An SRp75/hnRNPG complex interacting with hnRNPE2 regulates the 5' splice site of tau exon 10, whose misregulation causes frontotemporal dementia

Tau is a neuronal-specific microtubule-associated protein that plays an important role in establishing neuronal polarity and maintaining the axonal cytoskeleton. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism (FTDP-17), the early onset dementia observed in Down syndrome (DS; trisomy 21) and the dementia component of myotonic dystrophy type 1 (DM1). Splicing misregulation of adult-specific exon 10, which codes for a microtubule binding domain, results in expression of abnormal ratios of tau isoforms, leading to FTDP-17. Positions 3 to 19 of the intron downstream of exon 10 define a hotspot of splicing regulation: the region diverges between humans and rodents, and point mutations within it result in tauopathies. In this study, we investigated three regulators of exon 10 splicing: serine/arginine-rich protein SRp75 and heterogeneous nuclear ribonucleoproteins hnRNPG and hnRNPE2. SRp75 and hnRNPG inhibit splicing of exon 10 whereas hnRNPE2 activates it. Using co-transfections, co-immunoprecipitations and RNAi we discovered that SRp75 binds to the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region; and that hnRNPG and hnRNPE2 interact with SRp75. Thus, increased exon 10 inclusion in FTDP mutants may arise from weakened SRp75 binding. This work provides insights into the splicing regulation of the tau gene and into possible strategies for correcting the imbalance in tauopathies caused by changes in the ratio of exon 10.     

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.     

Identification of a cis-acting element involved in the regulation of BACE1 mRNA alternative splicing

β-site APP cleaving enzyme 1 (BACE1) is the transmembrane aspartyl protease that catalyzes the first cleavage step during proteolysis of the β-amyloid precursor protein, a process involved in the pathogenesis of Alzheimer disease. BACE1 pre-mRNA undergoes complex alternative splicing, and cis -acting elements important for its regulation have not been identified. We constructed and compared several BACE1 minigenes and found that BACE1 sequence from exon 3 through exon 5 was required for minigenes to undergo correct splicing. Minigene splicing was validated by showing specific splicing inhibition upon splice site mutation. Furthermore, we showed that mutation of the minigene at a predicted exonic splicing enhancer in exon 4 of BACE1 increased exon 4 skipping. Therefore, we have for the first time found evidence of a regulatory site involved in BACE1 alternative splicing, and these data indicate that minor sequence changes can dramatically alter BACE1 alternative splicing.     

Alternative splicing of fibronectin mRNAs in chondrosarcoma cells: role of far upstream intron sequences

The fibronectin (FN) gene encodes multiple mRNAs through the process of alternative splicing, and production of certain isoforms is characteristic of a given cell type. Chondrocytes produce FNs that completely lack alternative exon EIIIA, and loss of inclusion of the exon is tightly linked to chondrogenic condensation of mesenchymal cells. The inclusion of a second exon, EIIIB, is high in embryonic cartilage, but declines with age. Multiple exons are omitted to produce the (V + C)-form that is highly specific for cartilage and chondrocytes. A rat chondrosarcoma cell line, RCS, was identified that preserves key features of the cartilage-specific splicing phenotype. RCS cells, which exclude exon EIIIA, and HeLa cells, which include exon EIIIA similar to mesenchymal cells, were used to assess the contribution of intron sequences flanking exon EIIIA to splicing regulation. Deletion of most of the intron downstream of the exon had little effect on splicing in either cell type. However, deletions within upstream intron 32-A reduced inclusion of the alternative exon in both cell types. The sequences involved lie more than 200 nucleotides away from the exon, but could not be localized to a single region by deletion mapping. These intronic sequences contribute to the efficiency of exon EIIIA recognition, but not to cell-type specific regulation. The normally inhibitory factor polypyrimidine tract binding protein promotes exon EIIIA inclusion in a manner that is partially dependent on the regulatory sequences within intron 32-A.     

A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10

Mutations that stimulate exon 10 inclusion into the human tau mRNA cause frontotemporal dementia with parkinsonism, associated with chromosome 17 (FTDP-17), and other tauopathies. This suggests that the ratio of exon 10 inclusion to exclusion in adult brain is one of the factors to determine biological functions of the tau protein. To investigate the underlying splicing mechanism and identify potential therapeutic targets for tauopathies, we generated a series of mini-gene constructs with intron deletions from the full length of tau exons 9-11 mini-gene construct. RT-PCR results demonstrate that there is a minimum distance requirement between exon 10 and 11 for correct splicing of the exon 10. In addition, SRp20, a member of serine-arginine (SR) protein family of splicing factors was found to facilitate exclusion of exon 10 in a dosage-dependent manner. Significantly, SRp20 also induced exon 10 skipping from pre-mRNAs containing mutations identified in FTDP-17 patients. Based on those results, we generated a cell-based system to measure inclusion to exclusion of exon 10 in the tau mRNA using the luciferase reporter. The firefly luciferase was fused into exon 11 in frame, and a stop code was also created in exon 10. Inclusion of exon 10 prevents luciferase expression, whereas exclusion of exon 10 generates luciferase activity. To minimize baseline luciferase expression, our reporter construct also contains a FTDP-17 mutation that increases exon 10 inclusion. We demonstrate that the splicing pattern of our reporter construct mimics that of endogenous tau gene. Co-transfection of SRp20 and SRp55, two SR proteins that promote exon 10 exclusion, increases production of luciferase. We conclude that this cell-based system can be used to identify biological substances that modulate exon 10 splicing.     

