Transcript mRNA processing: no longer a headache!

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE
mRNA Processing:
No Longer a Headache
by
Kristen H. Short
Department of Biology
Manchester University, North Manchester, IN
Learning outcomes:
1) Differentiate between a pre-mRNA and a mature mRNA in
eukaryotes.
2) Explain the mechanism by which introns can be removed during
mRNA processing.
3) Explain how alternative splicing can allow a single pre-mRNA (and
thus a single gene) to give rise to several unique mature mRNAs.
4) Explain how monoclonal antibodies can be used to target molecules
in the body, contributing to drug development.
5) Evaluate the significance of alternative splicing in molecular biology
and physiology.
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One day in late May…
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Comic strip generated at http://www.MakeBeliefsComix.com. Used by permission of author and site creator Bill Zimmerman.
Two weeks later…
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Comic strip generated at http://www.MakeBeliefsComix.com. Used by permission of author and site creator Bill Zimmerman.
Lucy, I have to tell you what
I’ve learned about ALD403!
It’s an antibody that blocks
the protein calcitonin generelated peptide (CGRP), which
is encoded by the CALCA
gene. It’s being tested to see if
it reduces migraines in people
like me. I wonder if it will
work…
That’s neat, Dan! I hope it does
work for you! I’m confused
though, because my lab is
working on the CALCA gene,
which encodes the protein
calcitonin. You’re saying the
CALCA gene encodes the
protein CGRP. Are you sure you
didn’t get the protein
confused?
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No, I’m quite sure that ALD403
is a CGRP inhibitor. That makes
sense too, because when I
found out about this drug I did
some research and found that
CGRP plays an important role in
migraines. An inhibitor of that
protein could be a useful drug.
Hmm, maybe we need to do
some more background
research to figure out why
we’re getting confused. I’ll call
you next Monday!
Well, how can we be talking
about two different proteins
that are each supposedly
encoded by the same gene?
I’ll talk to you then!
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CQ#1: Can a single gene code for more than one
unique protein?
A. Yes
B. No
C. I don’t know
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Calcitonin
• 32 amino acid linear polypeptide
• Produced in thyroid gland
• Lowers blood calcium
Amino acid sequence of calcitonin:
Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-PheAsn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
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CGRP (calcitonin gene-related peptide)
• 37 amino acids
• Produced in neurons
• Acts as a vasodilator and functions in transmission of pain
Amino acid sequence of CGRP:
Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-SerArg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-SerLys-Ala-Phe
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If we look at the amino acid sequences
together…
Amino acid sequence of calcitonin:
Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-PheAsn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro
Amino acid sequence of CGRP:
Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-SerArg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-SerLys-Ala-Phe
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We’re obviously not talking
about the same protein.
Calcitonin is produced in
thyroid tissue, and CGRP is
produced in nervous tissue,
and they have different
functions. We already know,
too, that CGRP is the one
implicated in migraines.
They have completely different
amino acid sequences too. We
know that proteins with
different amino acid sequences
probably have different
structures and functions.
Let’s take a closer look at the
CALCA gene, in order to
understand the relationship
between these two proteins.
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The CALCA gene
5´--
1
2
Poly “A” sites
Calcitonin exon
CGRP exon
4
5
3
--3´
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Key features:
• 6 expressed sequences, or exons
• Exon 4 codes for 32 amino acids to form calcitonin
• Exon 5 codes for 37 amino acids to form CGRP
• Two polyadenylation, or poly-A sites, recognized by enzymes that can
facilitate cleavage and poly-A addition at the 3´ end of the corresponding
mRNA
• Intervening sequences, or introns
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CQ#2: Will the pre-mRNA (the initial product of
transcription of this gene) contain all the exons and
introns?
A. Yes
B. No
C. I don’t know
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I’m still wondering, though,
how this single gene can code
for different proteins, if only
one unique pre-mRNA can be
formed during transcription,
and it contains all the exons
and introns.
This is starting to clear a few
things up. Both calcitonin and
CGRP are encoded by the
CALCA gene, but they
correspond to different regions
of the gene.
Then let’s investigate how the
pre-mRNA is processed to form
the mature mRNA.
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CQ#3: Which of the following modifications to a premRNA molecule in eukaryotes will occur before it is
considered a mature mRNA molecule?
A.
B.
C.
D.
Addition of the 5´ cap
Addition of a poly-A tail
Splicing to remove introns
All of the above
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A closer look at pre-mRNA processing
Let’s consider this gene with three exons, numbered 1-3:
DNA
5´--
--3´
1
2
3
During and after transcription but before splicing, the pre-mRNA
receives two modifications:
RNA
5´-- G
AAAAA…..AAAA--3´
--3´
1
2
3
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A closer look at RNA splicing
Now let’s splice to remove introns:
5´--G
AAAAA…..AAAA--3´
1
………………..……AG
2
3
x
GU……A……AG
G………………………
The spliceosome recognizes
sequences at intron/exon
boundaries and cuts introns
out
With both introns removed, our mature mRNA now looks like this:
5´--G
AAAAA…..AAAA--3´
1
2
3
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What if exons are also sometimes removed?
