What
prompted the research on double-stranded RNA that led to your
highly cited 2000 Cell paper, "RNAi: double-stranded RNA
directs the ATP-dependent cleavage of mRNA at 21 to 23
nucleotide intervals" (Zamore PD, et al., Cell
101[1]: 25-33, 31 March 2000)?
I got into this field at very end of my post-doctoral
work. The Fire and Mello paper, the one for which Andy and
Craig won the Nobel Prize in 2006, came out in March 1998
(Fire A, et al., "Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis
elegans," Nature 391: 806-11). I was a post-doc
in the Bartel lab at the time. And so was Tom Tuschl. I
presented the Fire-Mello paper at journal club, and Tom was
giving the experimental progress report for his work, and he
proposed an in vitro system for RNAi. He also had
finished his post-doctoral work and had secured a faculty
position, and so he had some unstructured time ahead of him.
Out of the discussions that followed, we decided to attempt
the task of developing the in vitro system together.
We succeeded in doing that and published it in 1999 in
Genes and Development (Tuschl T, et al.,
"Targeted mRNA degradation by double-stranded RNA in vitro,"
Genes Dev. 13[24]: 3191-7, 15 December 1999).
What
exactly did your 2000 Cell paper report?
It was the discovery that small RNAs were produced from
the double-stranded RNA that triggers RNAi. They act as
guides to direct the cleavage of mRNA.
Why
was that so significant?
|
“In the span of a year, I went
from never working on RNAi to having
it be the consuming passion of my
life.” |
|
We figured out how small RNAs mediate RNAi.
Was
it obvious at the time that this was an incredibly significant
discovery?
Yes. We were unbelievably excited. I think our biggest
fear was that we would never do anything as exciting again
in our lives.
Did
that anxiety turn out to be true? Has there been anything since
that was so exciting?
The sensation that you’ve just done your most exciting
work can only really happen at the beginning of a really
marvelous adventure. So I think that description of it,
which I certainly felt at the time, is naïve. Every day that
I come to work and learn something new about how small RNA
functions in cells is incredibly exciting. But every day is
built on the day before. The point is that there were almost
no days before that 2000 paper. In the span of a year, I
went from never working on RNAi to having it be the
consuming passion of my life. I think that’s what happens
when you make an important contribution to the field and
then continue working in the field.
So while I don’t think I’ve ever been quite as excited as
when we were working on that paper, my lab has certainly
published a lot of research that I’m awfully proud of and,
in many ways, is much more thoughtful. Having said that, the
most exciting moment was not that work per se, but rather
the day we figured out we had an in vitro system that
worked. That was in 1999. When that happened, we knew how
much stuff was about to unfold—how much we could do and
learn. That technical breakthrough was probably the most
exciting thing that ever happened to me.
We did do one other thing that had me every bit as
excited at the time. That was a year after I started my own
lab and we discovered that Dicer, the enzyme that makes
siRNA [silencer RNA], also makes microRNA. That was equally
exciting, in part because it was done completely in my own
laboratory. That was published in Science in 2001 (Hutvágner
G, et al., "A cellular function for the
RNA-interference enzyme Dicer in the maturation of the let-7
small temporal RNA," Science 293[5531]: 834-8, 3
August 2001).
Once
you had that in vitro system up and running, was there
any anxiety that you might be scooped?
Not until about a month before the 2000 paper. Then we
started to hear rumors that Greg Hannon’s laboratory had
potentially similar data. Although it turned out to be
complementary data, not the same thing.
What
do you think was the most challenging aspect of this research?
Remaining self-critical. Not letting the excitement of
the data get in the way of being careful and skeptical about
our own interpretations and results. That, by the way, I
think is always the hardest part about anything you do that
might be important.
Was
there an element of serendipity to your research on RNAi?
The only thing serendipitous was the combination of being
in the right place at the right time with the right talents.
If figuring out the answer to how RNAi works required being
a great cell biologist, we wouldn’t have made a
contribution. This was a process in which expertise in the
in vitro study of RNA really gives you a leg up, and
that we had. But there is no substitute for being in the
right place at the right time.
Since
you published your seminal paper in 2000, how has your
understanding of these small RNAs developed?
We know a lot more about the proteins involved. We have
crystal structures for some of them. We understand that the
details are incredibly rich and complex, which reinforced
our view and, I think, the general view in the field, that
cells devote a considerable amount of energy to small
RNA-guided pathways. There are just a lot of proteins
involved in making small RNAs, in loading them into
complexes, and having these complexes do jobs in the cell.
We have also headed off in other directions. We’ve been
trying to understand related small RNA-guided pathways—those
that aren’t RNAi but use some of the same components, or
related components, but in apparently different ways. These
include microRNAs and rasiRNAs, which are the ones involved
in silencing transposons. I’m really enjoying that part of
our work.
