VU Research Portal From single genes to gene-networks Heetveld, S. 2018 document version Publisher's PDF, also known as Version of record Link to publication in VU Research Portal citation for published version (APA) Heetveld, S. (2018). From single genes to gene-networks: Cellomics meets neurodegenerative diseases. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. E-mail address: [email protected] Download date: 26. Sep. 2021 CHAPTER 3 Regulation of MAPT exon 10 splicing: a shRNA screen of the human spliceosome Sasja Heetveld Chapter 3 ABSTRACT Mutations in MAPT are a major cause of frontal temporal dementia (FTD) of which a subgroup alters alternative splicing of tau mRNA, primarily resulting in excess inclusion of exon 10. In this study we aimed to find regulators of tau exon 10 inclusion, which might provide potential targets for FTD therapeutics. To identify regulators of tau exon 10 splicing, we performed a short hairpin RNA (shRNA) library screen of the human spliceosome in BE(2)M17 neuroblastoma cell lines. The library contained 324 shRNA pools, targeting spliceosomal small nuclear ribonucleoproteins (snRNPs), snRNP-associated and non-snRNP splicing factors. Alterations in exon 10 inclusion were determined by quantitative PCR, which was optimized to specifically detect endogenous 4R and 3R tau transcript levels. The screen was conducted with an N=6, yielding >90% power to detect a 1.4 fold change in 4R tau transcript levels. We found several novel inhibitors as well as enhancers of tau exon 10 inclusion. Furthermore, we reconstructed a splicing network and found that a selection of these hits was connected at the protein level with high confidence. Together, this study provides a platform to further explore splicing factors as a potential therapeutic target for FTD. Key words: MAPT, alternative splicing regulation, shRNA screen 98 Regulation of MAPT exon 10 splicing INTRODUCTION In adult human brain alternative mRNA splicing of MAPT exon 2, 3 and 10 produces six tau isoforms ranging from 352 to 441 amino acids. Three of these isoforms contain 4 microtubule-binding repeats (4R), whereas the other three contain only 3 microtubule- binding repeats (3R). The additional repeat domain is encoded by exon 10. Frontotemporal dementia (FTD) is an early onset dementia that primarily affects frontal and temporal lobes. Mutations in MAPT are a major cause of disease and a large subgroup of these affects alternative mRNA splicing of exon 10.1-3 This includes intronic and exonic mutations. The intronic mutations are clustered at the exon 10/intron 10 junction that is predicted to form a stem-loop structure protecting the 5’ splice site (SS). 4,5 Subsequently, the altered RNA structure promotes more frequent use of the 5’ SS or modifies splicing factor or U1/E6 snRNP binding sites.6 Most of the mutations increase exon 10 inclusion and raise the normal 4R/3R isoform ratio from 1 to 2-3.1 Pre-mRNA splicing is catalyzed by the spliceosome, a highly complex, dynamic, and protein-rich ribonucleoprotein complex (RNP). During spliceosome assembly, activation, catalysis, and disassembly large RNP complexes are formed and ordered in a stepwise manner (Figure 1 and reviewed by Wahl et al., 2009). 7,8 The U1 snRNP binds the 5’ SS of the pre-mRNA and the A complex is formed after branch point recognition by U2 snRNP. Then, the U4/U6.U5 tri-snRNP complex joins generating the B complex. Subsequently, after major conformational and compositional rearrangements, the catalytically activated spliceosome is formed (the B* complex). The B* complex is converted into the C complex, in which the first of two catalytic steps occurs. The pre-MRNA is cleaved at the 5’-SS and a lariat-like structure is formed by the intron and 3’-exon. In the second catalytic step, after additional rearrangements, the two exons are ligated. The intron is released, the spliceosome dissociates and the snRNPs are recycled for new rounds of splicing. Both constitutive splicing and alternative splicing are carried out by the spliceosome and >90% of human protein-coding genes produce multiple mRNA isoforms.9 A network of trans-splicing factors that bind to cis-elements regulates alternative splicing. A cis-element is a region of RNA that regulates the splicing located on the pre- mRNA itself. These elements can be either intronic or exonic and can be positive (splicing enhancers) or negative (splicing silencers). Trans-acting factors are a group of proteins that include serine- and arginine-rich (SR) proteins and heterogeneous nuclear ribonuclear proteins (hnRNPs), as well as tissue specific factors. Many SR proteins have a specific affinity for cis-elements in the MAPT gene and regulate splicing of exon 10.10-12 In addition to these proteins, several non-SR proteins also play a role in the splicing of exon 10. Examples include: RNA-binding motif protein 4 (RBM4), Tra2ß, RNA helicase p68, hnRNP E2 and E3 and CUG- binding protein (CELF).13-19 99 Chapter 3 Figure 1. Pre-mRNA splicing by the major spliceosome. A simplified version of the stepwise interaction of the spliceosomal snRNPs (colored circles) in the removal of an intron from pre-mRNA containing two exons (blue) is shown. The U1, U2, U4/U6 and U5 snRNPs are the main elements of the major spliceosome. Assembly begins with binding of the U1 snRNP to the 5’ splice site (SS) of the intron. Subsequently, the U2 snRNP interacts with the pre-mRNA’s branch point (BP) sequence leading to the formation of the A complex. The U4/U6 and U5 snRNPs are recruited as a preassembled tri-snRNP, forming the B complex. This complex is catalytically inactive and is activated after major conformational and compositional rearrangements, releasing U1 and U4 (the B* complex). The activated spliceosome undergoes the first catalytic step of splicing, generating the C complex. Additional rearrangements occur and the spliceosome undergoes the second catalytic step. The spliceosome dissociates, releasing the mRNA in the form of an mRNP. The U2, U5 and U6 snRNPs are released and recycled for other rounds of splicing. 100 Regulation of MAPT exon 10 splicing Tau is a major target for therapeutic intervention in tauopathies, but no effective treatment has been developed to date.20 Several studies have explored possible therapeutic strategies for tau splicing. A high-throughput screen in HEK 293 cells using a fluorescent reporter identified several compounds that modify exon 10 inclusion.21 Reduction of tau exon 10 inclusion has been achieved in a tau minigene system and in endogenous tau RNA in rat neuronal pheochromocytoma cells.22 Modified antisense oligonucleotides were used targeting the 5’ or 3’ splice junctions of tau exon 10 and thereby blocking access of the splicing machinery to MAPT pre-mRNA. Additionally, spliceosome-mediated RNA trans-splicing (SMaRT) has been used to modify tau exon 10 splicing.23,24 SMaRT refers to the splicing between the 5’ SS of an endogenous target pre-mRNA and the 3’ SS of an exogenously delivered pre-trans-splicing RNA molecule. This system has been used in SH- SY5Y cells and COS cells transfected with a minigene containing FTD mutations. Cultured neurons of humanized MAPT (htau) mice have been transduced as well, expressing the human MAPT gene in a null mouse Mapt background. Of interest, the system was directly delivered using viral vectors into the brain of htau mice in vivo and modulated exon 10 inclusion at the endogenous level.25 In the majority of these studies, tau splicing was measured using mini-gene constructs and/or rodent models.21-25 Previous experiments showed that mini-gene size, especially the intron length between exon 10 and 11, is extremely important for correct splicing of exon 10.26,27 Moreover, splicing of exon 10 shows a species-specific difference that is crucial to neurodegeneration: exon 10 becomes constitutive in adult rodents.28,29 Therefore, we performed experiments at the endogenous level in the human neuroblastoma cell line BE(2)-M17. We aimed to investigate which components of the human spliceosome modify inclusion of MAPT exon 10 and used a qPCR based assay which has been optimized to specifically detect 4R and 3R tau. Here we show, that knockdown of splicing factors can change the 4R/3R tau isoform ratio in human neuroblastoma cells. Furthermore, we reconstructed a splicing network and found that a selection of hits was connected at the protein level. Together, this study provides a platform to further explore the therapeutic potential of modifying splicing factors for FTD. METHODS shRNA virus production Bacterial glycerol stocks containing the shRNA vectors (Sigma, TRC1) were grown overnight in Luria-Bertani media containing 100μg/ml of ampicillin (Sigma-Aldrich). Endotoxin free shRNA plasmids were extracted according to the manufacturer’s protocol (Zymo ZR Plasmid Miniprep Classic kit). Lentivirus was produced as follows: HEK 293T cells were maintained in 101 Chapter 3 6 T75 flasks at a density of 1.5 x 10 and incubated at 37°C with 5% CO2.