RNA splicing in the heart: Changing parts and performance - Front matter

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RNA splicing in the heart

Changing parts and performance

van den Hoogenhof, M.M.G.

Publication date

2018

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Other version

License

Other

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Citation for published version (APA):

van den Hoogenhof, M. M. G. (2018). RNA splicing in the heart: Changing parts and

performance.

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will be contacted as soon as possible.

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516079-L-os-hoogenhof 516079-L-os-hoogenhof 516079-L-os-hoogenhof

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RNA splicing in the heart

Changing parts and performance

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ISBN

978-94-028-0953-4 Design/Lay-out

Wendy Bour-van Telgen, Ipskamp Printing Enschede Print

Ipskamp Printing, Enschede Cover

Judith Segers, Kleinood Design, www.kleinood.me © Maarten van den Hoogenhof, 2018

All rights are reserved. No part of this book may be reproduced, distributed, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the author.

RNA splicing in the heart

Changing parts and performance

Academisch Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel

op dinsdag 3 april 2018, te 14:00 uur

door

Maarten Marinus Gerardus van den Hoogenhof

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RNA splicing in the heart

Changing parts and performance

Academisch Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus

prof. dr. ir. K.I.J. Maex

ten overstaan van een door het College voor Promoties ingestelde commissie, in het openbaar te verdedigen in de Agnietenkapel

op dinsdag 3 april 2018, te 14:00 uur

door

Maarten Marinus Gerardus van den Hoogenhof

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Promotiecommissie

Promotor: prof.dr. Y.M. Pinto AMC-UvA Copromotor: dr. E.E.J.M. Creemers AMC-UvA

Overige leden: prof. dr. J.D. Molkentin University of Cincinnati prof. dr. J. Backs University of Heidelberg prof. dr. L.J. de Windt Maastricht University prof. dr. V.M. Christoffels AMC-UvA

dr. R. Coronel AMC-UvA prof. dr. C.J.F. van Noorden AMC-UvA Faculteit der Geneeskunde

The research described in this thesis was supported by a grant of the Dutch Heart Foundation (CVON-ARENA-2011-11).

Financial support by AMC Medical Research and the Dutch Heart Foundation for the publication of this thesis is gratefully acknowledged.

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Introduction and Scope

of the thesis

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changing parts and performance

RNA splicing in the heart

M.M.G. van den Hoogenhof

Introduction and Scope

of the thesis

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8

Introduction

The main and primary function of the heart is to pump blood through the body to meet its demand of oxygen and nutrients. As these demands can change, so can the function of the heart. Physiological changes include acute changes in cardiac output, by changing heart rate or stroke volume, or if the change in demand persists, the heart can adapt with physiological hypertrophy. However, heart function (or performance) can also change due to pathological stimuli, for example after prolonged hypertension or increased work load after a myocardial infarction, or due to decreased capacity caused by a genetic defect. The molecular mechanisms that underlie changes in cardiac performance are enormously diverse, and certainly incompletely understood. However, since heart disease is a major health issue in the western world1_{, there is a growing need for additional insight in how the heart functions and adapts.} RNA splicing is such a process that can be altered to regulate cardiac performance. RNA is transcribed as a pre-mRNA, which still contains the ‘non-coding’ introns that need to be removed. The removal of these introns and joining of the exons is termed constitutive splicing. Alternative splicing, on the other hand, is the process in which different exons of a single gene can be in- or excluded in different ways in the mature mRNA transcript, and this allows for the creation of a much greater and more complex transcriptome. One of the main consequences of alternative splicing, is the formation of different protein or mRNA isoforms with altered or even opposing functions, with one of the most extreme examples being DSCAM (Down-syndrome cell adhesion molecule) in Drosophila Melanogaster. The DSCAM gene encodes an astonishing 38.000 mRNA different transcripts, each with a different exon composition, a number that even surpasses the total number of protein coding genes in the fly2_. An example of how alternative splicing can affect functional properties of the heart, is that of alternative titin (TTN) protein isoforms3_{. TTN is a giant protein that resides in the sarcomere (the contractile unit} of a cardiomyocyte, see Figure 1), and functions as a molecular spring. The passive tension of the cardiomyocyte largely depends on TTN, and changes in TTN isoform expression affect passive tension. In fetal hearts, the larger and more compliant TTN isoform N2BA is expressed, which after birth is gradually replaced with the smaller and stiffer N2B isoform4_{. This is needed to prevent overfilling of} the ventricles due to increased filling pressures in the postnatal heart4_{. In a diseased heart, a reverse} isoform switch happens, from the N2B towards the N2BA isoform. During diastole, this will be beneficial as it makes it easier to fill the ventricles, but during systole this is detrimental, as it will decrease contractility5_{. Moreover, missplicing of TTN due to mutations in the splicing factor RBM20, is causal for} heart disease6, 7_{. In that sense, alternative splicing can both be a cause and a consequence of disease.} A relatively new RNA species is that of circular RNAs (circRNAs). Despite their discovery in 19798_{, they} were long presumed to be accidental by-products of splicing without a function9_{. CircRNAs are formed} by the spliceosome, but instead of joining a 5’ splice site with a downstream 3’ splice site, it joins a 5’ splice site with an upstream 3’ splice site, which results in a covalently closed RNA circle. This specific form of splicing is termed ‘back-splicing’. In the last years, however, it has become increasingly clear that circRNAs are (highly) expressed, can be regulated, and can have important biological functions10-12_.

