There is the possibility, which we haven’t investigated yet, of using conditionally expressed constructs to cause cell death. … That is a future direction.
We have used constructs other than the white eye color gene — for example green fluorescent protein. We expressed it and an RNAi construct against [GFP], and one finds no fluorescence in the eye. But again, if one induces cell death, one can override the effect of RNAi on the GFP, as well. So it’s not specific to the white eye color gene and it does have an impact on RNAi as a general phenomenon.
Do you have any ideas as to what may be causing the inhibition of RNAi?
We don’t know for sure. We’ve done a mosaic analysis experiment where we have created lineages of cells that have undergone cell death and those that haven’t in the same eye. We see that the inhibition of RNAi is in the adjacent cells to those that have undergone cell death. So we postulate that there is some signal that is given off from the dying cells that goes to adjacent cells and interferes with RNAi.
It’s known from studies of apoptosis that dying cells do send signals to their adjacent cells, in particular, proliferation signals — it’s all part of the apoptosis machinery. Using inducers of apoptosis and inhibitors of apoptosis expressed together, [we have found that] … if you have a cell death construct by itself, you inhibit RNAi. If you inhibit the cell death with yet another construct, then the RNAi is reinstated but the proliferation signals are still occurring. So we can rule out that it is those particular signals that are inhibiting RNAi.
What do we think may be going on? Well, it’s known, for example, that in mammalian cells there is a response to, say, the infection of a virus … [in which] double-stranded RNA editing enzymes are induced … to edit double-stranded RNAs, which can no longer be a substrate for RNAi.
One possibility, which we would like to test, is that the dying [Drosophila]cells give off a signal to induce in the adjacent cells other mechanisms that can attack double-stranded RNA that then compete against RNAi for the double-stranded RNA as a substrate.
One might ask, “Why would such a mechanism exist?” Our rationale is as follows: there is evidence that RNAi is … probably working as an anti-viral agent because most viruses have machinery that inactivates RNAi. If [a cell’s RNAi machinery] fails, the virus will kill the cell. The host, then, may have evolved a mechanism to send a signal to adjacent cells to induce yet other mechanisms that can attack double-stranded RNA — in other words, the [RNA] from the virus.
That is potentially the way that such a system may have evolved, and that may be the underlying basis for why we see what we see. But that’s speculation and that is what we’d like to test.
How do you approach testing this?
One possibility is to build flies that are mutant for the [RNA editing enzyme] ADAR, [or adenosine deaminase acting on RNA], which may be what’s competing against RNAi. … There may be other aspects of the immune response in Drosophila that could be involved, as well. If the ADAR experiment is negative, then we would investigate other possibilities.
It’s known from other laboratories that putting the human ADAR enzyme into flies will, in fact, inhibit RNAi. So that’s the first good candidate to investigate.
What are your thoughts on the broader implications of this research?
This grant has some implications for the biotechnological application of RNAi. Cell death is a natural phenomenon and happening all the time during development, so if this is having some impact on RNAi in other species, particularly humans … then that knowledge is important for understanding how RNAi is going to react to signals from cell death.
Are you collaborating with anyone on this work?
This work is just from our laboratory.