Skip Navigation


Human Molecular Genetics Advance Access originally published online on August 28, 2007
Human Molecular Genetics 2007 16(22):2760-2769; doi:10.1093/hmg/ddm233
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/22/2760    most recent
ddm233v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Chapple, J. P.
Right arrow Articles by Gallo, J.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chapple, J. P.
Right arrow Articles by Gallo, J.-M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Expression, localization and tau exon 10 splicing activity of the brain RNA-binding protein TNRC4

J. Paul Chapple1,{dagger},{ddagger}, Karen Anthony1,{ddagger}, Teresa Rodriguez Martin1, Arvind Dev2, Thomas A. Cooper3,4 and Jean-Marc Gallo1,*

1 MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, De Crespigny Park, London SE5 8AF, UK, 2 Institute of Human Genetics, George August University, Goettingen, Germany, 3 Department of Pathology and 4 Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA

* To whom correspondence should be addressed at: MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, Box P037, De Crespigny Park, London SE5 8AF, UK. Tel: +44 2078480404; Fax: +44 2077080017; Email: jean-marc.gallo{at}iop.kcl.ac.uk

Received July 5, 2007; Revised August 19, 2007; Accepted August 19, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Elucidating the mechanisms of alternative splicing in the brain is a prerequisite to the understanding of the pathogenesis of major neurological diseases linked to impairment of pre-mRNA alternative splicing. The gene trinucleotide repeat-containing 4 (TNRC4) is predicted to encode a member of the CELF (CUG-BP- and ETR-3-like factors) family of RNA-binding proteins containing a 15–18-residue polyglutamine sequence. The TNRC4 transcript is selectively expressed in the brain. Using an anti-peptide antibody against the predicted sequence, we establish the presence of TNRC4 as a ~50 kDa protein in the brain. Full-length TNRC4 displays nuclear and cytoplasmic localizations in transfected cells, whereas a C-terminally truncated mutant is essentially confined to the cytoplasm. TNRC4 is not recruited into inclusions formed by polyglutamine-expanded ataxin-1 or huntingtin. TNRC4 activates tau exon 10 (E10) inclusion at high efficiency in transfected cells. TNRC4 contains two consecutive N-terminal RNA recognition motifs (RRMs) separated from the C-terminal RRM. Deletion and point mutant analysis show that the activity of TNRC4 on tau E10 splicing is mainly mediated by the RNA-binding activity of the second RRM and involves an intronic element of the tau pre-mRNA. The polyglutamine sequence has no effect on the activity of TNRC4 on tau E10 splicing. This study represents the first characterization of TNRC4 and provides further insight into the mechanisms of brain-specific alternative splicing and their possible pathological implications.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
An increasing number of neurological disorders are being linked to impairment of pre-mRNA alternative splicing (1–4). Prominent examples are cases of the rare dementia and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), caused by dominant mutations in the MAPT gene, encoding the microtubule-associated protein tau, affecting the splicing of E10 (5–7). Furthermore, a MAPT haplotype conferring susceptibility to Alzheimer's disease produces elevated levels of E10+ tau mRNA (8,9). E10 encodes the second of four microtubule-binding repeats in the C-terminus of the tau protein. Exclusion or inclusion of E10 gives rise to tau isoforms with three or four repeats that are expressed in approximately equal amounts in adult human brain.

Elucidating the mechanisms of alternative splicing in neurons is a prerequisite to the understanding of the pathogenic mechanisms underlying major neurological diseases and to the identification of relevant therapeutic targets. To date, at least sixteen splicing factors have been shown to be selectively expressed in the brain (10). Prominent examples of neuron-specific splicing factors include Nova-1 and Nova-2, which contain three KH (hnRNP K Homology) motifs (11). The majority of the proteins encoded by the RNA targets of Nova-2 have synaptic functions and a subset of these proteins are interacting partners (12). Thus, in neurons, a single splicing factor can regulate the activity of functional protein networks by coordinating exon usage.

Another splicing factor selectively expressed in the brain is TNRC4, the predicted product of the trinucleotide repeat-containing 4 (TNRC4) gene, also referred to as CELF3 or BRUNOL1. The TNRC4 gene was originally identified through the screening of a brain cDNA library for cDNAs containing CAG repeat sequences encoding proteins with long polyglutamine (polyQ) sequences (13). The aim of this screen was to identify new candidate disease genes, as a number of neurodegenerative diseases are caused by translated CAG repeat expansions in the causative gene (14). The CAG repeat of the TNRC4 gene is 15–18-repeat long but is unlikely to be the subject of pathogenic expansion as its length shows very little variation in the normal human population (15). TNRC4 is a member of the CELF (CUG-BP- and ETR-3-like factors) family of RNA-binding proteins (16,17). The first member of the CELF family identified, ETR-3, is related to Drosophila embryonic lethal abnormal vision protein (ELAV). Among the members of the CELF family, TNRC4 has a very restricted pattern of expression, being expressed in brain and testis only (16,18,19). TNRC4 expression is detected from an early stage of development, appearing at E9.5 in the mouse (19). CELF proteins have been implicated in the pathogenesis of myotonic dystrophy and they regulate alternative splicing of several transcripts known to be misregulated in myotonic dystrophy including cardiac troponin T (cTNT), the insulin receptor and the muscle-specific chloride channel (20). Interestingly, TNRC4 can promote the inclusion of E10 in transcripts from the MAPT gene (21). Like other members of the CELF family, TNRC4 contains two consecutive RNA recognition motifs (RRMs) in the N-terminus separated from a C-terminal RRM by a domain not conserved among other CELF proteins or divergent domain (16) TNRC4 is the only CELF protein containing a polyQ sequence.

