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

2. 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. 2

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

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

5. 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? 5

6. 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! 6

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

8. 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

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

10. 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-Leu-Ser- Arg-Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser- Lys-Ala-Phe 10

11. 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

12. The CALCA gene 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 12 CGRP exonCalcitonin exon 123456 5´-- --3´ Poly “A” sites

13. 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 13

14. 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. 14

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

16. --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: 16 123 5´----3´ 123 5´-- DNA RNA G AAAAA…..AAAA--3´

17. A closer look at RNA splicing Now let’s splice to remove introns: 17 123 5´--GAAAAA…..AAAA--3´ ………………..……AGGU……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 23 5´--G AAAAA…..AAAA--3´

18. 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. A.1 B.2 C.3 D.7 18 123 5´--GAAAAA…..AAAA--3´

19. 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 19

20. 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. 20

21. Alternative splicing of the CALCA gene 21 CGRP exonCalcitonin exon 123456 5´----3´ Poly “A” sites Calcitonin exon 1234 5´-G CGRP exon 12356 5´-G A..A-3´ Thyroid cellsNeuronal cells A..A-3´ DNA RNA

22. Translation and post-translational processing 22 CGRP exonCalcitonin exon 123456 5´-- --3´ Poly “A” sites Calcitonin exon 1234 5´-G A..A-3´ CGRP exon 12356 5´-G A..A-3´ Thyroid cellsNeuronal 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 RNA Protein

23. 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

24. 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 24

25. 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. 25

26. CQ#7: Theoretically, a medication that reduces the activity of CGRP and therefore prevents migraine headaches could … A.Target and block the CGRP directly B.Target and block CGRP receptors on cells C.Prevent transcription of the CALCA gene D.Prevent translation of the CALCA mRNA molecules E.All of the above are possibilities 26

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

28. 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

29. 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 29

30. 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! 30

31. 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