To examine the effects of activation of the RNAi machinery on Drosophila
lifespan, we chose a construct that produces a dsRNA with no target mRNA in the fly, an inverted repeat of 604 bp of the GFP
gene sequence (UAS-GFPRNAi
), which can efficiently knock-down GFP expression 
. To avoid confounding developmental effects, we used GeneSwitch drivers that are only induced upon feeding the flies the RU486 steroid drug 
. We focused on the effects in female flies since they are most commonly used in ageing studies. Driving the expression of the construct with two frequently used ubiquitous GeneSwitch drivers, actin
GeneSwitch (ActGS) and tubulin
) from day two of adulthood resulted in substantial and significant reduction in lifespan (). Feeding RU486 to control flies containing only the driver or only the transgene had no effect on their lifespan (). Hence activation of the RNAi machinery appears to decrease lifespan in Drosophila
Ubiquitous adult-induced expression of dsRNA targeting GFP for RNAi shortens lifespan in Drosophila females.
We examined the sequence of the GFPRNAi construct with the dsCheck program 
and found that it does not contain any 19-mers that perfectly match to a part of the Drosphila
genome. This indicated that the observed detrimental effect on lifespan was sequence-independent. To confirm this, we ubiquitously expressed an RNAi construct targeting the bacterial lacZ
) with the ActGS driver (), as well as activated the RNAi machinery not by inducing an exogenous dsRNA but by induction of Dicer, an endoribonuclease encoded by the Dcr2
gene (). Both these manipulations shortened lifespan, confirming that the reduction in lifespan results from sequence-independent effects of the activation of the RNAi machinery.
Ubiquitous adult-induced expression of dsRNA targeting lacZ for RNAi, or of Dicer2, shortens lifespan in Drosophila females.
RNAi has also been used in the worm to test if particular genes are involved in mediating the effects of dietary restriction, a phenomenon where lifespan is maximised at intermediate food concentrations 
. In Drosophila
, dietary restriction can be achieved by dilution of the yeast component of fly food 
. To assess if RNAi affected the response of the flies to dietary restriction, we investigated whether the flies subjected to RNAi differed from controls in their lifespan response. Ubiquitous expression of the GFPRNAi
construct with the tubGS
driver exacerbated the detrimental effect of RNAi on lifespan at higher and lower levels of yeast in the food, so that the two acted synergistically to shorten lifespan (). RNAi did not significantly shorten lifespan at its peak (0.5× yeast) but was detrimental at higher and lower yeast concentrations (), including the 1× yeast concentration that was tested above (). Analysing the data with Cox Proportional Hazard analysis 
confirmed this, and both the effect of food or transgene induction were significant and so was their interaction (p<0.0001). Hence, activation of the RNAi machinery appears to alter the lifespan response to different yeast concentrations in Drosophila
Lifespan-shortening effects of ubiquitous adult-induced expression of UAS-GFPRNAi are diet-dependent.
RNAi is potentially useful for tissue-restricted loss-of-function studies, so we wanted to examine if this, also, is necessarily detrimental to Drosophila
lifespan. To assess this, we tested two GeneSwitch drivers with tissue-restricted expression that are commonly used in ageing studies: S1106
, which drives expression in the midgut and abdominal fat body, and elavGS
, which drives expression in neuronal cells 
. Driving the expression of the GFPRNAi
construct with either driver had no negative effect on lifespan (). Hence, localised activation of the RNAi machinery does not necessarily have a detrimental effect on lifespan. However, driving GFPRNAi
showed a slight but significant increase in survival (), indicating that caution will need to be exercised when employing tissue-specific drivers as well, on a case-by-case basis.
Adult-induced expression of dsRNA targeting GFP for RNAi in midgut and abdominal fat body or in neurons does not shorten lifespan in Drosophila females.