Until now, TNRC4 transcripts only have been shown to be expressed in the brain. Here we establish the presence of the TNRC4 protein in the brain. We also define the domains of TNRC4 required for tau E10 splicing activation. This study represents the first characterization of TNRC4 and provides further insight into the mechanisms of brain-specific alternative splicing and their possible pathological implications.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Detection of TNRC4 in brain
Expression of TNRC4 in the brain has been demonstrated previously at the RNA level only (16,18,19). To ascertain that the transcript was translated into a bona fide protein, we generated an anti-peptide antibody against residues 294–306 (APDALYPNGVHPY) of the predicted sequence of human TNRC4. A BLAST search confirmed that this sequence was present in TNRC4 only. The anti-peptide antibody detected a protein of the molecular weight predicted for TNRC4 (465 amino acids, ~50 kDa) in Chinese hamster ovary (CHO) cells transfected with a full-length c-myc-tagged TNRC4 cDNA, co-migrating with the c-myc immunoreactive product (Fig. 1A). Anti-peptide reactivity was detected only in cells that had been transfected with the TNRC4 cDNA and not in non-transfected cells. The protein observed on western blot is therefore the direct product of the transfected cDNA. No immunoreactivity was observed using the pre-immune serum in transfected or non-transfected cells (data not shown).


Figure 1
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Detection of TNRC4 in human brain. (A) An anti-peptide antibody was raised against amino acids 294–306 of the predicted sequence of human TNRC4. The antibody detects a single band of the expected size (465 amino acids, ~50 kDa, green channel) in CHO cells transfected with a full-length c-myc-tagged TNRC4 cDNA, co-migrating with a c-myc immunoreactive product (red channel). (B) The TNRC4 antibody detects a single band in western blots of adult human brain cortex, co-migrating with TNRC4 from transfected CHO cells.

 
The TNRC4 anti-peptide antibody detected a single band in homogenates from adult human brain cortex, co-migrating with TNRC4 from transfected cells (Fig. 1B). Thus the TNRC4 transcript is translated as a protein of the expected molecular weight in human brain.

Subcellular localization of TNRC4
The subcellular localization of TNRC4 was determined by confocal microscopy imaging of CHO cells transfected with full-length, c-myc-tagged TNRC4. The protein displays both nuclear and cytoplasmic localizations (Fig. 2A), consistent with the subcellular distribution of other members of the CELF family, such as ETR-3 (22). ETR-3 possesses a typical arginine and lysine-rich nuclear localization signal (NLS) in the C-terminus of the protein, within RRM3 (22). This domain is conserved among CELF family members, including TNRC4. To determine whether the homologous domain of TNRC4 contained a functional NLS, we constructed a C-terminal deletion mutant, {Delta}RRM3, comprising amino acids 1–378, thus lacking RRM3 and N-terminal tagged with c-myc. In transfected CHO cells, {Delta}RRM3 is essentially confined to the cytoplasm (Fig. 2B), hence confirming the presence of an NLS in the C-terminus of TNRC4. The same distribution pattern was obtained whether the c-myc antibody or the TNRC4 anti-peptide antibody was used.


Figure 2
View larger version (69K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Nuclear and cytoplasmic localization of TNRC4. (A) CHO cells were transfected with full-length, c-myc-tagged, TNRC4 (FL TNRC4) and imaged by laser scanning confocal microscopy. TNRC4 is localized in the nucleus as well as in the cytoplasm. (B) A C-terminal TNRC4 deletion mutant lacking RRM3 ({Delta}RRM3), and N-terminally tagged with c-myc, is essentially confined to the cytoplasm and excluded from the nucleus. This is consistent with the presence of a strong NLS in the C-terminus of TNRC4. Scale bars, 20 µm.

 
TNRC4 is not recruited to aggregates of polyglutamine-expanded proteins
A number of neurodegenerative diseases are caused by the expansion of a polymorphic polyQ sequence in the disease protein. PolyQ-expanded pathogenic proteins aggregate into characteristic inclusion bodies that, in some cases, have been shown to sequester proteins containing polyQ sequences of moderate length (e.g. 10–15 residues) (23–25). TNRC4 contains a 15–18-residue polyQ sequence (Fig. 3A), and hence could potentially be sequestered into aggregates formed by polyQ-expanded, disease-causing proteins. To test this hypothesis, CHO cells were co-transfected with c-myc-tagged TNRC4, containing 15 glutamine residues, and EGFP-tagged ataxin-1, the product of the spinocerebellar ataxia type 1 (SCA1) gene with 82 glutamine residues. In co-transfected cells, polyQ-expanded ataxin-1 forms distinctive large nuclear aggregates (26,27) and TNRC4 has both nuclear and cytoplasmic localizations. However, laser scanning confocal microscopy imaging did not reveal recruitment of TNRC4 into ataxin-1 aggregates (Fig. 3B). Unlike ataxin-1, which forms exclusively intranuclear aggregates, another polyQ-expanded protein, huntingtin, forms both cytoplasmic and nuclear aggregates. We examined whether TNRC4 could be recruited into cytoplasmic huntingtin aggregates. EGFP-tagged huntingtin exon 1 containing 103 glutamine residues formed perinuclear, aggresome-like structures, in transfected cells, as described previously (28) but co-expressed TNRC4 was not recruited into these structures (Fig. 3C).


Figure 3
View larger version (64K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. TNRC4 is not recruited to aggregates of polyglutamine-expanded proteins. (A) Diagram of TNRC4 showing the position of the polyQ sequence. (B) Absence of co-localization of TNRC4 with polyQ-expanded ataxin-1. CHO cells were co-transfected with c-myc-tagged TNRC4, containing 15 glutamine residues and EGFP-tagged ataxin-1 with 82 glutamine residues (Atxn). TNRC4 does not co-localize with large nuclear aggregates formed by ataxin-1. (C) Absence of co-localization of polyQ-expanded huntingtin with TNRC4. CHO cells were co-transfected with c-myc-tagged TNRC4 and EGFP-tagged huntingtin exon 1 containing 103 glutamine residues (Htt). TNRC4 is not recruited into the perinuclear aggresome-like structures formed by huntingtin. Scale bars, 20 µm.

 
Mapping of TNRC4 domains required to activate tau E10 inclusion
The splicing activity of TNRC4 on a neuronal transcript was assayed by reverse transcription (RT)–PCR in transfected cells after co-expression with a tau minigene containing E10 and flanking intronic sequences. The minigene used, LI9/LI10, contains exons 9, 10 and 11 and the entire introns 9 and 10 (29). In the absence of TNRC4, the percentage of E10 inclusion in transcripts derived from LI9/LI10 was lower than 5% (Fig. 4A). E10 inclusion raised to 70–80% in cells co-transfected with full-length TNRC4, demonstrating the strong activity of TNRC4 on tau E10 inclusion (Fig. 4A and C).


