- I just wanna make a comment.
I think we're all very sad
that Tom's not with us.
I know Tom would have really enjoyed
being here today to
celebrate this with us.
So with that she now went to Cambridge
to do her postdoc and
then came back to Yale.
In 1970 she came to Yale with
Tom and has stayed ever since.
I'm sure you're gonna talk about your
research so I won't talk about that.
(congregation laughing)
Obviously she's very well known
for how RNA interacted with ribosomes
and then the snRNPs.
But I will say the
honors that she's earned,
cause I'm guessing Joan
won't go through that.
The list is so long that
she would have no time
to speak if I actually.
so I pulled out just a few highlights.
She's been elected to the
National Academy of Sciences,
National Academy of Medicine,
American Academy of Arts and Sciences.
She won the National Medal of Science.
She has honorary degrees from somewhere
between 10 and 15 universities,
some of which are Harvard,
Princeton, Columbia,
Rockefeller, Brandeis and Brown.
Joan won the Gairdner Award,
the Albany Medical Center Prize
and most recently the Lasker Award
that honored her research and her support
for the careers of women.
Even with all these accomplishments,
two years ago, she won the
William Clyde DeVane Award
for Teaching Excellence at Yale.
So I think that shows
really what she values.
So I think we are all here today
because of her contributions to science,
to mentoring and to the careers of women.
It's really combining all those three
that makes her so special.
I'm supposed to remind you all
that following this there
will be wine and some food
I guess over in the Harkness Ballroom.
Joan Steitz.
(congregation clapping)
- So, thank you all for being here
and I have to start by saying that
today has been really a real honor,
just reliving the spirit of the day.
They've also been many,
many exciting things
in science that I learned
and just the perspectives
that so many of you and the people
who came from a distance to be here
have brought to some of the issues
that we're still confronting today.
It seems like in a sense
I'm sort of overkill.
Cause so many of the things
that I've wanted to say
and communicate have
already been communicated.
But,
the next slide will tell
you an out line of my talk.
I told Bob not to be too heavy on this
because I do wanna tell you about
how I got to where I
got, namely Yale in 1970.
I'll go through each
one of these episodes.
Tom and I arrived at
Yale as I said, in 1970.
That was just prior to the
time I'd been studying RNA
but it was prior to the time
that we'd began studying non-coding RNA's
and that's what I wanna tell you about.
So this will be the
longest part of my talk.
I won't tell you about everything here
but the highlights of a lot of it.
Finally, at the end I wanna
present some more reflections
on mentoring and women in science
and what the problems are that
we still have to confront.
So I grew up in Minneapolis.
Both of my parents were teachers.
My father in particular
was very encouraging
of my interest in science.
He was also very encouraging
that I attend Antioch College.
He was actually an alumnus and
that was because he fervently
believed as a guidance counselor
that everybody needed to have experience
in what they were going to do as a career
and Antioch had a work study program,
where you not only studied on campus
but you went away and did
jobs various other places
in the country or the world.
The way I found molecular biology
at a time when it was just a nascent field
was because of one of those jobs.
I ended up in Alex Rich's lab at MIT
for two, three-month periods.
I found going through
records the other day,
this wonderful "Newsweek" article,
1963 that happens down here,
to have a picture of Alex
Rich in whose lab I was.
And of John Warner, who was
the senior graduate student
with who was my primary
mentor in Alex's lab.
The article is really pretty interesting
because even in 1963,
it was about the promises
and the hazards of future
genetic engineering.
But on a very different level,
you can imagine from the
way it would be written now.
So in that lab under John's direction,
the task that I was set to
was trying to denature
and renature ribosomes.
At that time it was known
that you could take DNA
and pull apart the two strands.
But it wasn't known about anything else
and people thought well maybe
the same thing would
happen with ribosomes.
That of course turned out to be ridiculous
because you heat ribosomes up,
the proteins fall off,
ribonuclease screw up the RNA
and the ribosomes never came back.
But nonetheless I got very
enthusiastic about science.
(congregation laughing)
Mostly I was really enthralled
because it was the
first time I had learned
about the structure of DNA.
Even though that had
occurred eight years prior
to my experience in the Rich lab,
it was still too new to
have made it into textbooks
or to have made it even into courses.
I just was blown by the idea
that there was an actual
molecule inside cells
that could convey the information
from one generation to another
and then actually be replicated.
Although I had this
wonderful job in Alex's lab
and also several others
while I was an undergraduate
I decided that I should
go to medical school.
The reason I decided that
was when I looked around
at least in my experience,
I had never seen a
woman science professor,
I had never seen a woman head of lab,
but I did know several women physicians.
So I applied to medical school
and was about to go off.
What changed that, was the summer before
I was to enter Harvard Medical School.
I ended up working in Minneapolis.
I wanted to be with my
parents for a short time.
In the lab of Joe Gall,
who many of you will know
as a fabulous cellular biologist
who spent most of his career here at Yale.
That was the summer when he was just
in the process of moving from
the University of Minnesota
where he'd been an
assistant professor to Yale
in order to really
establish his career here.
I wanna show you a wonderful picture of me
that was taken by Joe Gall
during a partial solar eclipse.
I think it's absolutely gorgeous.
So Joe was the first one who
set me on my own project.
