Name: Shane Rea
Position: Senior research associate, Institute for Behavioral Genetics, University of Colorado, Boulder
Background: Research associate, Institute for Behavioral Genetics — 2001-2007
Postdoc, Royal Victoria Hospital — 2000-2001
Postdoc, McGill University — 2000-2001
Casual research assistant, University of Queensland — 1998-2000
PhD, molecular biology, University of Queensland — 2000
BS, biochemistry/chemistry, University of Queensland — 1992
At the University of Colorado, Boulder, Shane Rea investigates the aging process and ways in which it might be altered. He will continue this focus in a new position that he recently accepted as assistant professor with the Barshop Institute for Longevity and Aging Studies at the University of Texas Health Science Center at San Antonio, where he expects to join in November.
Earlier this month, Rea and colleagues from UC Boulder published data in PLoS Biology suggesting that the life-extending effects of mitochondrial electron transport chain function in C. elegans is due to the knock down, not knock out, of certain genes.
This week, RNAi News spoke with Rea about the findings and the RNAi dilution system used to obtain them.
Let’s begin with an overview of the research that led to the PLoS Biology paper.
We were specifically trying to figure out why a collection of long-lived mutants in C. elegans were, in fact, long-lived when most of them had mitochondrial defects. If you think about it, [when creating these mutants] you seem to be knocking out the main energy production pathway in these animals, and yet here they were living longer than wild-type animals.
[Adding to our uncertainty was the fact that] some of those same genes, when knocked out in humans, were pathogenic. So the question we were trying to address was, ”Why are these mitochondrial mutants long-lived?”
And in trying to answer that question you turned to RNAi as an enabling technology?
What had happened was that there had been three large-scale RNAi screens done [in C. elegans] for increased longevity that had pulled out a lot of mitochondrial genes. Targeted removal of single mitochondrial-targeted genes made animals long-lived. … So we had taken some of those RNAi clones and, because of the ease of [doing] RNAi in C. elegans, started looking at RNAi-induced mitochondrial mutants, as opposed to using genetically defined mutants.
What we found is that it is knock down, not knock out, that seems to confer the long life. It turns out that there is an optimal level of transcript reduction that translates into long life.
To come to this conclusion you developed an RNAi dilution strategy?
The cool thing with worms is you can actually get bacteria to make double-stranded RNA, the worms can eat that bacteria, it crosses the gut, and then [the dsRNA] is processed into the small interfering RNAs that give the RNAi effect in a pretty much systemic manner. I say pretty much because there are certain tissues that seem to be a little bit [more] refractory to feeding RNAi than others.
So we cooked up this really simple method of just standardizing how we grew our bacteria, and we mixed them in a standard ratio — what we call a dilution series — where we used vector-only containing bacteria [and] bacteria expressing RNAi against our particular target gene. Essentially, we’re just diluting out the bacteria containing the target RNAi with vector-containing bacteria.
With very reproducible results we could titrate out the RNAi effect, and we saw very clearly that lifespan peaked within a certain window. But once we got out of that window, as you might predict, lifespan started shortening big-time.
We realized from that, that worms aren’t really that different from humans in the sense that if you knock down these genes too much, just like in humans, you get life-shortening effects. And that has some pretty interesting ramifications. Does it mean that in humans there is an optimal window in that if we knock down expression of the same mitochondrial genes by a little bit that we get a life-enhancing effect, just like the worms? Who knows? These are interesting things to ponder.
Interestingly, it is known that in certain human centenarian populations, specific mitochondrial mutations are more prevalent. Perhaps there is some connection here.
The genes that you focused in on in the work, are there mammalian counterparts?
For some of them, definitely there are orthologs.
With these findings, what is the next step? Are there plans to follow up on this or apply the dilution technique to other questions?
We think that the Mit mutants are turning on what we call compensatory pathways that are either acting to increase cellular ATP production via non-mitochondrial methods, or perhaps increasing the efficiency of mitochondria by enhancing the electron transport chain activity in the compromised mitochondria.
But we think as a consequence — and we don’t know if it’s direct or indirect — that as result of these compensatory processes, life extension occurs. The aim is to identify what these pathways are.
In the paper, we show that several cell-cycle-related phenomena are invoked at the same time as when lifespan starts to increase. It kind of points to the fact that an ATP deficit is being sensed in the nucleus, moving the focus away from the mitochondria into an entirely unexpected compartment of the cell.
The power of the [RNAi dilution] system is that [it should allow us] to investigate what these compensatory pathways are. That’s where we’re heading with the dilution system now. One way to do it is take different points along the [dilution] series and ask, “Are our favorite processes turned on or off?”
We can also use the series as a blind screening tool to find these pathways.