Figure 4
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Mapping of TNRC4 domains required to activate tau E10 inclusion. (A) TNRC4 promotes E10 inclusion in vitro. CHO cells were co-transfected with full-length TNRC4 and with the LI9/LI10 tau minigene, and E10 inclusion was assayed by RT–PCR. The percentage of E10 inclusion in transcripts derived from LI9/LI10 was lower than 5% and raised to 70–80% after co-expression with TNRC4. (B) Diagram of full-length TNRC4 and C-terminal deletion mutants. (C) E10 splicing activity of TNRC4 deletion mutants. CHO cells were co-transfected with the LI9/LI10 minigene and c-myc-tagged TNRC4 deletion mutants. Western blot analysis with anti-c-myc antibody confirmed that the deletion constructs produced proteins of the expected sizes and were expressed at similar levels (bottom gel). E10 splicing activity was assessed by RT–PCR. RRM3 is not essential but RRM1 and/or RRM2 as well as part of the divergent domains are required for the ability of TNRC4 to promote E10 inclusion. E10 inclusion is expressed as mean percentage±SEM.

 
TNRC4 comprises three conserved RRMs as well as a divergent domain separating the two adjacent N-terminal RRM1 and RRM2 from the C-terminal RRM3. To define which domain(s) of TNRC4 accounted for the activation of E10 splicing, we generated a series of c-myc-tagged C-terminal deletion mutants (Fig. 4B) and assayed their activity on tau E10 splicing. Western blots confirmed that proteins of the expected sizes were produced from the deletions constructs. Deletions mutants retaining up to residue 335 promoted E10 inclusion with an activity of up to 70%, almost identical to the activity of full-length TNRC4. In contrast, mutants 1–312 had an activity reduced to ~35% (Fig. 4C). Thus, RRM3 is not required but RRM1 and/or RRM2 as well as part of the divergent domains are sufficient for the ability of TNRC4 to promote E10 inclusion.

TNRC4 activates tau E10 inclusion through an RNA binding-dependent mechanism
The shortest TNRC4 deletion mutant promoting E10 inclusion to a significant extent contains both RRM1 and RRM2. The activity of TNRC4 can be mediated by, not mutually exclusive, RNA-binding-dependent mechanisms or protein–protein interactions. To establish whether RNA binding was implicated in TNRC4 activity, point mutations predicted to affect RNA binding were introduced in RRM1 and RRM2. Each RRM comprises two ribonucleoprotein motifs, (RNP1 and RNP2) responsible for RNA binding; RNP2 is highly conserved among CELF proteins (16,17). Changing conserved aromatic into non-aromatic amino acids can destabilize the structure of this motif (17). The conserved sequences of RNP2 in RRM1 and RRM2 are 6AIKLFVGQIPR16 and 94DEKLFVGMLGK104, respectively. Phe10/Gly12 and Phe98/Gly100 were mutated into alanine to abolish the RNA-binding activity of RRM1 and RRM2, respectively (Fig. 5A). Splicing assays were carried out in cells co-transfected with TNRC4 mutants and the LI9/LI10 minigene, as above. Western blotting confirmed that the two mutants were expressed at the same level as wild-type TNRC4. RNP2 mutation in RRM1 had a limited effect on TNRC4 activity, reducing it from 70 to 45% (Fig. 5B); in contrast, RNP2 mutation in RRM2 abolishes almost completely the activity of TNRC4 on tau E10 splicing (Fig. 5B). These results demonstrate that most of the activity of TNRC4 on tau E10 splicing is mediated by the RNA-binding activity of RRM2.


Figure 5
View larger version (26K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. TNRC4 activates tau E10 inclusion through an RNA-binding-dependent mechanism. (A) Diagram showing the position of point mutants destabilizing the RNP2 motifs of RRM1 and RRM2 of TNRC4. (B) Tau E10 splicing activity of TNRC4 point mutants. CHO cells were co-transfected with the LI9/LI10 minigene and TNRC4 mutants. Western blot analysis with anti-c-myc antibody confirmed that wild-type TNRC4 and the two mutants were expressed at similar levels (bottom gel). RNP2 mutation in RRM1 had a limited effect on TNRC4 activity but RNP2 mutation in RRM2 abolishes almost completely the splicing activity of TNRC4. E10 inclusion is expressed as mean percentage±SEM.

 
Role of the polyglutamine sequence in TNRC4 splicing activity
PolyQ sequences are found in a number of proteins with diverse functions, but more frequently in transcription factors. The length of the polyQ sequence influences the activity of some proteins, such as the androgen receptor (30,31). Although the length of the polyQ sequence of TNRC4 is not subject to variation in the normal population (15), we compared the effect of TNRC4 with one or 15 glutamine residues on tau E10 splicing. In cells co-transfected with the LI9/LI10 minigene, both forms of TNRC4 promoted the inclusion of E10 with a similar efficiency (Fig. 6). Thus, the polyQ sequence of TNRC4 does not appear to play a role in its function as a splicing factor.


Figure 6
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. Absence of polyglutamine sequence effect on TNRC4 splicing activity. CHO cells were co-transfected with the LI9/LI10 minigene and TNRC4 with 1Q or 15Q residues and the activity of TNRC4 on tau E10 splicing was assessed as in Figs 4 and 5. Both form of TNRC4 promoted the inclusion of E10 with a similar efficiency.