Prior to that working in labs,
I'd been helping other
people do their projects.
By August 1st, I was so enthralled
by making discoveries
in science of my own.
The problem I was working
on was trying to ask
whether the ciliary basal bodies
from tetrahymena had DNA or RNA in them.
Mitochondrial DNA had just been discovered
and it was suspected that other organelles
might have DNA or RNA in them.
So that was part of the
reason why I decided
that I shouldn't go to medical school
but that I really loved
making discoveries in science.
Because of the fact that I
had actually met Jim Watson
when I was in Alex Rich's lab at MIT,
I had a conduit into asking
whether I could switch
from the medical school to a PhD program.
As a result in the fall of 1963 I entered
a new PhD program at Harvard
called Biochemistry and Molecular Biology.
So as is still the case,
I spent my first year taking courses.
At the end of the first year,
I was still very much
interested in basal bodies
and what they did and what
they might have in them.
I went along to a very
famous cell biologist
in the Harvard Biology Department
and said I'd like to work in this lab.
He looked at me and he
said, "But you're a woman
and you'll get married
and you'll have kids
and then what good will
the PhD would've done you?"
I made it out of his office
before I burst into tears
and I went along to my second choice
thesis advisor Jim Watson.
And said will you take me.
Can you imagine.
This is history?
(congregation laughing)
Jim said, "Yes."
It wasn't until afterwards
that I found out
that I was the first
woman graduate student,
that he had ever said yes to.
Being in the Watson lab in that
year was extremely exciting.
Let me just put up one of these famous.
Every summer there was a
so-called rhino picture.
Those of you who've
been to Harvard bio-labs
know that there are two big rhinoceroses
outside the front door.
Jim would always gather
all the members of his lab
to have an annual picture take.
So here's Jim Watson and
Wally Gilbert was also sort of
co-runner of the lab at that point.
I think this was 1965.
Other people you might know,
this is Mario Capecchi.
Hiding back here is Peter Moore
of the Yale Chemistry Department.
I think that's probably about it.
But it was a very exciting
time in molecular biology.
The field was still very new,
maybe 50 labs total around the world.
We were in constant
communication with them.
They would come in, there would
be visitors that would come
to our daily teas and talk
about what they were doing.
Everybody wanted to exchange information
and share ideas with everybody else.
This was a time at which the basics
of protein synthesis
were being worked out,
the beginnings of
understanding DNA replication
and RNA transcription.
So it was a very, very exciting time.
And also I should say,
many of the things I'm gonna say later
and some of the things that I've said here
were things that I learned
in Jim Watson's lab
cause it was a tremendous experience
with many, many really
wonderful colleagues
and Jim turned out to
be a wonderful mentor.
When it came time for
me to choose a postdoc,
I was married to Tom.
This is a picture taken at
Harvard shortly before we left.
I went to the mecca of
protein crystallography,
Cambridge, England.
Because that was Tom's field
and that's what women did.
Luckily it turned out
to be not a bad place
in molecular biology either
because I ended up in the division
that was headed by Francis
Crick and Sydney Brenner.
(congregation laughing)
When I got there, I had a position
but I didn't have a project.
I went around and talked to a
lot of the people in the lab.
It was sort of a very loose
structure, not small groups,
but sort of big floors of
people doing the same things.
I found out about this
one intriguing project
that I knew many of my male postdoc peers
had considered and rejected
because it was too challenging.
But at that point, I had no expectations
of ever having to have
something to show in two years
to go back to the states to find a job.
I never expected that
I would be a lab head,
I just thought I would
be a research associate
in somebody else's lab the way women were.
I said this is a great project
why don't I take it on.
I'll tell you about what that
project was in just a moment.
So I did that, and after a
couple of years of struggle,
it did succeed and then it
was time for us to leave.
And let me now.
Go into coming to Yale.
So we actually spent about
half a year at Berkeley,
where my husband had
already accepted a position
and was teaching of course.
When we inquired about a
possible position for me,
we were told that all our
wives are research associates
in our labs, they love
being research associates.
Doesn't your wife wanna
be a research associate?
But armed with a couple of offers
of being an assistant professor,
which was still highly unusual
and it took a lot of courage
and thinking about it.
I felt that I did want
to address the challenge.
That was when both Tom and I
took up the job offer at Yale.
So we came here as you've heard in 1970
to a department called Molecular
Biophysics and Biochemistry
which was a brand new department
under the leadership of Fred Richards.
Fred had acquired something like six slots
and had hired a co-group
of young junior faculty
who although all the others were male
we nonetheless formed a
very closely knit group
and helped support each
other through the early years
of being on the faculty
and that was really great.
So what I wanna go on and
tell you about then is briefly
and hopefully won't take too long.
What I call adventures in
the noncoding RNA world.
Still what I wanna cover here,
is first of all this goes
back to my post-doctoral work
and I'll say a little bit about it.
Things that I wanna emphasize
is the role of serendipity
in this course in science.
Also how my fixation on base pairing,
which developed very early carried through
to so many of the things
that we ended up doing.
So I'll tell you at the
beginning about ribosomes
and base pairing and then
the story which I suspect
several of you at least have heard lupus
and the use of lupus antibodies
to discover the small particles in cells
that are involved in splicing.
