Hi, my name is Joan Steitz. I'm a professor at Yale.
And what I'd like to tell you about today is the importance of
serendipity in the discovery process in science.
The story I'm going to be telling you has to do with SNURPs
which are little particles inside all of our cells that are the building blocks of the spliceosome
which assembles on pre-messenger RNAs to remove the introns
and make the messenger RNA that can be translated by the ribosome.
And these little SNURPs, as you see in this slide,
have both RNA molecules and protein molecules in them.
The other way in which this story is unusual is that it's definitely not a bench to bedside story.
Instead it's very much a bedside to bench story
where we made use of something that physicians had known about for a long time
in order to make discoveries about the basic workings of cells.
So the story starts in the early 1970s and at that time of course we knew about the central dogma.
We knew that DNA made RNA, and RNA made protein.
What we didn't know about was introns or splicing.
People had worked out many of the steps in gene expression in bacterial cells
and most people thought that higher cells would be sort of similar.
Perhaps a little bit more complicated but no new principles.
How wrong we were!
What we did know in the early 1970s
was that there were several things that were peculiar about higher cells.
We knew that they had way too much DNA, a hundred to a thousand times more than
we thought they would need in order to encode the number of genes that we thought we had.
And the other things was that RNA,
lots of it got made in the nucleus but about ninety percent of it
got degraded and only ten percent of it ever made it out to the cytoplasm to be a messenger RNA.
So, what was going on?
And as a young assistant professor at Yale,
I had studied RNA structure and function in bacteria and bacteriophages
and I decided I wanted to take on this problem on my first sabbatical leave.
We knew that there were RNA binding proteins inside mammalian cell nuclei
that immediately bound to the newly synthesized messenger RNA and I thought if I had
antibodies against those proteins, I could use them as tools and maybe this would help me
figure out how the cell decides which molecules to degrade and which molecules to send out
to the cytoplasm as messenger RNAs.
So, for half a year, I tried to make antibodies
against these nuclear RNA binding proteins but this was without success
because these are very highly conserved proteins and therefore very non-immunogenic.
So I gave up and did something else for the rest of my sabbatical.
But it was the year 1977 in the summer that introns were discovered.
Evidence came together from laboratories from all over the world
that conclusively convinced everyone that our genes, in fact, were not like bacterial genes
but contained segments of nonsense, the so-called introns.
And that these introns were then spliced out once the entire gene was made into pre-messenger RNA
and that was what enabled the cell to send a fully formed message
`to the cytoplasm to be translated by the ribosomes.
So the question then was: what is the cellular machinery that very precisely recognizes
the ends of the introns and allows the splicing process to be exact?
When I went back to my lab in the fall of 1977 everybody wanted to work on this process.
We were an RNA lab, but frankly we were pretty clueless about what we should do next.
And then came the first piece of serendipity, in January of 1978 a new issue of the journal Nature
arrived in the lab with this rather complicated title, but the important point
was the underlined sentence here which says that patients with MCTD,
mixed connective tissue disease,
have antibodies against nuclear RNA protein complexes.
And this caught my attention because while I was trying to make antibodies and failing
several people had said that they thought they'd heard of some diseases where
patients made auto-antibodies, antibodies against their own cellular components,
that reacted with nuclear RNA protein complexes.
At that time I had in the lab a new M.D./Ph.D. student, Michael Lerner.
He was fresh from his medical school courses and I said,
"Michael, do you know anybody here at Yale Medical School
who might have patients with these diseases? And he said, ""Sure, I'll go see Harden."""
Harden was right across the street in the rheumotology section
and that very afternoon Michael came back
with several small vials of serum from patients with suspected auto-antibodies.
Now, the sobering part of this story is that if this were to happen today,
one would have to spend weeks if not months filling out human investigation forms,
getting it through committees in order to use even just a few milliliters of serum
`from a patient in an experiment.
So, this probably never would have happened.
But in any case, Michael started working with these sera and the idea was that the auto-antibodies
would then recognize the cellular component and we would use
them as tools to both characterize the, to purify and to characterize the function
of the target of the auto-antibodies.
And here, what you see in this picture is the idea that maybe RNA was in there and protein as well.
And we now know that those were SNURPs and that patients with lupus
and other autoimmune diseases often make antibodies against SNURPs.
However, the going was rough. As Michael purified, the cellular component kept disappearing.
We now know because ribonuclease
was in the preparations and kept eating away at the RNA component.
And then the second piece of serendipity happened.
And this was a seminar visit by Joan Brugee who is now professor at Harvard Medical School.
And she came and talked about a new reagent called Protein A that she was using
to capture immune complexes made with lysates of virus infected cells
and antibodies against those proteins.
And the advantage of that was it could be done very fast.
And you could also look at radioactively labeled cell lysates.
And what we see here is that, on the very far side, is the total RNAs in the nuclear extract
of HeLa cells labeled with P32.
Going from the very small ones at the bottom, about seventy nucleotides long,
up to U2, which is about two hundred nucleotides long.
Michael's own serum, which was used
as a control in the next lane immunoprecipitated no RNAs, but what you see
on the right-hand side here, is the pattern that he obtained with various
different auto-antibodies from various patients. And for the first time, we could see what the
RNAs were and then we could also use this to find out what the proteins were
in these little particles. So, what we now know,
is that these very highly conserved and very abundant targets of the auto-antibodies are,
in fact, the SNURPs that, by base pairing,
recognize specific sequences in the pre-messenger RNA,
gather together with a lot of other factors, to form the spliceosome that carries
out the two-step reaction that's involved in pre-messenger RNA splicing
to give, finally, the messenger RNA that can be translated into protein.
This isn't just test tube fiction, here you see a beautiful picture from Anne Byer's lab
in Virginia, showing DNA with nascent RNA transcripts coming off it,
particles building up at the five-prime and three-prime ends of an intron,
coalescing to form a spliceosome, with the intron looped out, which will then
shortly be removed by the actual chemical process of splicing.
So, what I've told you is about how serendipity
and being interested in RNA-protein complexes
in mammalian cells led to the discovery of SNURPs, which contain snRNAs and proteins,
and how that process is essential for making messenger RNA.
So, these snRNAs are, in fact, the first non-coding RNAs that we know to be important
in the regulation of gene expression.
Today we have microRNAs, lots, and lots of others.
And also, as we've understood about the spicing process,
we know that splicing is the reason why
we can have the same number of genes in our genome as the fruit fly Drosophila
and yet be more complicated. And this is because we do splicing in alternative ways,
therefore, making the most out of every gene, making multiple products from every gene.