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Introduction and Scope of the thesis

9 Driven by advances in next-generation sequencing, we have now begun to unravel the extent and complexity of alternative splicing. This is nicely illustrated by the fact that, upon discovery of alternative splicing in the 1970s13_{, it was thought that only 5% of genes were alternatively spliced}14_{. In the} beginning of this millennium, this number had already risen to 60%15_{, and now we know that over 95%} of all human genes are alternatively spliced16_{. The same holds true for proteins that are involved in} (alternative) splicing; the number of proteins that is able to bind RNA and affect splicing has also risen steadily in the last decade. Moreover, new RNA species like circRNAs, have only recently gained interest of the scientific field. Nevertheless, RNA biology is a relatively new field in (molecular) cardiology, and numerous questions remain to be answered. In this thesis, we have tried to add new insight into the regulation of alternative splicing, molecular mechanisms that underlie alternative splicing associated heart disease, and have examined potential roles of RNA-binding proteins and splicing factors in the heart.

Figure 1. A sarcomere; the contractile unit of a cardiomyocyte. From Weeland, van den Hoogenhof, Beqqali, and Creemers, JMCC, 20153_.

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10

Scope of this thesis

In Chapter 1, we summarize the current knowledge on the process of (alternative) splicing, with a focus on the heart, and discuss regulation, and maybe even more importantly, dysregulation of alternative splicing in the healthy and diseased heart. In Chapter 2, we investigate the molecular mechanisms of RBM20-induced cardiomyopathy, and examine specifically why patients with RBM20 mutations are at great(er) risk of arrhythmias. We compared clinical characteristics of RBM20 and TTN mutation carriers with DCM, and generated an Rbm20 knockout mouse to investigate downstream effects of Rbm20 dysfunction. We characterized the cardiac, transcriptomic, and electrophysiological phenotype of the Rbm20 knockout mouse, and provide proof-of-concept of a potential new therapeutic strategy for RBM20 mutation carriers. In Chapter 3, we investigate the finding that RBM20 also regulates a specific class of RNA molecules, namely circRNAs, arising from the TTN gene. We used RNA-sequencing data form healthy and diseased human hearts, and characterized the human circRNA landscape in the heart. Additionally, we link RBM20-dependent TTN splicing to circRNA production from the same gene. In Chapter 4, we dive deeper into Rbm20-regulated circRNA production, and more broadly try to answer the question whether alternative splicing is a general driver of circRNA production. We performed RNA-sequencing on human and mouse (wildtype and Rbm20 knockout) hearts, mapped alternative splicing events and overlaid these with expressed circRNAs at exon level resolution. In Chapter 5, we move to a different RNA-binding protein, Rbm24, which is known to act as a pivotal splicing factor in the developing heart. We used AAV9-mediated overexpression of Rbm24 to examine whether Rbm24 also plays a role in the early postnatal and adult mouse heart. In Chapter 6, we investigated yet another RNA-binding protein, Rbm38, as it closely resembles the pivotal cardiac splicing factor Rbm24. Rbm38 is, like Rbm24, expressed in the heart, and we examined whether Rbm38 is important for (normal) cardiac function. We generated an Rbm38 knockout mouse, and characterized the cardiac phenotype at baseline and after pressure overload-induced cardiac remodeling. In Chapter 7, we investigated to what extent hypoxia can alter alternative splicing in the heart. We made use of a hypoxic cell-culture model and performed RNA-sequencing on hypoxic cardiomyocytes. We discuss bioinformatic challenges and speculate about bioinformatic solutions and future directions. Finally, we end with a summary of the findings in this thesis and discuss future perspectives.

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Introduction and Scope of the thesis

11

References

1. Writing Group M, Mozaffarian D, Benjamin EJ, Go AS, Arnett DK, Blaha MJ, Cushman M, Das SR, de Ferranti S, Despres JP, Fullerton HJ, Howard VJ, Huffman MD, Isasi CR, Jimenez MC, Judd SE, Kissela BM, Lichtman JH, Lisabeth LD, Liu S, Mackey RH, Magid DJ, McGuire DK, Mohler ER, 3rd, Moy CS, Muntner P, Mussolino ME, Nasir K, Neumar RW, Nichol G, Palaniappan L, Pandey DK, Reeves MJ, Rodriguez CJ, Rosamond W, Sorlie PD, Stein J, Towfighi A, Turan TN, Virani SS, Woo D, Yeh RW, Turner MB, American Heart Association Statistics C and Stroke Statistics S. Heart Disease and Stroke Statistics-2016 Update: A Report From the American Heart Association. Circulation. 2016;133:e38-360.