This is and especially a nice story
because it takes clinical tools
and applies them to basic research
to make fundamental findings.
But just recently, they've
gone back to the clinic.
Which I something that
I find very satisfying.
I won't, this is in lighter print
because Susan Baserga has
already told you about
part of this story with the U3 snRNP.
But there are other base
pairing interactions
that we investigated.
I'll say a bit about
the second spliceosome
and then a bit about what we're doing
now with viral noncoding RNA's
and particularly about the
discovery of triple helices
in RNA which we're still struggling with.
So let me start with bacterial,
you have a ribosomes in based pairing.
What I had dome for my post-doctoral work,
I call Single-molecule ribosome profiling.
Because the experiment was
to take P32 labeled phage messenger RNA,
bind it to ribosomes under conditions
where you could initiate protein synthesis
but not actually translate.
So they just sit there at the beginning,
use ribonuclease to trim
off the ends of the RNA,
isolate the pieces that were
associated with ribosomes
and sequence them using the
methods for RNA sequencing
that had just been developed
by Fred Sanger at the MRC lab
in Cambridge where we were.
And I had come up in a couple of years
with these three sequences
which were very satisfying
because they all started
with an fMet codon
and then had the right sequences
for the phage specific proteins.
What I didn't notice at the beginning,
which became important later,
was the upstream of the AUG
but at variable distance.
We were disappointed that there was no
specific sequence there but you will note
that there are these purine-rich regions.
Those were only recognized
as being important in 1974
by John Stein and Linda Cano,
Australian scientist
who had stumbled across
the fact that these sequences
were potentially complementary
to the sequences the three
prime end of the ribosomal RNA
in the small ribosomal
subunit the 16s RNA.
This got me a job but the question is,
what did this mean?
Given Shine and Dalgarno's hypothesis,
the question was how could you possibly
prove that this was really
true that RNA-RNA base pairing
was a basis
of the initiation of protein
synthesis in bacteria.
I finally came up with this experiment
where you make these protected complexes,
you treat with a bacterial toxin,
that's bacteriasome bacteria make
to kill other bacteria called
Colicin E3 that cleaves 16s
RNA about 50 nucleotides
from the three prime end.
You then carefully
dissociate the ribosome,
take off all the proteins.
But do it under conditions
that does not destroy
RNA interactions, run it on a gel
and you can actually see the
complex made up of this short.
It was this region here,
the initiator region for this gene
and the and the three
prime end of 16s RNA.
So that was the first evidence
that this was really true
that there was base pairing.
I fell in love with base
pairing at that point
and if you'll see it's a theme
and everything else I'm gonna say.
If one thing's back now
to the late 1970s,
no I'm sorry to the early 1970s
of what was going on in molecular biology.
It was really a very weird time
because by then we knew the
basics of DNA replication,
RNA transcription, protein synthesis
and this had all been discovered
by working on bacteria
and their bacteria phages.
Because it was firmly
believed by the field
at the start of molecular biology,
just 10 or 15 years earlier.
That if you didn't start simple,
you would never be able
to figure anything out.
At that time I remember
when I was in Watson's lab,
there were even counter
writing comments made
about people trying to
work on mammalian cells
saying that they were working
on something so complicated
who would never be understood.
But in the early 1970s,
a lot of scientists thought
that everything important had
been discovered in bacteria.
When we did get to higher organisms,
there wouldn't be any
fundamental differences.
There would just be
more bells and whistles.
But there were some niggling things
that had been discovered by that time
and one of them as
represented in this slide.
Namely that the amount of DNA
in ourselves is way too much.
This is the amount of
DNA in a bacteriophage,
a couple hundred genes, the
amount of DNA tenfold as much
in a Bacterium.
A couple thousand genes
but we had a thousand
times more DNA than that
and what was all this extra DNA doing.
Another thing that was known
was that when mammalian cells
higher cells made messenger RNA,
that only about 10%
of the nucleotides incorporated
initially into RNA ever made it out
to the cytoplasm to be message.
The rest of it got degraded in the nucleus
and what was going on?
So during my first sabbatical from Yale,
which was 1976, 77
I decided I wanted to try
to tackle that problem
and how I wanted to do it
was to look at proteins
that bind to nascent RNA so
what we're looking at here
in this lovely picture
taken by Steve McKnight.
This is the DNA, here
are the RNA transcripts
and here these little
particles are the proteins
that are bound to the nascent RNA.
I thought that if I could make antibodies
against these proteins.
If they were binding the
RNAs that was being made
maybe they had something
to do with the decision
about which part of the
RNA molecules got out
and which stayed behind to
be degraded in the nucleus.
So I spent a whole half-year
isolating these things
injecting them into mice
and rats and chickens and everything
and never got any antibodies
because these are very
highly conserved proteins
and therefore very non-immunogenic.
I gave up on that project
and did something else
for the rest of my sabbatical
but as many of you will
recall it was in 1977
that evidence from many
labs around the world.
Phillip Sharp and Rick
Roberts won the Nobel Prize,
came together to tell us
that our genomes really are different,
we have exons that express that bit
introns the bits in between
and it's by a process of RNA splicing
that the messenger RNA is created.
I love this slide,
even though the three-letter code remains
(mumbles)
intact you see.