 
TNRC4 activity on tau E10 splicing involves an intronic element in tau pre-mRNA
To delineate the tau pre-mRNA domain involved in TNRC4 activity on E10 inclusion, splicing assays were conducted using tau minigenes containing reduced intronic sequences. The pSPLtau minigene comprises E10 and 34 and 85 bp of flanking 5' and 3' intronic sequences, respectively, in the exon-trapping vector pSPL3 (Fig. 7). In the absence of TNRC4, transcripts from pSPLtau incorporate E10 with an 80% efficiency (Fig. 7); E10 inclusion rises to 100% after co-expression with TNRC4. However, the difference in activity between wild-type TNRC4 and TNRC4 with the RNP2 mutation in RRM2 was only moderately significant (P=0.018, Fig. 7). Transcripts from a second minigene, pMAPT-4Rluc, comprising 571 and 707 bp of intronic sequences incorporate E10 with a ~30% efficiency, raising to ~95% after co-expression with TNRC4. In contrast, ~30% E10 inclusion was obtained in cells co-transfected with the RRM2 mutant. Thus TNRC4 promotes RRM2-dependent E10 inclusion in transcripts from pMAPT-4RLuc, but not from pSPLtau (Fig. 7). Taken together, these data suggest that the RRM2-dependent activity of TNRC4 on tau E10 inclusion involves intronic sequences located between positions –571 and –34 in intron 9 or between positions +85 and +707 in intron 10.


Figure 7
View larger version (22K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7. TNRC4 activity on tau E10 splicing involves an intronic element in tau pre-mRNA. The activity of TNRC4 on tau E10 inclusion was determined by RT–PCR in cells co-transfected with wild-type TNRC4 or RNP2 mutants and tau minigenes containing reduced intronic sequences. The difference in activity between wild-type TNRC4 and TNRC4 with the RNP2 mutation in RRM2 on pSPLtau transcripts was minimal. In contrast, TNRC4 strongly activates RRM2-dependent E10 inclusion in pMAPT-4Rluc transcripts. Thus, the activity of TNRC4 on tau E10 inclusion involves intronic sequences located between positions –571 and –34 in intron 9 or between positions +85 and +707 in intron 10.

 
Tau E10 splicing in Tnrc4 knock-out mice
To determine whether TNRC4 was involved in the regulation of tau E10 splicing in vivo, we analyzed tau isoforms in mice in which the Tnrc4 gene had been disrupted (19). Unlike human, rodents express only E10+ tau in adult brain and this was not changed after homozygous disruption of the Tnrc4 gene (data not shown), implying that TNRC4 is not essential for E10 inclusion. However, although tau is predominantly expressed in the brain, it is expressed in a number of other tissues, including testes (32,33). Interestingly, both E10+ and E10– tau isoforms are expressed in adult testes (33). As, in addition to the brain, TNRC4 is expressed in testes as well (19), we compared tau E10 splicing in the testes from adult Tnrc4+/– and Tnrc4–/– mice. RT–PCR analysis demonstrated the expression of E10+ and E10– tau mRNA in the testes from Tnrc4+/– and Tnrc4–/– mice, but the E10+/E10– ratio was reduced in Tnrc4–/– when compared with Tnrc4+/– animals (Fig. 8). Thus, the absence of TNRC4 expression results in a decrease of E10 inclusion in tau mRNA.


Figure 8
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 8. Analysis of tau splicing in testes from Tnrc4 knock-out mice. Tau isoform expression in testes from adult Tnrc4–/– and Tnrc4+/– mice was analyzed by RT–PCR. Both E10+ and E10– tau isoforms are expressed and the E10+/E10– ratio was reduced in Tnrc4–/– when compared with Tnrc4+/– animals.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Expression of TNRC4 transcripts has been detected in brain and testis (16,19). An antibody prepared against a peptide from the predicted sequence of the TNRC4 protein detected a protein of the expected molecular weight in brain. Thus, the TNRC4 transcript is translated into a bona fide protein in the brain. Unlike some disease-causing proteins, the length of the polyQ sequence does not appear to affect the function of TNRC4 as a splicing regulator. Furthermore, the presence of a polyQ sequence in TNRC4 is not sufficient to cause its recruitment in aggregates formed by expanded ataxin-1 or huntingtin.

When ectopically expressed in non-neuronal cells, TNRC4 displays cytoplasmic and nuclear localizations. A mutant with the C-terminal RRM, RRM3, deleted had a predominantly cytoplasmic localization, indicating the presence of a strong NLS in the C-terminus. This is consistent with the position of the NLS of ETR-3, which is in the arginine and lysine-rich domain within RRM3, typical of NLS (22). This domain, KRLKVQLKR, is conserved among members of the CELF family, including TNRC4. Dual nuclear and cytoplasmic localizations are a feature of other members of the CELF family, such as ETR-3 (22), indicating that TNRC4 is likely to be involved also in RNA-processing events other than splicing. ETR-3 has been implicated in cytoplasmic functions, such as translational control (17) and mRNA stability (34) as well as RNA editing (35). CELF proteins are related to the Hu family of regulators of mRNA stability. Of note, HuD binds to the 3'-untranslated region (UTR) of tau mRNA and stabilizes it (36,37).

TNRC4 activates tau E10 inclusion in vitro at high efficiency. Analysis of RNA-binding deficient mutants showed that the effect of TNRC4 on tau splicing was mediated mainly by the RNA-binding activity of RRM2. Similarly, the splicing activation or repression activity of CELF4 and ETR-3 on some specific substrates is mediated by RRM1/RRM2 (38,39). On the other hand, both RRM1 and RRM2 of ETR-3 and CELF4 can activate cTNT exon 5 inclusion (38). RRM3 does not appear to contribute to the activity of TNRC4 on tau splicing. This is consistent with the lack of activity of RMM3 of ETR-3 and CELF4 on cTNT exon 5 inclusion (38). Deletion mutant analysis showed that a domain located between residues 312 and 335 was necessary for maximum activity of TNRC4 on tau E10 inclusion. This suggests that the divergent domain might mediate the interaction of TNRC4 with other proteins regulating its activity or involved in target recognition, as shown for other CELF proteins (38). An alternative possibility is that this domain contains a weak NLS. Indeed, it may appear contradictory that TNRC4 deletion mutants can still activate tau E10 splicing in the absence of the C-terminal NLS. However, this has been observed also for CELF4 and ETR-3 (38,39), thus supporting the presence of a second, weak NLS, in the divergent domain, consistent with the existence of such a signal in ETR-3 (22).

