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RBM4 Interacts with an Intronic Element and Stimulates Tau Exon

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RBM4 Interacts with an Intronic Element and Stimulates Tau Exon THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 34, pp. 24479–24488, August 25, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. RBM4 Interacts with an Intronic Element and Stimulates Tau Exon 10 Inclusion* Received for publication, April 12, 2006, and in revised form, June 7, 2006 Published, JBC Papers in Press, June 15, 2006, DOI 10.1074/jbc.M603971200 Amar Kar‡, Necat Havlioglu§, Woan-Yuh Tarn¶, and Jane Y. Wu‡1 From the ‡Department of Neurology, Lurie Comprehensive Cancer Center, Center for Genetic Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, the §Department of Pathology, Saint Louis University, St. Louis, Missouri 63103, and the ¶Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan Tau protein, which binds to and stabilizes microtubules, is Involved in maintaining cell morphology, axonal extension, and critical for neuronal survival and function. In the human brain, vesicle transport, Tau is critical for the formation and function tau pre-mRNA splicing is regulated to maintain a delicate bal- of neurons (1–3) (for recent reviews, see Refs. 4–9). The ance of exon 10-containing and exon 10-skipping isoforms. expression of the tau gene is under complex regulation at mul- Splicing mutations affecting tau exon 10 alternative splicing tiple steps, including both post-transcriptional and post-trans- lead to tauopathies, a group of neurodegenerative disorders lational levels. In the human brain, six tau isoforms are including dementia. Molecular mechanisms regulating tau expressed as a result of alternative splicing of exons 2, 3, and 10 alternative splicing remain to be elucidated. In this study, we (10–12). Alternative splicing of exon 10 (Ex10), which encodes have developed an expression cloning strategy to identify splic- for one of the four MT-binding domains, gives rise to tau iso- ing factors that stimulate tau exon 10 inclusion. Using this forms containing either four MT-binding repeats (Tau4R, expression cloning approach, we have identified a previously Ex10ϩ) or three MT- binding repeats (Tau3R, Ex10-). In the unknown tau exon 10 splicing regulator, RBM4 (RNA binding adult human brain, the regulated splicing of exon10 results in a motif protein 4). In cells transfected with a tau minigene, RBM4 ratio of Tau4R to Tau3R of approximately 1. overexpression leads to an increased inclusion of exon 10, Genetic studies have revealed a number of mutations in whereas RBM4 down-regulation decreases exon 10 inclusion. the human tau gene in patients with tauopathy. More than 30 The activity of RBM4 in stimulating tau exon 10 inclusion is different mutations have been associated with frontotempo- abolished by mutations in its RNA-binding domain. A putative ral dementia with parkinsonism linked to chromosome-17 intronic splicing enhancer located in intron 10 of the tau gene is (FTDP-17) (13–15). This is an autosomal dominant disorder required for the splicing stimulatory activity of RBM4. Immu- with clinically heterogeneous manifestations that include nohistological analyses reveal that RBM4 is expressed in the behavioral, cognitive, and motor abnormalities. FTDP-17 human brain regions affected in tauopathy, including the hip- mutations can be classified into two groups, missense muta- pocampus and frontal cortex. Our study demonstrates that tions that affect Tau protein activity and splicing mutations RBM4 is involved in tau exon 10 alternative splicing. Our work that alter the ratio of distinct tau splicing isoforms (for also suggests that down-regulating tau exon 10 splicing activa- recent reviews, see Refs. 4–9). Almost all splicing mutations tors, such as RBM4, may be of therapeutic potential in tauopa- characterized so far affect the regulation of tau exon 10 splic- thies involving excessive tau exon 10 inclusion. ing. In vitro experiments suggest that Tau4R and Tau3R pro- teins bind and stabilize MTs in different manners (16–18). This delicate balance between exon 10ϩ to exon 10Ϫ tau Microtubule-associated protein tau regulates the organiza- isoforms is crucial for neuronal function in learning and tion and stability of microtubules (MTs)2 in the neurons. In memory. However, the underlying mechanism remains to be humans, the Tau protein is encoded by a single gene on chro- elucidated (13, 19, 20) (reviewed in Ref. 5). mosome 17. The tau gene is expressed at a high level in neurons A large number of genes in the human genome utilize alter- and at lower levels in glia and certain nonneuronal cells. native splicing to generate functionally distinct gene products. Understanding how these alternative splicing events are regu- * This work was supported by National Institutes of Health Grants AG17518, lated is an important issue in functional genomics. In the past 2 EY014576, and GM070967 (to J. Y. W.), the Society for Progressive Supranu- decades, a number of alternative splicing regulators have been clear Palsy, Muscular Dystrophy Association (to J. Y. W.), and by