So knowing that there was a process
this moved the question
to what was the cellular
machinery that could possibly be
very discriminatorily
identifying the junctions
between the sense and the nonsense
and cutting out the
nonsense, throwing it away
and joining back together the
sense bits of the message.
When I came back to Yale
in the fall of 1977,
everybody in my lab honored
to work on this problem,
we were an RNA lab.
But quite frankly, we
were sort of clueless
as how to proceed.
So then happened the first
piece of serendipity.
In late January 1978,
this article appeared
among the short articles
at the back of nature
of a new nature and I've just copied part
of the first paragraph here
and I've underlined the sentence patience
with MCTD makes connective tissue disease,
have high titers of antibody
to nuclear ribonucleoprotein
which also gives a
nuclear speckle pattern.
The reason that that sentence
caught my attention was,
well I've been trying to
make antibodies and failing.
Several people had said to me
you know I think I've heard some sort of
patients that make antibodies
and gets things in the
nucleus that have RNA
and protein in them and you
know maybe that's what you want.
But at that point I didn't
know how to proceed further.
But when this article came
I then knew MD-PhD student
in my lab Michael Lerner,
fresh from all his courses.
I said to Michael do you
know anybody here at Yale
who might have patients that
would have these antibodies?
Michael said,
"Sure, I'll go across the
street and see Hardin."
Hardin turned out to be John Hardin
in the Rheumatology section
and his lab was literally
right across Cedar Street.
Michael went over there
that very afternoon
and came back with several vials of blood
and started to work with them.
Now you'll all realize that
if that had happened today
instead of in 1978,
we would have had to
spend months if not years
filling out human investigation forms,
but that wasn't true back in 1978.
So he got a hold of the serum
that might have these antibodies in them
and started to work with them.
It was rough slogging, he
was working with liver cells
which are bathed in serum.
Serum has ribonuclease, so
as he tried to purify things
they were just getting degraded.
Also we had very, very
bad ways of assesing,
trying to use the antibodies as a probe
of assesing whether we were
actually achieving purification.
I stepped ahead of
myself a little bit here.
We also of course read
the medical literature
and realize that these
things cause auto antibodies
are simply abnormal immunoglobulins
that are directed against
one's own cellular components
and that if a patient
has these auto antibodies
it's likely that immune complexes
will be formed in the serum
because cells are breaking down
and these immune complexes can then
lodge in various tissues.
Fine capillaries under
the skin cause rashes
that give the rise to the name Lupus
and the hair follicles
make your hair fall out
in all of your joints etc.
We also learned that the antibodies
that had been characterized at that time,
were against things that are very abundant
and very conserved in cells.
You've already heard
about the central dogma
of molecular biology but
here's a picture of DNA
making RNA and ribosomes.
In some patients, many
patients had been identified
as making anti DNA antibodies
some making anti ribosome antibodies.
But there was also this other
large class of antibodies
where the target antigen it was nuclear,
it was speckled, was
unknown what was in it
and that was what Michael
Lerner was trying to purify
using the very primitive techniques
that were available at that time.
Then another piece of
serendipity happened.
MBMB had a job advocate by
the name of Joan Brugge.
Some of you may know
that she's just retired
after many years, the
chair of cell biology
at Harvard Medical School.
So she did not come to
Yale she went to Harvard.
But she came and she talked
about her postdoc project
where she had been investigating
virus infected cells
and had used a brand new product
that had come on the
market called Pansobin
which is just another name
for Protein A Sepharose.
That this was a wonderful reagent
for pulling out antigen
antibody complexes.
So that point Michael
discarded liver cells,
went to heal it cells,
labeled them with P 32.
Did the procedure using the Protein A
and it was all of a sudden
able to get profiles
that looked like this.
So here we see what total RNA
from a HeLa cell looks like
looking at molecules from tRNA
size 75 on up to about 180
a lot of different molecules.
Michael's own serum,
happily did not immunoprecipiate anything
but this mysterious serum that
a lot of people with lupus
in fact had this
specificity called anti SM.
Gave a sub pattern of some of those bands
and it was also this Anti-RNP serum
that was mentioned in that article,
that gave just one particular band.
U2 and U1 were known as small nuclear RNAs
they'd been investigated in the labs
of Harris Bush and Sheldon Penman.
U4, U5 and U6 were molecules
that we later identified.
So we knew what
this RNAs were
doing the same experiment
with S 35 methionine.
Michael was able to
show proteins associated
with the U1 and U2 and other four
four or five, six snRNPs.
We knew that the epitopes
were on the proteins
it was just what you see here.
At that very exciting time
we were able to publish in 1980
that it was suspected that
at least this particular
snRNP might be in splicing.
That was because the sequence
at the five prime end of that RNA
was beautifully complementary
by base pairing to the list
of five prime splice sites
that was beginning to accumulate
as people sequence more and more genes.
There were only about 10 genes
in the database at that point that it fit.
The other piece of evidence that we had
that was at least consistent with the idea
that at least the U1 snRNP
might be involved in splicing,
was simply the results
of taking antibodies
and using them on cultured cells.
These were anti ribosome
antibodies from a lupus mouse
and you see that ribosomes
are in the cytoplasm
and also are getting
assembled in the nucleoli
which Susan mentioned to you.
Whereas the anti-RNP serum
that's specific for U1 snRNP.