2. Kornblihtt AR, Schor IE, Allo M, Dujardin G, Petrillo E and Munoz MJ. Alternative splicing: a pivotal step between eukaryotic transcription and translation. Nat Rev Mol Cell Biol. 2013;14:153-65.

3. Weeland CJ, van den Hoogenhof MM, Beqqali A and Creemers EE. Insights into alternative splicing of sarcomeric genes in the heart. J Mol Cell Cardiol. 2015;81:107-13.

4. Opitz CA, Leake MC, Makarenko I, Benes V and Linke WA. Developmentally regulated switching of titin size alters myofibrillar stiffness in the perinatal heart. Circ Res. 2004;94:967-75.

5. Nagueh SF, Shah G, Wu Y, Torre-Amione G, King NM, Lahmers S, Witt CC, Becker K, Labeit S and Granzier HL. Altered titin expression, myocardial stiffness, and left ventricular function in patients with dilated cardiomyopathy. Circulation. 2004;110:155-62.

6. Beqqali A, Bollen IA, Rasmussen TB, van den Hoogenhof MM, van Deutekom HW, Schafer S, Haas J, Meder B, Sorensen KE, van Oort RJ, Mogensen J, Hubner N, Creemers EE, van der Velden J and Pinto YM. A mutation in the glutamate-rich region of RNA-binding motif protein 20 causes dilated cardiomyopathy through missplicing of titin and impaired Frank-Starling mechanism. Cardiovasc Res. 2016;112:452-63.

7. Guo W, Schafer S, Greaser ML, Radke MH, Liss M, Govindarajan T, Maatz H, Schulz H, Li S, Parrish AM, Dauksaite V, Vakeel P, Klaassen S, Gerull B, Thierfelder L, Regitz-Zagrosek V, Hacker TA, Saupe KW, Dec GW, Ellinor PT, MacRae CA, Spallek B, Fischer R, Perrot A, Ozcelik C, Saar K, Hubner N and Gotthardt M. RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing. Nat Med. 2012;18:766-73.

8. Hsu MT and Coca-Prados M. Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells. Nature. 1979;280:339-40.

9. Nigro JM, Cho KR, Fearon ER, Kern SE, Ruppert JM, Oliner JD, Kinzler KW and Vogelstein B. Scrambled exons. Cell. 1991;64:607-13.

10. Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, Gregersen LH, Munschauer M,

Loewer A, Ziebold U, Landthaler M, Kocks C, le Noble F and Rajewsky N. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature. 2013;495:333-8.

11. Ebbesen KK, Hansen TB and Kjems J. Insights into circular RNA biology. RNA Biol. 2017;14:1035-1045.

12. Piwecka M, Glazar P, Hernandez-Miranda LR, Memczak S, Wolf SA, Rybak-Wolf A, Filipchyk A, Klironomos F, Cerda

Jara CA, Fenske P, Trimbuch T, Zywitza V, Plass M, Schreyer L, Ayoub S, Kocks C, Kuhn R, Rosenmund C, Birchmeier C and Rajewsky N. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science. 2017;357.

13. Chow LT, Gelinas RE, Broker TR and Roberts RJ. An amazing sequence arrangement at the 5’ ends of adenovirus 2

messenger RNA. Cell. 1977;12:1-8.

14. Sharp PA. Split genes and RNA splicing. Cell. 1994;77:805-15.

15. Modrek B, Resch A, Grasso C and Lee C. Genome-wide detection of alternative splicing in expressed sequences of

human genes. Nucleic Acids Res. 2001;29:2850-9.

16. Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP and Burge CB. Alternative

RNA splicing in the heart: Changing parts and performance - Front matter

UvA-DARE (Digital Academic Repository)

RNA splicing in the heart

Changing parts and performance

van den Hoogenhof, M.M.G.

Publication date

2018

Document Version

Other version

License

Other

Link to publication

Citation for published version (APA):

van den Hoogenhof, M. M. G. (2018). RNA splicing in the heart: Changing parts and

performance.

General rights

It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s)

and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open

content license (like Creative Commons).

Disclaimer/Complaints regulations

If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please

let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material

inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter

to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You

will be contacted as soon as possible.

RNA splicing in the heart

Changing parts and performance

RNA splicing in the heart

Changing parts and performance

Academisch Proefschrift

RNA splicing in the heart

Changing parts and performance

Academisch Proefschrift

Promotiecommissie

TABLE OF CONTENTS

Introduction and Scope

of the thesis

changing parts and performance

RNA splicing in the heart

M.M.G. van den Hoogenhof

Introduction and Scope

of the thesis

Introduction

Scope of this thesis

References