a scholar identified. Many of these trans-acting factors were initially award from the Leukemia Society of America (to J. Y. W.). The costs of pub- lication of this article were defrayed in part by the payment of page identified using biochemical approaches (reviewed in Refs. charges. This article must therefore be hereby marked “advertisement”in 21–23). In this study, we have developed an expression cloning accordance with 18 U.S.C. Section 1734 solely to indicate this fact. approach using a tau exon 10 splicing green fluorescent protein 1 To whom correspondence should be addressed: Northwestern University (GFP) reporter, Tau4R-GFP, in which GFP expression was Feinberg School of Medicine, Center for Genetic Medicine, 303 E. Superior St., Lurie 6-117, Chicago, IL 60611. Tel.: 312-503-0684; Fax: 312-503-5603; dependent on the tau exon 10 inclusion. Using this system to E-mail: [email protected]. screen a human brain cDNA library, we have identified a pre- 2 The abbreviations used are: MT, microtubule; Ex10, exon 10; FTDP-17, fron- viously unknown tau exon 10 splicing activator, RBM4, a pro- totemporal dementia with parkinsonism linked to chromosome-17; RT, reverse transcription; GFP, green fluorescent protein; RNAi, RNA interfer- tein recently shown to play a role in alternative splicing of ence; HA, hemagglutinin; RRM, RNA recognition motif. ␣-tropomyosin (24). Our experiments show that overexpres- AUGUST 25, 2006•VOLUME 281•NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 24479 Alternative Splicing Regulation of Tau Exon 10 plates. cDNAs were prepared from each pool using an optimized medium scale plasmid preparation method combining polyethylene glycol precipitation with the endo- toxin-free DNA preparation kit (Qiagen) to ensure the high quality of DNA preparations for transfec- tion. The typical transfection effi- ciency is ϳ50–60%, as assessed by GFP expression in the control cells transfected with a GFP plasmid. For the first round of transfection, 40 combined pools (combined from 10 primary pools in a grid fashion to allow easy identification of indiv- idual positive primary pools) of cDNAs were transfected in cells sta- bly expressing the Tau GFP reporter gene. Using this grid design, 400 cDNA pools were screened in 40 primary transfections in 15-cm tis- sue culture dishes and further screened in subsequent rounds of transfections. Because HEK293 cells had a high efficiency of transfection and a low background GFP expression, we chose this cell line for preparing a stable cell line expressing the Tau reporter gene. Stable HEK293 cells expressing Tau4R-GFP reporter were selected with G418 and used for expression of cDNA clones fol- lowing transfection with pooled cDNAs from the adult brain cDNA FIGURE 1. A, a schematic diagram of the Tau4R-GFP reporter constructs. The tau splicing reporter gene is library. During the first two rounds designed in such a way that GFP is only expressed when the exon 10 is included (for a detailed description of of transfection, it appeared more the reporter gene construction, see “Experimental Procedures”). B, a diagrammatic representation of the cDNA library screening protocol based on GFP expression following progressive division and subdivision of cDNA efficient to visually examine individ- pools and transfection of cDNA pools into the stable cell line expressing Tau4R-GFP reporter. ual transfected culture plates under an inverted fluorescent microscope than using a cell sorter. The primary sion of RBM4 stimulates tau exon 10 inclusion, and RNA inter- positive pools that gave significant activation of GFP expression ference (RNAi)-mediated knock-down of RBM4 expression in were identified, and cDNAs in these pools were further divided. transfected cells reduces tau exon inclusion. Immunohisto- For each primary positive pool, 20 subpools were prepared and chemical staining indicates that RBM4 is expressed in the brain individually transfected into stable cells expressing the Tau4R- regions involved in tauopathy. Our results support a role of GFP reporter gene in 10-cm dishes. The positive subpools were RBM4 in regulating tau exon 10 splicing. again identified using inverted fluorescent microscopy and sub- divided for another round of transfection until individual single EXPERIMENTAL PROCEDURES cDNA clones were isolated. After seven rounds of retransfec- Expression Cloning Using the Tau Reporter Gene—We pre- tion, we were able to isolate single cDNA clones that have sig- pared a human adult brain cDNA library using the adult brain nificant effects on tau exon 10 splicing. mRNA purchased from Clontech. The quality of this cDNA Plasmids and Antibodies—Mammalian expression plasmids library was tested using reverse transcription-PCR and small for HA-tagged wild type or mutant RBM4 (25) and for raver scale screening before the library was subdivided into 400 were described previously (26). The plasmid expressing RNAi cDNA pools. It was estimated that each pool contained ϳ5000 for RBM4 was described (24). Monoclonal anti-HA antibody clones. Each of these 400 primary pools was amplified for 6–8 h was purchased from Covance.
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