Lit up the nucleoplasm which
is where the chromatin is.
Where the RNA is being transcribed
and where's splicasing is
taking place not in cytoplasm
and not in the nucleolus.
So we had this wonderful idea,
we were very excited about it.
It was difficult to get evidence
and I just wanted to show you
this article which appeared in science
in June of 1982.
Big problems faced in RNA processing
and it goes on to talk about how maybe
people were excited about the possibility
of snRNPs being involved in splicing.
But the gloss is off the small nuclear RNA
is temporarily at least.
Nonetheless we decided to persevere
and many colleagues in the
RNA field came to the rescue
with ingenious experiments.
For instance,
the labs of Alan Weiner,
he was then at Yale
and Christine Guthrie UCSF,
performed genetic suppression experiments
where they would take
a PRM RNA substrate
and make mutations in
these important sequences
which we knew by consensus
we're at the splice sites
or at the branch site, get
splicing negative phenotypes
and then engineer into those
same cells a U1 or a U2 RNA
that had a compensatory mutation
and show that splicing could be regained.
That of course was very powerful evidence
that this base pairing interactions
were going on with those sites.
We and many other labs that
over the next several years
were able to provide evidence
that not only the five
prime end of U1 here
in this highly conserved sequence in U2
were involved in recognizing
five prime splice sites
in the branch site
but that U4 and U6, U4
serves as a chaperone
bringing U6 into the spliceosome.
U6 then replaces U1 at the
five prime splices site
for the catalytic steps of the reaction
and that in U5 there were our sequences
in this highly conserved loop
that are complementary to
the axons of the five prime
and three prime splice site in stead of
serve again by base pairing
to hold this all together.
So by the late 1980s then
it was pretty much accepted
that there was this extra
step in gene expression
and that these snRNAs in
their pronatious form snRNPs
really were involved and
that they were essential
for the process of gene
expression in mammalian cells.
I just wanna show you this nice picture
of people in the lab at about that time.
This is Michael Lerner,
he now works in Biotech in California.
Sandy Waller is hiding
here, she was a colleague
at Yale for many, many years.
A lot of other people.
That day we were being
visited by Sid Altman
because after we thought we
knew he wants snRNP was doing
we didn't know what the other related ones
that shared protein components were doing
and we thought that they.
Maybe the U2 snRNP might
be Sid Altman's RNA P
for which he later won the Nobel Prize
but that turned out not to be the case.
Okay, so Tom marked us on,
let me just fill in with a few things.
I love these beautiful pictures
that have come out of Oscar Miller's lab
and his trainees like Ann Beyer
that actually showed DNA
with RNA transcripts,
particles building up at what knows.
What one knows have to be the five prime
and three prime splice
sites and then to form
what we would now call this lysosome,
the huge body with all
the additional factors
looping out the intron
before the splicing reaction
is actually performed.
Finally I just wanna fast-forward
to the last couple of years
when we've had these amazing insights,
into the high-resolution
three-dimensional structures
of the spliceosome.
This is from Reinhard Luhrmann's
lab, that's one of them.
The others are Kiyoshi Nigai's
lab and Yigong Shi's lab.
Where we can actually see the spliceosome
at the various steps in the reaction.
What I find so wonderfully satisfying
is all of these RNA, RNA
based pairing interactions
that were initially predicted by genetics
and by Biochemistry.
There they are, exactly as predicted
in one many of the structures
that have emerged in the
last couple of years.
So where did this all go?
Let me now come back just briefly
to the back to the
bedside part of the story.
Obviously splicing can occur in many ways,
alternative splicing namely forming
more than one messenger RNA
from the same set of introns
actually occurs in about 95% of our genes
and as a result perhaps
this isn't surprising
that splicing mutations
account for about 30%
of all genetic diseases.
One of those diseases where was,
there's been a very, very
satisfying outcome as a result
I like to think of understanding about
the splicing mechanism and how cells do it
is Spinal Muscular Atrophy or SMA.
This causes degeneration
of the lower motor neurons
of the spinal cord leading
to atrophy and paralysis.
It's the most common genetic cause
of childhood mortality worldwide.
Positional cloning in 1995
by Judith Melki in France revealed,
however that we have two copies
of this critical gene, the SMN gene
because humans have a 500 kb
inverted segment on chromosome 5
but when you have homozygous deletion
or mutation of the functional
one of these genes.
The child is afflicted with
spinal muscular atrophy
despite the presence of another gene
which only differs in 11 positions
in the entire gene called SM 2.
They are different types
of spinal muscular atrophy
which correlate with different levels
of this protein being made
with the worst type
where children never sit
and die very, very early.
Other completely horrible consequences
if they have slightly
more of this protein.
What is SMN protein?
It turns out to be a
snRNP assembly factor.
You can't make your snRNPs
and make them properly
unless you have an adequate
dose of the SMN protein.
That was mostly discovered
by getting in Dreyfuss' lab
at the university of Pennsylvania.
It further turns out that
what happens with this
second copy of the gene
is because of a couple
of these 11 mutations,
that in that gene alternative splicing
does occurs in properly.
Exon 7, one of the eight
exons in the protein
is not included usually in the splicing.
So what various other labs,
this is now our work have
done much more recently
in addition to studying all the things
that interact with exon
7 in a positive way
to cause its inclusion to
identify various elements
that are recognized by negative factors.
