mRNA - PowerPoint Presentation

Slide1

mRNA

Processing: No Longer a Headache

byKristen H. ShortDepartment of BiologyManchester University, North Manchester, IN

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCESlide2

Learning outcomes:

Differentiate between a pre-mRNA and a mature mRNA in eukaryotes.Explain the mechanism by which introns can be removed during mRNA processing.

Explain how alternative splicing can allow a single pre-mRNA (and thus a single gene) to give rise to several unique mature mRNAs.Explain how monoclonal antibodies can be used to target molecules in the body, contributing to drug development.Evaluate the significance of alternative splicing in molecular biology and physiology.2Slide3

One day in late May…

3

Comic strip generated at http://www.MakeBeliefsComix.com. Used by permission of author and site creator Bill Zimmerman. Slide4

Two weeks later…

4

Comic strip generated at http://www.MakeBeliefsComix.com. Used by permission of author and site creator Bill Zimmerman. Slide5

Lucy

, I have to tell you

what

I’ve learned about ALD403

! It’s

an antibody that blocks the protein calcitonin gene-related 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?

5Slide6

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

.

Well

, how can we be talking about two different proteins that are each supposedly encoded by the same gene?

Hmm

, maybe

we

need to do some more background

research to

figure out why we’re getting confused

. I’ll

call you next

Monday!

I’ll talk to you then!

6Slide7

CQ#1: Can a single gene code for more than one unique protein?

YesNoI don’t know

7Slide8

Calcitonin

32 amino acid linear polypeptideProduced in thyroid glandLowers blood calciumAmino acid sequence of calcitonin:Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro

8Slide9

CGRP (calcitonin gene-related peptide)

37 amino acidsProduced in neuronsActs as a vasodilator and functions in transmission of painAmino acid sequence of CGRP:

Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe9Slide10

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-Phe-Asn-Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-ProAmino acid sequence of CGRP:

Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe10Slide11

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

.

L

et’s take

a closer look at the

CALCA gene, in order to understand

the relationship between these two proteins

.

11Slide12

The CALCA gene

Key features:6 expressed sequences, or exonsExon 4 codes for 32 amino acids to form calcitoninExon 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 mRNAIntervening sequences, or introns12

CGRP exon

Calcitonin exon

1

2

3

4

5

6

5´--

--3´

Poly “A” sitesSlide13

CQ#2: Will the pre-mRNA (the initial product of transcription of this gene) contain all the exons and introns?

YesNo

I don’t know13Slide14

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.

14Slide15

CQ#3: Which of the following modifications to a pre-mRNA molecule in eukaryotes will occur before it is considered a mature mRNA molecule?

Addition of the 5´ cap

Addition of a poly-A tailSplicing to remove intronsAll of the above15Slide16

--3´

A closer look at pre-mRNA processing

Let’s consider this gene with three exons, numbered 1-3:During and after transcription but before splicing, the pre-mRNA receives two modifications:

16

1

2

3

5´--

--3´

1

2

3

5´--

DNA

R

NA

G

AAAAA…..AAAA--3´Slide17

A closer look at RNA splicing

Now let’s splice to remove introns:

17

1

2

3

5´--G

AAAAA…..AAAA--3´

………………..……AG

GU……A……AG

G………………………

x

The spliceosome recognizes sequences at intron/exon boundaries and cuts introns out

With both introns removed, our mature mRNA now looks like this:

1

2

3

5´--G

AAAAA…..AAAA--3´Slide18

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.12

3

7

18

1

2

3

5´--G

AAAAA…..AAAA--3´Slide19

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 proteinHelps 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 development19Slide20

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.

20Slide21

Alternative splicing of the CALCA gene

21

CGRP exon

Calcitonin exon

1

2

3

4

5

6

5´--

--3´

Poly “A” sites

Calcitonin exon

1

2

3

4

5´-G

CGRP exon

1

2

3

5

6

5´-G

A..A-3´

T

hyroid cells

Neuronal cells

A..A-3´

DNA

R

NASlide22

Translation and post-translational processing

22

CGRP exon

Calcitonin exon

1

2

3

4

5

6

5´--

--3´

Poly “A” sites

Calcitonin exon

1

2

3

4

5´-G

A..A-3´

CGRP exon

1

2

3

5

6

5´-G

A..A-3´

T

hyroid cells

Neuronal cells

Calcitonin (32 amino acids)

CGRP (37 amino acids)

(Other peptides corresponding to other translated exons are removed during post-translational processing and not shown here)

DNA

R

NA

ProteinSlide23

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.

True

False23Slide24

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.True

False24Slide25

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 pre-mRNA is spliced before

translation.

25Slide26

CQ#7: Theoretically, a medication that reduces the activity of CGRP and therefore prevents migraine headaches

could …Target and block the CGRP directly

Target and block CGRP receptors on cellsPrevent transcription of the CALCA genePrevent translation of the CALCA mRNA moleculesAll of the above are possibilities

26Slide27

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 activityTreats migraine headaches by reducing CGRP activity, reducing pain

27

Let’s pretend this is CGRP:

Epitope: site where antibody binds to antigen

Y

Antibody: protein that binds very specifically with antigenSlide28

CQ#8: If an antigen is a protein, then whether or not an antibody binds with the antigen probably depends on …

The amino acid sequence of the antigen

The secondary, tertiary, or quaternary structure of the antigenWhether or not there is a mutation in the antigenAntibody binding would depend on any/all of the above items28Slide29

CQ#9: Would you expect ALD403 to block activity of the calcitonin protein in the body?

YesNo

It’s unlikely, but we couldn’t be sure without more information29Slide30

That makes sense. The

antibody is highly specific so its effects should be limited to CGRP

.

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.

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!

If the medicine was able to shut down transcription of the CALCA gene, that

might

be a different

story!

30Slide31

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?31