NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE

NATIONAL CENTER FOR CASE STUDY TEACHING IN SCIENCE m. RNA Processing: No Longer a Headache by Kristen H. Short Department of Biology Manchester University, North Manchester, IN

Learning outcomes: 1) Differentiate between a pre-m. RNA and a mature m. RNA in eukaryotes. 2) Explain the mechanism by which introns can be removed during m. RNA processing. 3) Explain how alternative splicing can allow a single pre-m. RNA (and thus a single gene) to give rise to several unique mature m. RNAs. 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. 2

One day in late May… 3 Comic strip generated at http: //www. Make. Beliefs. Comix. com. Used by permission of author and site creator Bill Zimmerman.

Two weeks later… 4 Comic strip generated at http: //www. Make. Beliefs. Comix. com. Used by permission of author and site creator Bill Zimmerman.

Lucy, I have to tell you what I’ve learned about ALD 403! 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? 5

No, I’m quite sure that ALD 403 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! 6

CQ#1: Can a single gene code for more than one unique protein? A. Yes B. No C. I don’t know 7

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-Phe-Asn -Lys-Phe-His-Thr-Phe-Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro 8

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-Ser. Arg-Ser-Gly-Val-Lys-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser. Lys-Ala-Phe 9

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-Pro Amino acid sequence of CGRP: Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Ser. Arg-Ser-Gly-Val-Lys-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser. Lys-Ala-Phe 10

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. 11

The CALCA gene 5´-- 1 2 Poly “A” sites Calcitonin exon CGRP exon 4 5 3 --3´ 6 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 m. RNA • Intervening sequences, or introns 12

CQ#2: Will the pre-m. RNA (the initial product of transcription of this gene) contain all the exons and introns? A. Yes B. No C. I don’t know 13

I’m still wondering, though, how this single gene can code for different proteins, if only one unique pre-m. RNA 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-m. RNA is processed to form the mature m. RNA. 14

CQ#3: Which of the following modifications to a prem. RNA molecule in eukaryotes will occur before it is considered a mature m. RNA molecule? A. B. C. D. Addition of the 5´ cap Addition of a poly-A tail Splicing to remove introns All of the above 15

A closer look at pre-m. RNA processing Let’s consider this gene with three exons, numbered 1 -3: DNA --3´ 5´-- 1 2 3 During and after transcription but before splicing, the pre-m. RNA receives two modifications: RNA AAAAA…. . AAAA--3´ 5´-- G 1 2 3 16

A closer look at RNA splicing Now let’s splice to remove introns: AAAAA…. . AAAA--3´ 5´--G 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 m. RNA now looks like this: 5´--G AAAAA…. . AAAA--3´ 1 2 3 17

What if exons are also sometimes removed? CQ#4: How many unique mature m. RNAs can theoretically be formed from this single pre-m. RNA? Assume that this pre-m. RNA 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 m. RNA. AAAAA…. . AAAA--3´ 5´--G A. B. C. D. 1 2 3 7 1 2 3 18

Alternative splicing • Refers to splicing of pre-m. RNA molecules from the same gene in different ways, to produce a variety of different mature m. RNA 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 19

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 m. RNAs through alternative splicing of the same pre-m. RNA, maybe. Right, and different mature m. RNAs 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. 20

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 21

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 22

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 23

CQ#6: True or false: If we extracted m. RNA from thyroid and neuronal cells, we would find the same sets of mature m. RNA molecules in both types of cells. A. True B. False 24

Right. I’m still curious, though, about the ALD 403 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 prem. RNA is spliced before translation. 25

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 m. RNA molecules All of the above are possibilities 26

ALD 403: 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 Y Let’s pretend this is CGRP: Antibody: protein that binds very specifically with antigen Epitope: site where antibody binds to antigen 27

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 28

CQ#9: Would you expect ALD 403 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 29

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! ALD 403 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! 30

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