Then they've gone in with
antisense oligonucleotides
that span these regions
again using base pairing.
Found ones that if you bind
them to the pre-messenger RNA
they will then prevent the
binding of the negative factors
for including exon 7 in this result
of there being there exon 7
will be included in the transcript.
Many of you will know
that this has resulted
in the first successful treatment
for infantile onset SMA.
Use of these oligonucleotides
in the first one
and the first ones to come
out was called nusinersin.
Just a couple of years ago,
the phase three clinical
trials were so encouraging
that they had to stop them quite shortly
and give nusinersin to everybody.
Now we have examples like
this comes from the
Cold Spring Harbor book
that they publish this little girl
who at the age of two had
lost the ability even to sit.
Could basically do nothing.
Within six months she was walking
and this has continued to be
a very effective treatment for SMA.
I should cite Adrian Craner's
lab at Cold Spring Harbor
as being instrumental in a lot of this.
So that's the sort of more global effect
of learning about splicing.
More locally, we had a lot of fun.
(congregation laughing)
You can all guess there's one person
we've heard from today in this picture
but this was a crew that
arrived at a Halloween party.
They have exons and introns
and each one is a different snRNP.
Here's a poly-A tail.
(congregation laughing)
So this is the sort of intermediate
in my discussion of auto antibodies.
Because as I told you and brushed over,
we ended up discovering
that the building blocks
of the spliceosome.
We also discovered using auto antibodies
other classes of RNA
like the Ro RNPs that
Sandy Wolin worked on
and show they are involved
in RNA surveillance.
I already mentioned
when I said a few words
about Nancy Andrews that EBER1 and EBER2
which are immunoprecipitated
in EBV infected cells
by anti-Ro antibodies turn out now
to be the marker that's used clinically
for the presence of EBV in tumors.
So I wanna go on and just very quickly
talk about a couple more things.
I told you I wasn't gonna
talk about base pairing
being defining in the
processing of histone messages
or in the modification for ribosomal RNAs
but we spent a lot of time on that.
Early 1990s, it turned out
that the databases were growing
and not all the splice rate sequences
were matching up with what was expected
for the major class of APRI mRNAs
in there in pro sequences.
There seemed to be
another consensus arising
that ended up being called a AT-AC
because on the DNA level,
they had a U with the
five prime splice site
and AC at the three prime splice site.
So they're called attack introns
found in a variety of different proteins
and nobody understood what was going on
and how these could be spliced
if the consensus sequences
matching U1, U2 etc weren't there.
But then what occurred
to both Rick Padgett at the
Cleveland Clinic and to us
was that too low abundance
RNAs that also formed SM
snRNPs like the splicing snRNPs
that had been isolated by Karen
Montzka Wassarman in my lab.
Luckily named U11 and U12
because U11 turns out
to be the homolog of U1
and U12 is a homolog of U2
and if she'd named them backwards
that would have been impossible confusion.
That they had sequences
that seemed to be matching
these new consensus sequences.
At first we thought that
well maybe just the ends
of the intron are identified
by these two minor snRNPs
that are present about 1/100
the level of the major snRNPs
That the 4, 5, 6 which
was at the catalytic core
of the spliceosome would be the same
as four major class splicing.
But then it turned out that in fact
there are minor versions of U4 and U6
that were identified by Wong Yutai
when she was a postdoc in the lab.
To carry farther what
turns out to be the case
is that we do have these
two kinds of spliceosomes.
The minor class which is about
1 in 100 introns
is recognized by this spliceosome
containing these minor components.
The one snRNP that is conserved
between the two is U5.
And U5 I should say,
is right at the catalytic
core of the spliceosome
so it's not surprising
that it might have been
the one that was retained.
The spliceosome has interesting
phylogenetic distribution
that sort of indicates that
it probably formed fairly early on.
Because it can be found
in plants and vertebrates.
Even cnidarians and insects.
It's not found in fungi,
it's not found C.elegans
suggesting that they lost it
but that this is a very
ancient mode of splicing
that probably co arose with
the major class of splicing.
So why is this relevant
in a medical school?
Well it turns out again
they're human diseases
that are associated with defects
in this minor spliceosome.
The first disease that was identified
as having its U4 attack component mutated
has this incredible name.
Anyhow it's a developmental disease
that affects young children.
By now there are seven different diseases
all developmental diseases.
So this is a special spliceosome
that somehow seems to be
acting more on messenger RNAs
that are required early in development.
Again people are thinking hard
about anti-sense therapeutics
that might be able to also deal
with these particular diseases.
At this point I wanted
to finish off the science
by mentioning briefly
that starting back in 1981
with Nancy Andrews work.
We've been investigating non-coding RNAs
made by various herpes viruses
that co fall into this
Gammaherpes virus class
and includes Kaposi's sarcoma herpesvirus,
hepsviridae virus and herpesvirus saimiri
are more distantly related
to herpes simplex and CMV.
These viruses have large
double stranded DNA genomes,
they have both the latent phase
where the DNA exists
as a circular epistome
and the nucleus makes very few proteins
The lytic phase where lots
more viruses are made.
Many of them, are these ones
all make non-coding RNAs
of several types and we've
been trying to figure out
what those might do.
