Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M505557200 on September 12, 2005

J. Biol. Chem., Vol. 280, Issue 45, 37572-37584, November 11, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/45/37572    most recent
M505557200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buratti, E.
Right arrow Articles by Baralle, F. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buratti, E.
Right arrow Articles by Baralle, F. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

TDP-43 Binds Heterogeneous Nuclear Ribonucleoprotein A/B through Its C-terminal Tail

AN IMPORTANT REGION FOR THE INHIBITION OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR EXON 9 SPLICING*

Emanuele Buratti1, Antonia Brindisi1, Maurizio Giombi, Sergio Tisminetzky, Youhna M. Ayala, and Francisco E. Baralle2

From the International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy

Received for publication, May 20, 2005 , and in revised form, September 7, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TDP-43 is a highly conserved nuclear factor of yet unknown function that binds to ug-repeated sequences and is responsible for cystic fibrosis transmembrane conductance regulator exon 9 splicing inhibition. We have analyzed TDP-43 interactions with other splicing factors and identified the critical regions for the protein/protein recognition events that determine this biological function. We show here that the C-terminal region of TDP-43 is capable of binding directly to several proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with well known splicing inhibitory activity, in particular, hnRNP A2/B1 and hnRNP A1. Mutational analysis showed that TDP-43 proteins lacking the C-terminal region could not inhibit splicing probably because they were unable to form the hnRNP-rich complex involved in splicing inhibition. Finally, through splicing complex analysis, we show that splicing inhibition mediated by TDP-43 occurs at the earliest stages of spliceosomal assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In its most basic form, the splicing process has the task of removing from the primary RNA transcript all of those sequences (introns) that will not be present in the mature mRNA (1–3). Alternative splicing, i.e. the inclusion/exclusion of selected exonic sequences in particular tissues or developmental stages, has been heavily exploited by evolution to generate multiple mRNA transcripts from the same pre-mRNA sequence (4–6). However, all of this flexibility also means that the splicing process is prone to mistakes following even minor changes (7), and alterations of splicing are being increasingly reported as the underlying cause of many genetic diseases (8–12).

At the molecular level, the removal of introns and the joining of exons are catalyzed by the spliceosome, which contains several hundred different proteins in addition to the five spliceosomal small nuclear RNAs (13, 14). This complex arrangement of factors has two functions: first, to define the exact boundaries of an exon; and second, to catalyze the cut-and-paste generation of the mature mRNA. However, many external factors can also contribute to its workings, such as RNA secondary structure (15), transcription rates (16), the presence of splicing enhancer and silencer elements (17, 18), and even external stimuli (19, 20). It is the combinatorial effect of all of these factors that will decide when, where, and to what degree a specific sequence will be included or not in the mature mRNA (17). Recently, the finding that at least 5% of all human alternative exons are derived from the highly repeated dimeric retrotransposons Alu elements has focused a lot of attention on the potential splicing modulatory ability of repeated nucleotide sequences (21).

In previous works, we have focused our attention on clarifying the pathological role played by (ug)m-repeated sequences near the 3'-splice site of cystic fibrosis transmembrane conductance regulator (CFTR)3 exon 9 (22–24), as they have been known to promote skipping and to correlate well with disease penetrance (25). In particular, we have shown that (ug)m repeats promote skipping of this exon through their interaction with TDP-43, a protein that, although first described as a DNA-binding protein, has all of the characteristics of a heterogeneous nuclear ribonucleoprotein (hnRNP) (23, 26, 27). Significantly, depletion of this protein in transient transfections using antisense oligonucleotides (23) or in in vitro assays (24) results in increasing CFTR exon 9 inclusion, whereas add-back experiments in a depleted nuclear extract restore exon inhibition (24, 28). In keeping with this, it has been recently shown through minigene analysis that CFTR exon 9 inclusion in a wide panel of different cell lines correlates well with the endogenous levels of TDP-43 in each respective cell line (29). It should be noted, however, that an alternative explanation based on RNA secondary structure has been offered by Hefferon et al. (30) in which the role of TDP-43 binding to the ug repeats has been questioned.