E10 contains several splicing regulatory elements that have been extensively characterized. A splicing enhancer within E10 serves as a binding site for Tra2ß and SF2/ASF (40,41); in turn, Tra2ß activity is antagonized by the serine–arginine-rich (SR) protein, SRp54, hence providing an additional level of regulation (42). SRp30c and SRp55 inhibit E10 inclusion by binding to silencer elements at the 5' end of E10 (21,43). Intronic elements regulating E10 splicing have not been as extensively characterized as exonic elements and few trans-acting factors binding to tau pre-mRNA intronic elements have been identified. Some elements are close to E10, such as a regulatory domain at the 5' end of intron 10, comprising an intronic splicing silencer and an intronic splicing modulator (44). The SR protein 9G8 binds to the intron 10 silencer and strongly inhibits E10 inclusion (45). Other regulatory elements are distant from E10; for instance, the RNA-binding protein, RBM4 (RNA-binding motif protein 4), promotes E10 inclusion by binding to an element about 100 nt downstream of the exon 10–intron 10 junction (46).

Splicing assays using minigenes with reduced intronic sequences flanking E10 showed that the activity of TNRC4 on tau E10 splicing involved intronic elements. This is consistent with the known binding of CELF proteins to intronic elements (47) and also indicates that TNRC4 is unlikely to act directly or indirectly on a splicing regulatory element within E10. The elements mediating TNRC4 activity on tau E10 splicing are located either in intron 9 between positions –571 and –34 or in intron 10 between positions +85 and +707. Elements to which CELF proteins bind are not precisely defined. For example, ETR-3, the best characterized member of the CELF family binds preferentially to sequences with UG repeats and UGUU motifs (47), but some CELF proteins, such as CELF6, can influence splicing of some substrate in the absence of such motifs (18,48). Defining the preferred binding sites of TNRC4 would require the use of methods such as systematic evolution of ligands by exponential enrichment (SELEX).

Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansion in the 3'-UTR of the DMPK gene (20) and involves abnormal activities of the RNA-binding proteins, Muscleblind, and CUG-BP. DM1 patients display a tau pathology correlated with abnormal splicing of tau E2 (49,50) and E10 (51). However, the connection of the latter with the phenotype is uncertain. Interestingly, the splicing of tau E2 is regulated by a CELF protein, ETR-3 (52). Thus, an abnormal activity of CELF proteins, including TNRC4, on tau splicing may be a factor contributing to the neuronal pathology of DM1.

Analysis of tau splicing in testes from Tnrc4–/– mice suggests that TNRC4 is involved in the regulation of tau E10 splicing in vivo, and we are currently investigating the precise role of TNRC4 in tau splicing in the brain. Taken together, our results identify TNRC4 as a brain RNA-binding protein and suggests a role for TNRC4 in multiple RNA processing events as well as its possible implication in the pathogenesis of tauopathies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Plasmids
A human TNRC4 full-length cDNA clone (16) was tagged with the c-myc epitope sequence at the N-terminus. Deletions and point mutations of TNRC4 were generated using the QuickChange system (Stratagene). The MAPT minigenes LI9/LI10, pMAPT-4RLuc and pSPLtau (for details, see Results and Fig. 7) have been described previously (29,53,54) and were obtained from Drs Jianhua Zhou, Michael Wolfe and Michel Goedert, respectively. Expression vectors encoding ataxin-1 with 82 glutamine repeats and huntingtin exon 1 with 103 glutamine residues tagged with EGFP were obtained from Drs Huda Zoghbi and David Rubinsztein, respectively.

Cell culture and transfection
CHO-1 cells were maintained in DMEM supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, Invitrogen), 100 U/ml penicillin/100 µg/ml streptomycin and 2 mM L-glutamine, in a humidified atmosphere of 95% air/5% CO2 at 37°C. Cells were transiently transfected with Lipofectamine 2000 reagent (Invitrogen). Briefly, cells plated on 35 mm dishes were grown to 60–70% confluence and exposed to the DNA/liposome complex for 5 h in Optimem (Invitrogen) before being returned to normal culture medium. Cells were routinely analyzed 24 or 48 h after transfection. Typically, 3 µg of each plasmid was used in each transfection.

Splicing assays
Tau E10 splicing was assayed by RT–PCR in cells transiently co-transfected with a tau minigene and TNRC4 constructs essentially as described previously (55). Briefly, total RNA was isolated from transfected cells using Trizol reagent (Invitrogen) and treated with DNase I (Promega). RT was performed using the MultiscribeTM RT kit (Applied Biosystems) and oligo-dT. The RT conditions were 10 min at 25°C, 30 min at 48°C and a final step of 5 min at 95°C. The sequence of the primers used for PCR amplification is listed in Table 1. PCR was carried out under the following conditions: 95°C for 5 min, 40 cycles of 30 s at 94°C, 30 s at 68°C, 45 s at 72°C and a final step of 10 min at 72°C. RT–PCR products were separated by electrophoresis in 2% (w/v) agarose gels and stained with ethidium bromide. Densitometric analysis of the gels was performed using the Innotech software (Alpha Innotech, San Leandro, CA, USA). The splicing activity was expressed as percentage of E10 inclusion and statistical comparisons were made with the Student's t-test.


View this table:
[in this window]
[in a new window]

  		Table 1. Sequence of the primers used in tau E10 splicing assays

 
Analysis of tau isoforms from Tnrc4 knock-out mice
Tnrc4 knock-out mice (originally referred to as Brunol1 knock-out mice) have been described previously (19). RNA was purified from the brain and testes of Tnrc4 knock-out mice as described (19). Tau isoform ratio was analyzed as above using a forward primer in exon 9 and a reverse primer in exon 13 (Table 1). The identity of the two RT–PCR products as E10+ and E10– mouse tau was confirmed by sequencing. For sequencing, RT–PCR products were excised from agarose gels and cloned into the pCR2.1 vector using the TA cloning system (Invitrogen). Sequencing of both strands was performed using T3 and T7 primers.