We have continued mechanistic studies of how the RNAi
pathway works. That has taken me in some very quantitative
directions and keeps reminding me that I should have worked
harder when I was taking math in college. We are going to
have to become much better biophysicists in the near future.
We’ve also started looking at a lot of really cool biology.
We’ve found connections between RNAi and lifespan regulation
in flies, stem cell biology in flies, and genome stability.
That’s an aspect that I never could have anticipated—the
incredible depth of biology associated with RNAi.
So
suddenly you’re seeing applications of this research everywhere?
That’s right. That’s part of the reason for the
incredibly frantic pace in the field. Suddenly unexplained
phenomena and data all over the world are starting to make
sense to people when put in the context of this new cellular
pathway.
How
does that frenetic pace affect your own life and your own lab?
The main effect for me, personally, is that I’m a lot
more tired than I used to be. And I worry a lot more about
protecting my students from the pressures of such a
competitive field.
How
does one go about creating successful scientists while
simultaneously protecting them from the competitive pressures?
It’s just like childrearing and protecting your kids from
the scary things in life. It doesn’t mean pretending they
don’t exist. It’s giving them the tools to cope with them
successfully. For my students, I try to teach them how to be
thoughtful in designing and carrying out experiments, so
they use their time productively. I also try to foster a
spirit of community in the lab, where everyone supports each
other. In that way, when the competitive pressures become
overwhelming, people can turn to others in the lab for help,
both in terms of emotional support and in simply getting
help to work on a particular project. This is probably why
we always have multiple authors on most of our papers. It
puts the science first rather than the ego.
I also try to teach my students to have respect for the
other labs in the fields, the labs they’ll end up competing
against. It’s important to realize that any competition is
secondary to the beauty of the work that the lab does. And
one of the wonderful things about our field is that despite
the fact that it’s so competitive, there are a lot of really
nice people in it, people whose company I enjoy. It makes
the research a lot more fun.
Could
this be in part because the field is so fertile and there are so
many discoveries to go around?
True. That’s probably because there are still about 100
cookies per lab. When you get into a field where there are
100 labs per cookie, it’s more of a problem. But those
fields fix themselves too, because the smart people leave.
Nobody should stay in a field like that.
Five
years from now what do you think we’ll have learned about these
small RNAs?
I’m pretty sure we’re going to know almost the entire
small RNA content of every major model organism plus humans.
And we’re going to have crystal structures of most of the
major complexes in the pathway. We’re still going to be
struggling with describing in detail the molecular
interactions and conformational changes that lead to the
regulatory events in the various small RNA pathways. I think
we will be grappling with that for a long time, since it’s
such a hard problem. I think the amount of biology
associated with small RNA is going to increase
exponentially. People will still be scratching their heads
trying to understand why this type of regulatory system is
used for so many different things. What’s advantageous about
it for the cell?
Also I think there will be a much greater appreciation in
the field about the evolutionary significance of making
pathways so robust, making them free of noise. Right now
there are people who appreciate it, but most molecular
biologists don’t think much about the role of reducing noise
in bio-regulatory pathways, certainly not in terms of the
critical importance for evolution—that organisms able to
make their regulatory pathways less noisy are inherently
more successful. Although in some circumstances, that noise
actually drives evolution forward. Those are areas in which
small RNA are going to prove to be really important.
What
do you mean by noise in the context of regulatory pathways?
Regulatory mistakes. For example, cells in which certain
genes need to be off 100 percent of the time, but they’re on
a very little bit, despite regulatory pathways designed to
turn them off. I think there’s growing evidence that this is
one of the jobs that RNA does, especially microRNA. It
cleans up that noise. It helps make "off" really look like
"off."
If
you could do one experiment and funding, time, and resources
were no object, what would it be?
Single-molecule studies of conformational dynamics in
RNAi pathways. I want to look at a single molecule of RISC,
the complex that carries RNAi, and follow it through all the
different conformational changes it undergoes to become
loaded with small RNA—fully active, bind to target, cut
target, and release the piece. Something truly spectacular
and unexpected is happening in that cycle.
Once we understand that, we will know why cells have
machinery to create RISC. They have whole pathways designed
just to assemble RISC, and we’ll understand why the
so-called Argonaute proteins are uniquely evolved for small
RNA-guided pathways. At the core of every small RNA-guided
pathway that’s ever been discovered, there are always these
Argonaute proteins holding onto the small RNA and mediating
its function. I think those kind of single-molecule studies
would tell us why these Argonaute proteins are so special.
Phillip D. Zamore, Ph.D.
Department of Biochemistry and Molecular Pharmacology
University of Massachusetts Medical School
Worcester, MA, USA
n
the interview below, Special Topics correspondent Gary Taubes
talks with Dr. Phillip Zamore about his highly cited work in
gene silencing. Dr. Zamore is ranked at #9 in our analysis of
the topic, with 23 papers cited a total of 3,805 times. In