CQ#4: How many unique mature mRNAs can theoretically be
formed from this single pre-mRNA?
Assume that this pre-mRNA can be spliced to include any
combination of exons in their original 5´-3´ order, all introns are
removed, and that exon 1 does not have to be the first and exon 3
does not have to be the last one in the mature mRNA.
5´--G
A.
B.
C.
D.
1
2
3
7
AAAAA…..AAAA--3´
1
2
3
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Alternative splicing
• Refers to splicing of pre-mRNA molecules from the same gene in
different ways, to produce a variety of different mature mRNA
molecules, each of which contains a different combination of exons
and introns, and therefore codes for a unique protein
• Helps to explain why humans can produce more than 100,000
proteins with only an estimated 19,000 protein-coding genes
• Can account for differences in gene expression in different tissues, or
at different stages of development
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So now we can explain how we
might be talking about two
different proteins that are both
produced from transcription of
the same gene. They produce
different mature mRNAs
through alternative splicing of
the same pre-mRNA, maybe.
Right, and different mature
mRNAs code for different
proteins, of course. So the
CALCA gene could code for
multiple different proteins.
Yes, let’s take a more detailed
look at the CALCA gene and its
alternative splicing now.
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Alternative splicing of the CALCA gene
Poly “A” sites
DNA
5´--
1
2
3
Calcitonin exon
CGRP exon
4
5
Thyroid cells
--3´
6
Neuronal cells
RNA
5´-G
Calcitonin exon
1
2
3
4
A..A-3´ 5´-G
A..A-3´
CGRP exon
1
2
3
5
6
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Translation and post-translational processing
Poly “A” sites
DNA
5´--
1
2
3
Calcitonin exon
CGRP exon
4
5
Thyroid cells
--3´
6
Neuronal cells
RNA
5´-G
Calcitonin exon
1
Protein
2
3
A..A-3´ 5´-G
A..A-3´
CGRP exon
4
1
2
3
Calcitonin (32
amino acids)
(Other peptides corresponding to
other translated exons are
removed during post-translational
processing and not shown here)
5
CGRP (37
amino acids)
6
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CQ#5: True or false: If we extracted DNA from thyroid
and neural tissues, we would find the CALCA gene in
both types of cells.
A. True
B. False
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CQ#6: True or false: If we extracted mRNA from
thyroid and neuronal cells, we would find the same
sets of mature mRNA molecules in both types of cells.
A. True
B. False
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Right. I’m still curious, though,
about the ALD403 drug I’m
taking in the clinical trial. I’m
going to look for more
information about how it
works, and what effect it has
on proteins associated with the
CALCA gene.
So cells in different tissues of
the body all contain the CALCA
gene, but they can produce
different proteins from it,
depending on how the premRNA is spliced before
translation.
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CQ#7: Theoretically, a medication that reduces the
activity of CGRP and therefore prevents migraine
headaches could …
A.
B.
C.
D.
E.
Target and block the CGRP directly
Target and block CGRP receptors on cells
Prevent transcription of the CALCA gene
Prevent translation of the CALCA mRNA molecules
All of the above are possibilities
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ALD403: blocks activity of CGRP directly
• An antibody (similar to antibodies produced by your own immune
system)
• Binds with CGRP, the antigen, to block its activity
• Treats migraine headaches by reducing CGRP activity, reducing pain
Antibody: protein that
binds very specifically
with antigen
Let’s pretend
this is CGRP:
Epitope: site where
antibody binds to antigen
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CQ#8: If an antigen is a protein, then whether or not
an antibody binds with the antigen probably depends
on …
A. The amino acid sequence of the antigen
B. The secondary, tertiary, or quaternary structure of the
antigen
C. Whether or not there is a mutation in the antigen
D. Antibody binding would depend on any/all of the above
items
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CQ#9: Would you expect ALD403 to block activity of
the calcitonin protein in the body?
A. Yes
B. No
C. It’s unlikely, but we couldn’t be sure without more
information
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That makes sense. The
antibody is highly specific so its
effects should be limited to
CGRP.
Yes. My doctor said that earlier
clinical trials have shown this
drug to be effective at treating
migraines, without too many
side effects. Understanding
alternative splicing certainly
helps us understand how this
all works!
ALD403 does not interfere with
calcitonin activity, because the
proteins have entirely different
structures, so the antibody
does not bind to calcitonin the
way it binds to CGRP.
If the medicine was able to
shut down transcription of the
CALCA gene, that might be a
different story!
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Discuss with your group:
What is the significance of alternative splicing? Why is it important for
us to study and understand this process?
What does this example illustrate about the relationship between a
gene and a protein?
What questions have arisen for you during today’s class? How could we
answer them?
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