I'm Philip A. Sharp,
an Institute professor at MIT
in the Koch Institute for Integrative Cancer Research.
I want to talk about RNA splicing
and the discovery of split genes,
but I want to set it in the context
of the question of "What is a gene?",
because we use the word gene when we talk to each other,
but when we start thinking about it
in the context of specific biochemical structures,
genes are very complicated,
and we really find it very difficult
to define a gene,
and this can be quite important
in terms of understanding biology.
Many years ago,
when I began in molecular and cell biology,
the gene was really quite simple,
and it's illustrated here
by a diagram from Jacob and Monod,
which was published in 1961,
illustrating the lac operon,
and the structure of the genes in the lac operon
and the messenger RNA that comes from those genes,
and then the proteins you see
at the bottom of this illustration,
of galactosidase, permease, and others,
that are encoded by that messenger RNA.
And, in 1961,
this explained what a gene was.
A gene was, like Z in that diagram,
a linear sequence encoding and transferring information
into an RNA in a linear fashion,
and then a protein being coded from it.
And we even understood
in this year, in 1961,
that there were sequences nearby, operons,
that were cis to the gene
that were critical to the expression of a gene.
So, in the late 60s/early 70s,
when I entered cell biology,
everyone suspected that they understood a gene,
to the point that Monod actually made the statement
that what is true of bacteria is true of an elephant.
Well, by and large, that is the case,
but an elephant is a little more complex than a bacteria,
as we will see.
Cell biologists have long suspected,
even in the early 70s,
that genes in mammalian cells
were somewhat more complicated,
maybe there were mysteries here.
And this influenced by own thinking
as I moved to Cold Spring Harbor
to begin to study animal viruses.
And some of the hints that there might be
a difference in the genetic structure
in mammalian cells, cells with nuclei,
was two general facts.
One is, if you look at the nucleus of a cell,
you see the outline of nucleus,
and this is the nucleolus...
we knew that in the nucleus of a mammalian cell,
there was a thousand-fold higher levels of DNA.
It was a thousand-fold more complex
than in bacteria.
But it didn't seem that mammalian cells
would need a thousand-fold more genes.
So we had a lot more DNA in the nucleus of the cell
than we suspected we needed for genes.
And then another issue was that
when we metabolically labeled RNA in the nucleus of a cell,
it was much larger than the RNA
that we found in the cytoplasm of the cell,
and there was this dichotomy.
It was larger by a factor of five
than the size of the message in the cytoplasm.
So, there was this paradox...
long nuclear RNA, smaller cytoplasmic RNA...
the long nuclear had a cap at the 5' end,
and we'll talk a little bit more about that in a moment,
had polyA at the 3' end, yet it was longer,
and the message had the same modifications
and it was shorter.
So, in 1971,
I moved to Cold Spring Harbor to start
learning how to work with animal viruses,
particularly adenoviruses,
DNA viruses that caused cancer in animals.
And one of the viruses that we worked with
was adenovirus 2,
and this is a micrograph of adenovirus 2.
It's got a beautiful virus with capsid proteins,
primarily of the hexon protein,
that constitutes most of this capsid.
It has its fibers at the end,
that you see here, that are to attach to cells.
It's long enough as a genome
to have 35 genes encoded,
so it was a wonderful viruses to work with
to probe the cell biology of cells,
as well as to study its ability
to cause cancer in animals.
So, we started, in 1970,
at Cold Spring Harbor,
determining the structure of the genome of adenovirus 2.
And that led us to the development of
restriction endonuclease maps,
and these are two examples
of restriction endonuclease maps of the adenovirus genome.
One is the EcoRI, here at the top,
and the other is HindIII, at the bottom.
So, we mapped the cleavage sites
of these restriction enzymes across the genome,
and therefore were able to start
preparing a map of the relationship of RNA
to genome structure.
And I want you to recall two restriction fragments.
One is this HindIII 'A' fragment, here,
and another is this EcoRI fragment, right there.
And this hexon messenger RNA
- we knew that the messenger RNA
mapped across the genome in this position -
and we wanted to compare the structure
of the messenger RNA from this hexon protein
to the nuclear RNA that was expressed
in the nucleus of the cell,
and we were going to do this by electron microscopy,
which I had learned at Cold Spring Harbor...
I mean, at Cal Tech, when I was a postdoc
with Norman Davidson.
So, when we did that,
and we in this case is
Sue Berget and Claire Moore
- Sue a postdoc, Claire a technician in the lab -
and we did an R-loop mapping
between the messenger RNA for hexon
and the genome.
What we observed is shown in these series of diagrams.
What is shown all the way over here to right
is the HindIII A fragment in total,
and then this is a blowup of a piece of that R-loop.
And what you see here is this section
of the electron micrograph is the RNA/DNA hybrid.
And what was puzzling to us was,
here, a little tail at the 3' of the message.
And what was really puzzling
was this little this little tail at the 5' end of the message.
Now, the 3' end tail, we understood,
that was the polyA tract,
but there shouldn't have been a tail at the 5' end of the message.
It should have annealed to the DNA sequences.
And we spent a large amount of time
trying to do controls to show
that that tail really didn't come from the DNA sequences,
and therefore that led us to the question of,
well, where did these sequences come from?
I asked you to remember the EcoRI fragment
because that allowed us to do
a very direct experiment
to test whether the upstream sequences
in the adenovirus genome
had the sequences to this tail.
And what we observed when we made the RNA/DNA hybrid
between the long fragment and the message for the hexon
is illustrated here.
This shows you a diagram of it, here.
This is the actual micrograph, there.