A nonsense exon in the Tpm1 gene is silenced by hnRNP H and F

As well as generating protein isoform diversity, in some cases alternative splicing generates RNAs that harbor premature termination codons and that are subject to nonsense-mediated decay (NMD). We previously identified an apparent pseudo-exon in the rat α-tropomyosin (Tpm1) gene as a probable genuine alternatively spliced exon that causes NMD when spliced into Tpm1 RNA. Here, we report the analysis of cis-acting splicing regulatory elements within this “nonsense exon.” Guided by the data set of predicted splicing enhancer and silencer elements compiled by Zhang and Chasin, we made a series of mutations through the nonsense exon and found that like authentic exons it is densely packed with enhancer and silencer elements. Strikingly, 11 of 13 tested mutations behaved as predicted computationally. In particular, we found that a G-rich silencer at the 5′ end, which is crucial for skipping of the nonsense exon, functions by binding hnRNP-H and F.     

SRp54 (SFRS11), a regulator for tau exon 10 alternative splicing identified by an expression cloning strategy

The tau gene encodes a microtubule-associated protein that is critical for neuronal survival and function. Splicing defects in the human tau gene lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), an autosomal dominant neurodegenerative disorder. Genetic mutations associated with FTDP-17 often affect tau exon 10 alternative splicing. To investigate mechanisms regulating tau exon 10 alternative splicing, we have developed a green fluorescent protein reporter for tau exon 10 skipping and an expression cloning strategy to identify splicing regulators. A role for SRp54 (also named SFRS11) as a tau exon 10 splicing repressor has been uncovered using this strategy. The overexpression of SRp54 suppresses tau exon 10 inclusion. RNA interference-mediated knock-down of SRp54 increases exon 10 inclusion. SRp54 interacts with a purine-rich element in exon 10 and antagonizes Tra2beta, an SR-domain-containing protein that enhances exon 10 inclusion. Deletion of this exonic element eliminates the activity of SRp54 in suppressing exon 10 inclusion. Our data support a role of SRp54 in regulating tau exon 10 splicing. These experiments also establish a generally useful approach for identifying trans-acting regulators of alternative splicing by expression cloning.     

Nova autoregulation reveals dual functions in neuronal splicing

The Nova family of neuron-specific RNA-binding proteins were originally identified as targets in an autoimmune neurologic disease characterized by failure of motor inhibition. Nova-1 regulates alternative splicing of pre-mRNAs encoding the inhibitory neurotransmitter receptor subunits GABA(A)Rgamma2 and GlyRalpha2 by directly binding intronic elements, resulting in enhancement of exon inclusion. Here we identify exon E4 in the Nova-1 pre-mRNA itself, encoding a phosphorylated protein domain, as an additional target of Nova-dependent splicing regulation in the mouse spinal cord. Nova binding to E4 is necessary and sufficient for Nova-dependent exon exclusion. E4 harbors five repeats of the known Nova-binding tetranucleotide YCAY and mutation of these elements destroys Nova-dependent regulation. Furthermore, swapping of the sites from Nova-1 and GABA(A)Rgamma2 indicates that the ability of Nova to enhance or repress alternative exon inclusion is dependent on the position of the Nova-binding element within the pre-mRNA. These studies demonstrate that in addition to its previously described role as a splicing activator, Nova autoregulates its own expression by acting as a splicing repressor.     

RNA helicase p68 (DDX5) regulates tau exon 10 splicing by modulating a stem-loop structure at the 5' splice site

Regulation of tau exon 10 splicing plays an important role in tauopathy. One of the cis elements regulating tau alternative splicing is a stem-loop structure at the 5' splice site of tau exon 10. The RNA helicase(s) modulating this stem-loop structure was unknown. We searched for splicing regulators interacting with this stem-loop region using an RNA affinity pulldown-coupled mass spectrometry approach and identified DDX5/RNA helicase p68 as an activator of tau exon 10 splicing. The activity of p68 in stimulating tau exon 10 inclusion is dependent on RBM4, an intronic splicing activator. RNase H cleavage and U1 protection assays suggest that p68 promotes conformational change of the stem-loop structure, thereby increasing the access of U1snRNP to the 5' splice site of tau exon 10. This study reports the first RNA helicase interacting with a stem-loop structure at the splice site and regulating alternative splicing in a helicase-dependent manner. Our work uncovers a previously unknown function of p68 in regulating tau exon 10 splicing. Furthermore, our experiments reveal functional interaction between two splicing activators for tau exon 10, p68 binding at the stem-loop region and RBM4 interacting with the intronic splicing enhancer region.     

Position-dependent splicing activation and repression by SR and hnRNP proteins rely on common mechanisms

Alternative splicing is regulated by splicing factors that modulate splice site selection. In some cases, however, splicing factors show antagonistic activities by either activating or repressing splicing. Here, we show that these opposing outcomes are based on their binding location relative to regulated 5′ splice sites. SR proteins enhance splicing only when they are recruited to the exon. However, they interfere with splicing by simply relocating them to the opposite intronic side of the splice site. hnRNP splicing factors display analogous opposing activities, but in a reversed position dependence. Activation by SR or hnRNP proteins increases splice site recognition at the earliest steps of exon definition, whereas splicing repression promotes the assembly of nonproductive complexes that arrest spliceosome assembly prior to splice site pairing. Thus, SR and hnRNP splicing factors exploit similar mechanisms to positively or negatively influence splice site selection.     

RBM4 interacts with an intronic element and stimulates tau exon 10 inclusion

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.