TNRC4 antibody preparation
An antibody to TNRC4 was prepared commercially (Affiniti/Biomol International) in rabbits against the peptide APDALYPNGVHPY corresponding to residues 294–306 of the predicted sequence of human TNRC4. This sequence is conserved between the human, rat and mouse proteins. The peptide was conjugated to keyhole limpet hemocyanin before immunization and the antiserum was affinity-purified against the immunizing peptide.

Western blotting
Cells were homogenized in electrophoresis sample buffer [0.0625 M Tris–HCl, pH 6.8, 5% (v/v) ß-mercaptoethanol, 2% (w/v) SDS, 10% (v/v) glycerol, 0.00125 (w/v) Bromophenol blue] containing a protease inhibitor cocktail (Complete®, Roche). Proteins were resolved in 7.5% (w/v) SDS–polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked in 10% (w/v) skimmed milk in PBS for 1 h at room temperature and then incubated overnight at 4°C with the monoclonal anti-c-myc antibody 9E10 or with the TNRC4 polyclonal antibody. Immunoreactivity was detected using IRDye800-conjugated anti-rabbit (Rockland Laboratories) or Alexa Fluor-680-conjugated anti-mouse (Molecular Probes/ Invitrogen) antibodies and the membranes were imaged using an Odyssey Infrared Imaging System (Li-Cor).

Immunofluorescence microscopy
Cells grown on cover slips were rinsed in PBS at 37°C and fixed in methanol at –20°C for 10 min. Following fixation, cells were rehydrated in PBS for 10 min and blocked in PBS containing 5% (v/v) FBS for 30 min at room temperature. Cells were sequentially incubated with the 9E10 anti-c-myc antibody followed by Alexa Fluor-488 or Texas Red anti-mouse antibody (Molecular Probes/Invitrogen). All antibody incubations were carried out in 5% (v/v) FBS at room temperature for 30 min. Between incubations, cells were washed in PBS over 20 min and counterstained with Hoechst 33258 (180 mM). Cover slips were mounted in fluorescent mounting medium. Cells were imaged using a Zeiss LSM510 META laser scanning confocal microscope.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
This work was supported by the Biotechnology and Biological Sciences Research Council, the Medical Research Council, the Alzheimer's Research Trust and the Psychiatry Research Trust and by the Muscular Dystrophy Association and NIH (R01AR45653) TO T.A.C.


   ACKNOWLEDGEMENTS
 
We thank Drs Jianhua Zhou, Michael Wolfe and Michel Goedert for tau minigenes and Drs Huda Zoghbi and David Rubinsztein for ataxin-1 and huntingtin constructs. We thank Ms Selina Wray for human brain samples. We also thank Drs Janice Robertson and Lubov Timchenko for helpful discussions.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back

{ddagger} Present address: Centre for Endocrinology, William Harvey Research Institute, Barts and the London, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

  1. Faustino N.A., Cooper T.A. Pre-mRNA splicing and human disease. Genes Dev. (2003) 17:419–437.[Free Full Text]

  2. Garcia-Blanco M.A., Baraniak A.P., Lasda E.L. Alternative splicing in disease and therapy. Nat. Biotechnol. (2004) 22:535–546.[CrossRef][Web of Science][Medline]

  3. Gallo J.-M., Jin P., Thornton C.A., Lin H., Robertson J., D'Souza I., Schlaepfer W.W. The role of RNA and RNA processing in neurodegeneration. J. Neurosci. (2005) 25:10372–10375.[Free Full Text]

  4. Licatalosi D.D., Darnell R.B. Splicing regulation in neurologic disease. Neuron. (2006) 52:93–101.[CrossRef][Web of Science][Medline]

  5. Hutton M., Lendon C.L., Rizzu P., Baker M., Froelich S., Houlden H.H., Pickering Brown S., Chakraverty S., Isaacs A., Grover A., et al. Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature (1998) 393:702–704.[CrossRef][Medline]

  6. Poorkaj P., Bird T.D., Wijsman E., Nemens E., Garruto R.M., Anderson L., Andreadis A., Wiederholt W.C., Raskind M., Schellenberg G.D. Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol. (1998) 43:815–825.[CrossRef][Web of Science][Medline]

  7. Spillantini M.G., Murrell J.R., Goedert M., Farlow M.R., Klug A., Ghetti B. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl Acad. Sci. USA (1998) 95:7737–7741.[Abstract/Free Full Text]

  8. Caffrey T.M., Joachim C., Paracchini S., Esiri M.M., Wade-Martins R. Haplotype-specific expression of exon 10 at the human MAPT locus. Hum. Mol. Genet. (2006) 15:3529–3537.[Abstract/Free Full Text]

  9. Myers A.J., Pittman A.M., Zhao A.S., Rohrer K., Kaleem M., Marlowe L., Lees A., Leung D., McKeith I.G., Perry R.H., et al. The MAPT H1c risk haplotype is associated with increased expression of tau and especially of 4 repeat containing transcripts. Neurobiol. Dis. (2006) 25:561–570.[CrossRef][Web of Science][Medline]

  10. McKee A.E., Minet E., Stern C., Riahi S., Stiles C.D., Silver P.A. A genome-wide in situ hybridization map of RNA-binding proteins reveals anatomically restricted expression in the developing mouse brain. BMC Dev. Biol. (2005) 5:14.[CrossRef][Medline]

  11. Jensen K.B., Dredge B.K., Stefani G., Zhong R., Buckanovich R.J., Okano H.J., Yang Y.Y., Darnell R.B. Nova-1 regulates neuron-specific alternative splicing and is essential for neuronal viability. Neuron (2000) 25:359–371.[CrossRef][Web of Science][Medline]

  12. Ule J., Ule A., Spencer J., Williams A., Hu J.S., Cline M., Wang H., Clark T., Fraser C., Ruggiu M., et al. Nova regulates brain-specific splicing to shape the synapse. Nat. Genet. (2005) 37:844–852.[CrossRef][Web of Science][Medline]

  13. Margolis R.L., Abraham M.R., Gatchell S.B., Li S.H., Kidwai A.S., Breschel T.S., Stine O.C., Callahan C., McInnis M.G., Ross C.A. cDNAs with long CAG trinucleotide repeats from human brain. Hum. Genet. (1997) 100:114–122.[CrossRef][Web of Science][Medline]

