The Experiment that Discovered RNA Splicing

And it’s a technique that uses something called
an R loop. You take a double-stranded DNA and you melt it a little bit and
then anneal to it a messenger RNA. So you can imagine, this is one strand of
the DNA and the other strand of the DNA looks like this. So these are all base pairs coming down here and this is normal DNA. And normally
this would be hybridized to this. And the messenger RNA hybridizes to this strand of the DNA. Now the thing about eukaryotic messenger
RNAs is that at the three prime end they have a tail, poly A residues. Just a whole
strain of poly A residues. And at the five prime end, we had predicted that there should be a
primer, there was going to be an extra piece of sequence that wouldn’t match
here, but was coded somewhere else in the adenovirus genome. And I had a clue, I had
an idea in my head of where this was coded. And so what we did was to make a
single-stranded piece of DNA that would hybridize specifically to this. And so the idea is that this would tell us that there was this extra piece of RNA
at the five prime end of the messenger RNA, that is, at the start of the
messenger RNA. And this I envisioned was the primer that somehow became joined to this in
order to make the messages and to provide the signals we were seeing. And so we made this single strand, we made some messenger RNA, that
was just a crude preparation because there were many genes and we thought
they were likely all to look the same way. And then we made the double-stranded DNA
and so we we gave this to Tom [Broker] and to Louise [Chow] and i think Louise
actually did the experiment, so she did the annealing, made the R loop, added the single-stranded piece of DNA and lo and behold, when she looked in the
electron microscope, she saw something that looked exactly like this. And it was
just — the only problem with it was that, in fact, this had folded back in some way. And so it turned that — I’ve not really drawn it properly, let me draw it properly, okay — that the bit at the five prime end
actually mapped to two different segments of this piece of DNA the we’d put in as the marker. And so if we draw a linear, if you
think of a linear adenovirus genome, we’ve got this as the main body of the messenger RNA and down here, there were actually two pieces, which
were actually, we call them leaders, and here was another leader, and these two things, these three things
rather, somehow all become joined together and that’s what this would show, this
diagram was showing, this, all come together. And so, then when we started to map these a
little bit further, we found that, in fact, there was one additional leader, so that there in fact [is] a tripartite
leader that got joined to this gene. But this same leader also got joined
to a gene out here and a gene out here and a gene . . . and so on. All of the late
adenovirus gene all had the same structure. And that was the discovery of splicing. This
was how splicing was discovered. This was really the key experiment the convinced
everybody that what we’ve been saying for about a year was correct and eventually led to the Nobel Prize.

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