Here you might think
that it would be easier
to find functions for
a viral non-coding RNA
than for host non-coding RNAs.
Host non-coding RNAs could
be doing absolutely anything
but for a virus you would think
that they would be doing something
to enhance the viral life cycle
or perhaps counteract the responses
that the host organism raises
against the viral infection.
Moreover since viral genomes
are relatively small,
if a virus is gonna devote some of that
precious genetic material
to making a non-coding RNA
ought to be doing something important.
As a result, we've devoted a lot of time
over the last few years.
I just wanna mention
here herpesvirus saimiri
like these other viruses
is associated with many
human and primate cancers.
In this case we have the curious situation
which we still don't understand.
That if squirrel monkeys a
natural host are infected by HVS
then you get a productive lytic infection.
But if it goes into New
World monkeys like marmosets
you get aggressive T-cell
leukemia and lymphomas
and a latent response.
So this is just a chart,
I just wanna sort of lay this on you,
saying that given the
dates of discovery here
and the number of questions
we have left over here.
It's clear that even finding functions
for viral non coding RNAs is just hard.
On the other hand the last
decade or so we've seen an uptick
which I think is because
technology has caught up
and finally now we have methodologies
where we can look for
associations between something
like a non-coding RNA
that has bound proteins
and other things in cells
and that's given us a real leg up.
So over here in green
are some of the things
that we've actually been
able to figure out so far
but we still have lots of question marks.
One nice one, this is a project
that Haidy worked on
when she was in the lab.
Has now led us to an investigation
of a process occurring in uninfected sores
called Target Directed
MicroRNA Degradation,
that we think may be very important
in shaping whole micro
RNA populations in cells
because of the fact that the
targets they interact with
can in some cases cause their degradation.
I just wanna mention one
more thing on the splicing
and then we get to my nine last comments.
That investigation of a
non-coding RNA made by KSHV
has led us into the realm
of triple helices in RNA molecules.
Turns out that based on fiber diffraction
back in 1957.
Alex Rich, David Davies
and Gary Feldon Feld
predicted that two strands of poly U
and one strand of poly A
would make triple helices
and they predicted that the triple pair
would look like this.
They were absolutely right,
but it wasn't until
Rachael Mitton-Fry postdoc in the lab
with lots of help from Tom's lab
was able to obtain a structure
for a naturally occurring
triple helix in 2010,
that we actually saw that
this thing does occur
in naturally-occurring RNA molecules
and one of them was in a
this non-coding RNA copain
made by KSHV.
Where what happens is that
the poly A tail gets engaged
in a triple helix here
making Watson Crick base
pairs and hooks 10 base pairs
just as predicted back in 1957
for a store short stretch.
That seems to reduce the decay
of the RNA making it rise
to very, very high levels.
Because it sort of clamps down
and prevents the decay of the poly A tail
which is usually the
first step in the decay
of RNAs in the nucleus.
So we're still proceeding with that
looking for more such structures.
Other structures
involving the poly A tail.
I have a few minutes left,
I hope to talk a little bit about
my reflections on mentoring.
You heard a wonderful panel
with many other things
that we've already we've
already talked about.
I just wanted to present you with my list
that I made a fourth meeting.
So I didn't know what
was gonna come up here.
You will have heard about
these things as I just said
from both the panel and the speakers.
This is my list of advice for mentees.
If making discoveries in
science gives you joy go for it.
Keep exposing yourself
to more diverse aspects
of science than your
own little outer area.
This is why I encourage
people to go to all sorts
of seminars and why we have
the famous C wing seminars
where people in the department
whether they're working on cryo-EM
or splicing or neurobiology
or nematode genetics.
Come together and talk about
what's going on in those labs.
Go to meetings,
Connecting with other
scientists is important
because things can come
flying in from left field.
Take on the most challenging questions.
It's always a question of determining
what is the most challenging
and important question.
That's something I don't
really have an answer to
but I think everybody
sort of has a feeling.
Everyone even undergrads
should have their own project.
That way they take ownership
and have this just joy of discovery
where they're working on something.
They develop a gel and they
know for the first time
that the answer to this
question is what they're seeing.
They're the only person in
the world that knows this
until they tell somebody else about it.
It's wonderful.
Stick to it even if public
opinion is against you
I showed you that lovely nature blurb.
Diverse people come up with
the most creative solutions to problems.
There are actually lots
of studies that show this.
This is the reason that in
my lab we have subgroups
to solve everybody's weekly problems.
There's more people there than just me
and a particular person
who's doing the experiment.
There are four or five other people there
and they provide suggestions
and I think we move
faster because of that.
Somebody already said this,
when you start a new lab
keep the most important
problems for yourself
because new people coming in
don't really know what
they're doing at the beginning
and if you wanna move
ahead you have to do that.
Take vacations.
Choose a supportive partner,
this is so important.
We've already heard examples of this.
Then finally teach both by
mentoring undergraduates
this is very important
and in the classroom
Here I just wanna add one slide.
I was engaged and I'm very proud
of it being one of the co-authors
of the fourth edition of the classic
"Molecular Biology of the
Gene" the textbook for us.
The first edition was in 1965
I think.
No sorry it's 1967 by Jim Watson
It's me and Watson and
Nancy Hopkins of MIT
and Jeff Roberts of Cornell.