And what is striking about this micrograph,
if you look in this region,
is there are three loops,
and that immediately told us that
that tail had sequences from three different regions
of the adenovirus genome.
And we show it here in a diagram
- an A loop, B loop, and C loop.
And that told us that the RNA for this hexon message
was actually being joined together
out of four different segments,
and it's diagrammed here in this diagram
of the RNA/DNA hybrid,
You can see the hexon RNA message,
that was the end of that electron micrograph,
you see the C loop,
the B loop, and the A loop
being created by the annealing
of these sequences off in the genome here,
a', b', c',
to the hexon message down here.
And that immediately told us
that this RNA for the hexon message
was actually coming from a long RNA
that was being transcribed from a, b, and c,
all the way through the genome
and then spliced together
to link these three segments to the body of the message.
So, that told us that the secret
between the long nuclear RNA
and the shorter cytoplasmic RNA
was this process of RNA splicing,
the removal of sequences
from the middle of the messenger RNA,
a suggestion no one had made before,
and it told us, more generally,
that all the genes, or most the genes,
in these complex mammalian cells
are expressed by this process of RNA splicing.
So, in 1977,
when this work was announced and published,
we created this diagram that illustrates,
in the same way that Jacob and Monod did,
the flow of information from DNA
into the messenger RNA,
but in this case in mammalian cells,
as contrasted to bacterial cells.
And what you see here, then,
is the promoter
where the RNA is initiated,
the cap is synthesized,
and from the work of Erin Shatkin and Bernie Moss,
we knew a great deal about the chemistry of the cap,
and then over here where there was polyA cleavage,
cutting of the RNA at a polyA site,
and a polyA added,
making a precursor RNA
that then is spliced
before the RNA is transported to the cytoplasm, here.
So, we diagrammed, then,
the flow of information from DNA
into messenger RNA in mammalian cells.
Now, this diagram is a little simplified,
because most genes in mammalian cells
have ten different introns, not just one,
and therefore there's much more splicing.
And in many cases that splicing is nascent.
While the RNA is being synthesized from the DNA...
doesn't occur in a polyadenylated precursor,
but in about one in ten cases
- one in ten introns -
it is removed posttranscriptionally,
with this polyadenylated precursor RNA.
So, we outlined the pathway
of the synthesis of messenger RNA
in higher cells,
and we knew those cells had a much more complex structure.
Now, just for completeness,
and because no one is interested
in adenovirus anymore,
just let me illustrate the complexity
by which a virus
uses this process of RNA splicing to express itself.
You see, as we said before,
there are three different leaders,
exons A, B, and C,
we saw in the previous diagram,
and those are spliced to all of these messenger RNAs
across the genome.
So, there's a long RNA made
from the left end of the genome across the genome
and all of these sets of RNAs
are processed from it.
And, for example, this is a polyadenylation site
where the hexon message
is actually produced by splicing,
and this is another polyadenylation site
where another major messenger RNA is used.
Each of these involves a long precursor
across the whole genome,
alternative polyadenylation at different sites,
and the splicing of those three leaders
to the end of the message.
But when we look at the other transcriptional units in the virus,
here's one, that's the E4, E3, E2, and E1
on the left side, over here,
you see, again, very complex splicing
of alternative polyadenylation
and alternative splicing
accounting for the complexity of the message
from this adenovirus genome.
This has led us to think about what is actually a gene,
and when you start thinking about a gene,
then you ask yourself,
what's the unit of information in a gene
that actually accounts for the functional unit of a gene?
So, this is my favorite gene.
This is from Larry Zipursky,
who's at UCLA.
It's the Dscam gene,
it's expressed in the nervous system of Drosophila,
and it produces 38,000 different proteins
from one gene.
Now, each of these little bars, here,
this is a genomic DNA diagram...
each of these little bars here is an exon,
and what you note, here,
is that there are something like 33 different exons,
and you can alternatively splice
one of these into each precursor messenger RNA.
And this has 48 different exons
for exon 6
and 12 for exon 4.
And these are all used in permutation,
producing 38,000 different messenger RNAs
and different proteins.
So, when we talk about this gene,
is it one of those exons
being spliced to another to make one functional unit
that goes into the protein?
Or is it the whole transcription unit,
and all 38,000 different proteins,
with different functions, within the gene?
And each of those exons
could potentially be a gene.
So, this leads us to the question
that the cellular environment
can modify the nature of the messenger RNA
in the transfer of information
from DNA to protein.
In one cell,
the DNA sequence is interpreted one way.
In another cell, it's interpreted in another way,
through alternative splicing and alternative polyadenylation,
and something I haven't spoken about, RNA editing.
So, a given genomic sequence
is actually not a faithful representation
of the functional information expressed from a gene,
and therefore as we look at our genome,
and now we have the genome sequence,
it was published in 2003,
fifty years after Watson and Crick,
and we say to each other, "We have 23,000 genes."
Well, we do have 23,000 transcription units,
which for example accounts for
where the transcription begins
on the left side of this diagram
and moves to the right,
but you see alternative promoters,
alternative polyadenylation sites,
alternative splicing in the middle of the gene, here,
and you see in some cells
you would use this information in the gene,
and in other cells, by alternative splicing,
you would skip that information.
So, what's a gene?
It's a very difficult question to answer.
When we get to the level of biochemistry,
are we looking at functional units
in a genetic transmission sort of thing?
We use the term gene, among ourselves, all the time,
but when we start specifically probing a gene,
the gene is actually a very difficult thing to define.
So, I want to thank you for your attention
and listening to this discussion
of what is a gene.