RBM4 Interacts with an Intronic Element and Stimulates Tau Exon 10 Inclusion

Abstract

Tau protein, which binds to and stabilizes microtubules, is critical for neuronal survival and function. In the human brain, tau pre-mRNA splicing is regulated to maintain a delicate balance of exon 10-containing and exon 10-skipping isoforms. Splicing mutations affecting tau exon 10 alternative splicing lead to tauopathies, a group of neurodegenerative disorders including dementia. Molecular mechanisms regulating tau alternative splicing remain to be elucidated. In this study, we have developed an expression cloning strategy to identify splicing factors that stimulate tau exon 10 inclusion. Using this expression cloning approach, we have identified a previously unknown tau exon 10 splicing regulator, RBM4 (RNA binding motif protein 4). In cells transfected with a tau minigene, RBM4 overexpression leads to an increased inclusion of exon 10, whereas RBM4 down-regulation decreases exon 10 inclusion. The activity of RBM4 in stimulating tau exon 10 inclusion is abolished by mutations in its RNA-binding domain. A putative intronic splicing enhancer located in intron 10 of the tau gene is required for the splicing stimulatory activity of RBM4. Immunohistological analyses reveal that RBM4 is expressed in the human brain regions affected in tauopathy, including the hippocampus and frontal cortex. Our study demonstrates that RBM4 is involved in tau exon 10 alternative splicing. Our work also suggests that down-regulating tau exon 10 splicing activators, such as RBM4, may be of therapeutic potential in tauopathies involving excessive tau exon 10 inclusion.Microtubule-associated protein tau regulates the organization and stability of microtubules (MTs) 2 in the neurons. In humans, the Tau protein is encoded by a single gene on chromosome 17. The tau gene is expressed at a high level in neurons and at lower levels in glia and certain nonneuronal cells. Involved in maintaining cell morphology, axonal extension, and vesicle transport, Tau is critical for the formation and function of neurons (1-3) (for recent reviews, see . The expression of the tau gene is under complex regulation at multiple steps, including both post-transcriptional and post-translational levels. In the human brain, six tau isoforms are expressed as a result of alternative splicing of exons 2, 3, and 10 (10-12). Alternative splicing of exon 10 (Ex10), which encodes for one of the four MT-binding domains, gives rise to tau isoforms containing either four MT-binding repeats (Tau4R, Ex10+) or three * This work was supported by National Institutes of Health Grants AG17518, EY014576, and GM070967 (to J. Y. W.), the Society for Progressive Supranuclear Palsy, Muscular Dystrophy Association (to J. Y. W.), and by a scholar award from the Leukemia Society of America (to J. Y. W.).1To whom correspondence should be addressed: Northwestern University Feinberg School of Medicine, Center for Genetic Medicine, 303 E. Superior St., Chicago,; E-mail: jane-wu@northwestern.edu. 2 The abbreviations used are: MT, microtubule; Ex10, exon 10; FTDP-17, frontotemporal dem...

Functional Pre- mRNA trans-Splicing of Coactivator CoAA and Corepressor RBM4 during Stem/Progenitor Cell Differentiation

“…Tau has previously been identified as a RBM4 target gene (29), and this study indicates that both CoAA and RBM4 cooperatively regulate Tau alternative splicing. Tau is involved in the intracellular filamentous deposits that define multiple neurodegenerative diseases, including Alzheimer disease, progressive supranuclear palsy, corticobasal degeneration, Pick disease, and the inherited frontotemporal dementia and parkinsonism linked to chromosome 17, collectively known as tauopathies (50,51).…”

“…To test this model on a physiological target gene, we chose to analyze Tau, as RBM4 has been previously identified by another group through a library screen that binds to Tau mRNA and promotes Tau exon 10 inclusion (29). The microtubule-associated protein Tau is important for neuronal function.…”

LARK activates posttranscriptional expression of an essential mammalian clock protein, PERIOD1

“…In humans, RBM4a (human LARK1), appears to have a role as a modulator of alternative splicing of mRNAs (27,38,39); however, it is unlikely that mLARK regulates mPer1 expression at the level of splicing, because mLARK1 directly interacts with the mPer1 3Ј UTR, not with a splicing junction, and up-regulates mPER1 protein expression. The most parsimonious explanation is that mLARKs act on mPER1 translation, because mLARKs activate mPER1 expression in a Cap/poly(A)-dependent manner (Fig.…”

RNA Helicase p68 (DDX5) Regulates tau Exon 10 Splicing by Modulating a Stem-Loop Structure at the 5′ Splice Site

“…We made use of a mutant tau pre-mRNA minigene in which the RBM4 binding site was mutated, tau9-11mtRBM4BS (65). As shown in Fig.…”

“…HEK293 cells transfected with plasmids expressing HA-tagged wild-type p68 or LGLD mutant p68 or Flag-tagged RBM4 or Raver were lysed with a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM DTT, 1ϫ protease inhibitor mixture [Roche Molecular Biochemicals]) as described previously (65). Lysates were immunoprecipitated with specific antibodies and protein A/G agarose beads for 2 h at 4°C.…”