So the final thing I wanna
say just a few words about
This pretty much mimmics what I said.
Last June,
at the first symposium
for women in science here
at Yale Medical School.
Is that the problem that we face
women in science, women in
medicine is the lack of equity
as one particularly proceeds
up the academic ladder.
Here I absolutely love this
graph which was published by
Karla Neugebauer who is now at
(murmurs)
in MBMB.
But long before when she
was still in Germany,
long before she arrived at Yale.
That plots as a function of time,
all the way from starting
out students to professors.
The percentage of women in those positions
and the percentage of
men in those positions
and although this is German
you can equate it to postdocs
and junior faculty and professors.
Now in the U.S we're doing a little bit
about twice as well as this
for around 20% to 23% I
think in the life sciences
at American universities nowadays.
So it's also later.
But still if a graph is gonna have
this scissors curve appearance.
Which means that at
each step along the way
starting with approximate equivalents
of men and women getting PhDs.
Man has a better chance of going on
to the next step that woman does.
This is inequitable
and leads to lack of representation
in science at the higher levels.
I learned a lot about this,
when I was asked in 2005
to join the National Academies committee
that wrote this report published in 2006
"Beyond Bias and Barriers."
Fulfilling the potential of women
in academic, science and engineering.
I learned it from Anderson Huntington.
I learned about a lot of feelings
that I had a lot of reactions
that I'd had earlier on
is a part of being on the
committee that wrote this report.
Reading lots of the literature
and cognitive psychology
about implicit bias
and other things that were going on
that could account for the situation.
What do I think it's
the biggest problem now?
I think that everybody
knows about implicit bias.
They know what it is
and they know what is
of trying to prevent it.
But the phenomenon that I think
not enough people know about
is this second phenomenon
called social identity
or stereotype threat.
This is a beautiful paper from 2007 group
with the Stanford Psychology department.
Recognition by a person
that he, she may be devalued
in a setting because of social identity.
Specifically just being in the minority.
So that then encompasses
where women mostly are
in science and in medicine
you're almost always
in the middle already.
As documented in this paper,
what that elicits in
a person male, female,
or anybody is a state of quote vigilance.
You can imagine their
physiological reactions
but there are also cognitive reactions
that are documented in
this particular paper.
Those are negative reactions
and people are uncomfortable
when there in this state.
I think that that's a very,
very large part of the problem.
For me I can think back
and I'll show you a
picture in just a moment
of all she committees that I was on
in the 80s and 90s where I was
the only woman in the room.
Basically didn't dare open
my mouth and say anything
because I was undergoing this.
Just understanding that
that's what's happening to me
and then having the opportunity
to tell larger groups of people about it.
You can't solve a problem
if you don't know what the problem is
but if you do know what the problem is,
you can at least try
to think constructively
and creatively about it.
So I think this is a
huge part of the problem
and it's documented in this study
even 30% women in group is not enough.
You have to have 50% women
in order for women to feel
completely comfortable.
The same thing is true of men
in a couple of situations.
Finally this is something we
can all do something about
and it was just briefly mentioned
in the panel I think today
that women seem to
worry unrealistically about
the far distant future
rather than thinking about the next step.
If I made it this far and
I'm doing it well this far
that next step shouldn't
be so impossible for me.
So discard those feelings of,
Oh am I gonna be like so
and so in another 20 years.
20 years is too far away it's
not worth worrying about,
worry about the next step.
I just promised I would
show you a picture.
So this was in 1984 when I became a member
of the National Academy of Sciences.
Look at the population here, they're about
(congregation laughing)
they're 20 people in this picture,
I think there's maybe
one other woman there.
But this is often the situation
or at least it was back then
on scientific committees.
The National Academy of Sciences
luckily has gotten slightly better
but we still have a long
way to go there as well.
So I'm to the thank yous.
I don't have pictures of,
I can't cite everybody.
Thank you all for the help of
those of you who've talked
and before me have given.
So I'm just to say thank you to my lab
which has always been wonderful.
I have one picture of
my lab this past fall.
These are the wonderful people
that are currently in my lab.
It's because of having such
wonderful people in the lab
that we've been able to accomplish
what we've accomplished
scientifically over the years.
I also need to say thanks to.
This is reiterative but all those people.
Finding that by mentoring.
That there's almost as much joy
in a new discovery
if you could share it
with the younger colleague
as there is if you make it yourself.
That's how I've come to
know and really treasure.
My colleagues in the MBMB Department
have been very valuable.
I've mentioned some of the
medical school collaborators.
The international RNA community
has been a much more egalitarian community
than many sub communities
in modern science.
I'm very grateful to Dan and George,
with whom I've had this long-standing
program project grant
and I wanna finish off
by giving the most important thanks.
To my husband Tom,
who's unfortunately no longer with us
and my son John who's now
38 years old.
(congregation laughing)
So he doesn't look like that anymore
but we happen to be in Cambridge, England
when John was five weeks old
at Fred Singer's second Nobel
Prize for DNA sequencing
the first being for protein
sequencing was announced.
So they're the people who've
really provided me with
wonderful support and
thank you to all of you
for listening and let's go drink.
(congregation laughing)
(congregation clapping)
Thank you.
(congregation clapping)