  14. Gatchel J.R., Zoghbi H.Y. Diseases of unstable repeat expansion: mechanisms and common principles. Nat. Rev. Genet. (2005) 6:743–755.[Web of Science][Medline]

  15. Butland S.L., Devon R.S., Huang Y., Mead C.L., Meynert A.M., Neal S.J., Lee S.S., Wilkinson A., Yang G.S., Yuen M.M., et al. CAG-encoded polyglutamine length polymorphism in the human genome. BMC. Genomics. (2007) 8:126.[CrossRef][Medline]

  16. Ladd A.N., Charlet N., Cooper T.A. The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol. Cell. Biol. (2001) 21:1285–1296.[Abstract/Free Full Text]

  17. Good P.J., Chen Q., Warner S.J., Herring D.C. A family of human RNA-binding proteins related to the Drosophila Bruno translational regulator. J. Biol. Chem. (2000) 275:28583–28592.[Abstract/Free Full Text]

  18. Ladd A.N., Nguyen N.H., Malhotra K., Cooper T.A. CELF6, a member of the CELF family of RNA-binding proteins, regulates muscle-specific splicing enhancer-dependent alternative splicing. J. Biol. Chem. (2004) 279:17756–17764.[Abstract/Free Full Text]

  19. Dev A., Nayernia K., Meins M., Adham I., Lacone F., Engel W. Mice deficient for RNA-binding protein brunol1 show reduction of spermatogenesis but are fertile. Mol. Reprod. Dev. (2007) 74:1456–1464.[CrossRef][Medline]

  20. Ranum L.P., Cooper T.A. RNA-mediated neuromuscular disorders. Annu. Rev. Neurosci. (2006) 29:259–277.[CrossRef][Web of Science][Medline]

  21. Wang J., Gao Q.S., Wang Y., Lafyatis R., Stamm S., Andreadis A. Tau exon 10, whose missplicing causes frontotemporal dementia, is regulated by an intricate interplay of cis elements and trans factors. J. Neurochem. (2004) 88:1078–1090.[CrossRef][Web of Science][Medline]

  22. Ladd A.N., Cooper T.A. Multiple domains control the subcellular localization and activity of ETR-3, a regulator of nuclear and cytoplasmic RNA processing events. J. Cell Sci. (2004) 117:3519–3529.[Abstract/Free Full Text]

  23. Perez M.K., Paulson H.L., Pendse S.J., Saionz S.J., Bonini N.M., Pittman R.N. Recruitment and the role of nuclear localization in polyglutamine-mediated aggregation. J. Cell Biol. (1998) 143:1457–1470.[Abstract/Free Full Text]

  24. Lee W.-C.M., Yoshihara M., Littleton J.T. Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease. Proc. Natl Acad. Sci. USA (2004) 101:3224–3229.[Abstract/Free Full Text]

  25. Narain Y., Wyttenbach A., Rankin J., Furlong R.A., Rubinsztein D.C. A molecular investigation of true dominance in Huntington's disease. J. Med. Genet. (1999) 36:739–746.[Abstract/Free Full Text]

  26. Skinner P.J., Koshy B.T., Cummings C.J., Klement I.A., Helin K., Servadio A., Zoghbi H.Y., Orr H.T. Ataxin-1 with an expanded glutamine tract alters nuclear matrix-associated structures. Nature (1997) 389:971–974.[CrossRef][Medline]

  27. Cummings C.J., Mancini M.A., Antalffy B., DeFranco D.B., Orr H.T., Zoghbi H.Y. Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet. (1998) 19:148–154.[CrossRef][Web of Science][Medline]

  28. Wyttenbach A., Sauvageot O., Carmichael J., Diaz-Latoud C., Arrigo A.P., Rubinsztein D.C. Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin. Hum. Mol. Genet. (2002) 11:1137–1151.[Abstract/Free Full Text]

  29. Yu Q., Guo J., Zhou J. A minimal length between tau exon 10 and 11 is required for correct splicing of exon 10. J. Neurochem. (2004) 90:164–172.[CrossRef][Web of Science][Medline]

  30. Mhatre A.N., Trifiro M.A., Kaufman M., Kazemi-Esfarjani P., Figlewicz D., Rouleau G., Pinsky L. Reduced transcriptional regulatory competence of the androgen receptor in X-linked spinal and bulbar muscular atrophy. Nat. Genet. (1993) 5:184–188.[CrossRef][Web of Science][Medline]

  31. Butler R., Leigh P.N., McPhaul M.J., Gallo M. Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophy. Hum. Mol. Genet. (1998) 7:121–127.[Abstract/Free Full Text]

  32. Ashman J.B., Hall E.S., Eveleth J., Boekelheide K. Tau, the neuronal heat-stable microtubule-associated protein, is also present in the cross-linked microtubule network of the testicular spermatid manchette. Biol. Reprod. (1992) 46:120–129.[Abstract]

  33. Gu Y., Oyama F., Ihara Y. Tau is widely expressed in rat tissues. J. Neurochem. (1996) 67:1235–1244.[Web of Science][Medline]

  34. Mukhopadhyay D., Houchen C.W., Kennedy S., Dieckgraefe B.K., Anant S. Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein, CUGBP2. Mol. Cell (2003) 11:113–126.[CrossRef][Web of Science][Medline]

  35. Anant S., Henderson J.O., Mukhopadhyay D., Navaratnam N., Kennedy S., Min J., Davidson N.O. Novel role for RNA-binding protein CUGBP2 in mammalian RNA editing. CUGBP2 modulates C to U editing of apolipoprotein B mRNA by interacting with apobec-1 and ACF, the apobec-1 complementation factor. J. Biol. Chem. (2001) 276:47338–47351.[Abstract/Free Full Text]

  36. Aranda-Abreu G.E., Behar L., Chung S., Furneaux H., Ginzburg I. Embryonic lethal abnormal vision-like RNA-binding proteins regulate neurite outgrowth and tau expression in PC12 cells. J. Neurosci. (1999) 19:6907–6917.[Abstract/Free Full Text]

  37. Aronov S., Marx R., Ginzburg I. Identification of 3'-UTR region implicated in tau mRNA stabilization in neuronal cells. J. Mol. Neurosci. (1999) 12:131–145.[Web of Science][Medline]

  38. Singh G., Charlet B., Han J., Cooper T.A. ETR-3 and CELF4 protein domains required for RNA binding and splicing activity in vivo. Nucleic Acids Res. (2004) 32:1232–1241.[Abstract/Free Full Text]

  39. Han J., Cooper T.A. Identification of CELF splicing activation and repression domains in vivo. Nucleic Acids Res. (2005) 33:2769–2780.[Abstract/Free Full Text]

  40. Jiang Z., Tang H., Havlioglu N., Zhang X., Stamm S., Yan R., Wu J.Y. Mutations in tau gene exon10 associated with FTDP-17 alter the activity of an exonic splicing enhancer to interact with Tra2ß. J. Biol. Chem. (2003) 278:18997–19007.[Abstract/Free Full Text]

  41. D'Souza I., Schellenberg G.D. Arginine/serine-rich protein interaction domain-dependent modulation of a tau exon 10 splicing enhancer: altered interactions and mechanisms for functionally antagonistic FTDP-17 mutations {Delta}280K and N279K. J. Biol. Chem. (2006) 281:2460–2469.[Abstract/Free Full Text]

  42. Wu J.Y., Kar A., Kuo D., Yu B., Havlioglu N. SRp54 (SFRS11), a regulator for tau exon 10 alternative splicing identified by an expression cloning strategy. Mol. Cell. Biol. (2006) 26:6739–6747.[Abstract/Free Full Text]

  43. Wang Y., Wang J., Gao L., Lafyatis R., Stamm S., Andreadis A. Tau exons 2 and 10, which are misregulated in neurodegenerative diseases, are partly regulated by silencers which bind a complex comprised of SRp30c and SRp55 that either recruits or antagonizes htra2ß1. J. Biol. Chem. (2005) 280:14230–14239.[Abstract/Free Full Text]

  44. D'Souza I., Schellenberg G.D. Tau exon 10 expression involves a bipartite intron 10 regulatory sequence and weak 5' and 3' splice sites. J. Biol. Chem. (2002) 277:26587–26599.[Abstract/Free Full Text]

  45. Gao L., Wang J., Wang Y., Andreadis A. SR protein 9G8 modulates splicing of tau exon 10 via its proximal downstream intron, a clustering region for frontotemporal dementia mutations. Mol. Cell. Neurosci. (2007) 34:48–58.[CrossRef][Web of Science][Medline]

  46. Kar A., Havlioglu N., Tarn W.Y., Wu J.Y. RBM4 interacts with an intronic element and stimulates tau exon 10 inclusion. J. Biol. Chem. (2006) 281:24479–24488.[Abstract/Free Full Text]

  47. Faustino N.A., Cooper T.A. Identification of putative new splicing targets for ETR-3 using sequences identified by systematic evolution of ligands by exponential enrichment. Mol. Cell. Biol. (2005) 25:879–887.[Abstract/Free Full Text]

  48. Gromak N., Matlin A.J., Cooper T.A., Smith C.W. Antagonistic regulation of alpha-actinin alternative splicing by CELF proteins and polypyrimidine tract binding protein. RNA. (2003) 9:443–456.[Abstract/Free Full Text]

  49. Vermersch P., Sergeant N., Ruchoux M.M., Hofmann-Radvanyi H., Wattez A., Petit H., Dwailly P., Delacourte A. Specific tau variants in the brains of patients with myotonic dystrophy. Neurology (1996) 47:711–717.[Abstract/Free Full Text]

  50. Sergeant N., Sablonniere B., Schraen-Maschke S., Ghestem A., Maurage C.A., Wattez A., Vermersch P., Delacourte A. Dysregulation of human brain microtubule-associated tau mRNA maturation in myotonic dystrophy type 1. Hum. Mol. Genet. (2001) 10:2143–2155.[Abstract/Free Full Text]

  51. Jiang H., Mankodi A., Swanson M.S., Moxley R.T., Thornton C.A. Myotonic dystrophy type 1 is associated with nuclear foci of mutant RNA, sequestration of muscleblind proteins and deregulated alternative splicing in neurons. Hum. Mol. Genet. (2004) 13:3079–3088.[Abstract/Free Full Text]

  52. Leroy O., Dhaenens C.M., Schraen-Maschke S., Belarbi K., Delacourte A., Andreadis A., Sablonniere B., Buee L., Sergeant N., Caillet-Boudin M.L. ETR-3 represses Tau exons 2/3 inclusion, a splicing event abnormally enhanced in myotonic dystrophy type I. J. Neurosci. Res. (2006) 84:852–859.[CrossRef][Web of Science][Medline]

  53. Donahue C.P., Muratore C., Wu J.Y., Kosik K.S., Wolfe M.S. Stabilization of the tau exon 10 stem loop alters pre-mRNA splicing. J. Biol. Chem. (2006) 281:23302–23306.[Abstract/Free Full Text]

  54. Hasegawa M., Smith M.J., Iijima M., Tabira T., Goedert M. FTDP-17 mutations N279K and S305N in tau produce increased splicing of exon 10. FEBS Lett. (1999) 443:93–96.[CrossRef][Web of Science][Medline]

  55. Rodriguez-Martin T., Garcia-Blanco M.A., Mansfield S.G., Grover A.C., Hutton M., Yu Q., Zhou J., Anderton B.H., Gallo M. Reprogramming of tau alternative splicing by spliceosome-mediated RNA trans-splicing: Implications for tauopathies. Proc. Natl Acad. Sci. USA (2005) 102:15659–15664.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
P. Stoilov, C.-H. Lin, R. Damoiseaux, J. Nikolic, and D. L. Black
A high-throughput screening strategy identifies cardiotonic steroids as alternative splicing modulators
PNAS, August 12, 2008; 105(32): 11218 - 11223.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
16/22/2760    most recent
ddm233v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (1)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Chapple, J. P.
Right arrow Articles by Gallo, J.-M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Chapple, J. P.
Right arrow Articles by Gallo, J.-M.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?