In addition to CFTR exon 9 splicing, the isolation of TDP-43 as a highly specific (ug)m-binding protein is relevant to other genes in which (ug)m-repeated sequences have been described at the 3'-splice site, such as in the case of apolipoprotein AII exon 3 (31) and intron 2 of the human cardiac Na+-Ca2+ exchanger (32). In addition, ug-repeated sequences have been predicted to function as intronic splicing enhancer elements in the fish Fugu rubripes (33). All of these examples indicate that this kind of dinucleotide repeat may well play an important biological role in other systems besides the CFTR gene, something that may well be reflected also in the observation that (ug)m-binding proteins have been found in many different organisms. In fact, (ug)m-binding proteins have been described in Chlamydomonas reinhardtii, in which they participate in the regulation of circadian-controlled processes (34); in Caenorhabditis elegans, in which a novel ug-specific binding protein (SUP-12) has been shown to control muscle-specific splicing of the actin-depolymerizing factor/cofilin gene (35); and also in humans (36–38). Interestingly, this kind of repeated dinucleotide sequence can play a functional role irrespective of the direction that its gets transcribed. In fact, (ca)m repeats have also been described to have an effect on splicing by promoting splicing inclusion of exon 13 in the endothelial nitricoxide synthase gene (39, 40). Also in this case, the trans-acting factor binding to these repeats has been identified and shown to correspond to the hnRNP L nuclear protein. Not unexpectedly and in a way analogous but opposite to TDP-43, the length of the (ca)m repeats correlates with hnRNP L binding and exon inclusion (39).

However, besides its role in CFTR exon 9 splicing, very little is known concerning the normal biological function of TDP-43. At present, experimental data show only that it is a highly conserved protein, as closely related proteins have been found in Drosophila and Caenorhabditis (28, 41), that its Xenopus homolog may be involved in the timing of events in mitosis (42), and that it may act as a bridge between the various nuclear bodies possibly through an interaction with the SMN protein (43). Recently, its expression has also been shown to be up-regulated in cells infected by respiratory syncytial virus (44). Analogously, very little is also known regarding the mechanism through which TDP-43 can inhibit splicing; and at the moment, the available data show only that its C-terminal tail plays an essential role in inhibiting this process (28, 41). Here, we have investigated the reason for this observation; and in this work, we show that the C-terminal region of TDP-43 is capable of interacting with several proteins of the hnRNP family, in particular, hnRNP A2/B1, hnRNP A1, hnRNP C1/C2, and hnRNP A3. This interaction is probably essential for the splicing inhibitory activity of TDP-43.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
RNA Secondary Structure Determination—RNA secondary structure determination using limited V1 RNase, T1 RNase, and S1 nuclease digestion has been described in detail (45).

Glutathione S-Transferase (GST) Overlay, HPLC Analysis, and Nanoelectrospray Mass Spectrometry—Western blots containing both HeLa nuclear and cytoplasmic extracts (150 µg) were incubated for 2 h with GST-TDP-43 (10 µg of protein in 20 ml of phosphate-buffered saline (PBS) and 10% (w/v) nonfat dried milk) for 1 h. The blots were washed four times with PBS plus 0.2% Tween 20 and incubated for 1 h with a commercial anti-GST antibody (Sigma) at a dilution of 1:2000. The blots were then washed again four times with PBS plus 0.2% Tween 20 and incubated for 1 h with horseradish peroxidase-conjugated anti-goat antibody (DakoCytomation A/S) at a dilution of 1:2000. After four final washes, the Western blots were developed by ECL (Amersham Biosciences). Fractionation of nuclear extracts was performed on an HPLC Agilent 1100 series using a Phenomenex C4 300A reverse phase column (250 x 4.6 mm). The proteins were eluted using a solution of 95% acetonitrile and 0.1% trifluoroacetic acid in a 20–70% linear gradient over 38 min. The different fractions were first speed-dried for 2 h to reduce volume and then concentrated using Microcon YM-10 filters. Each fraction was divided in two and loaded onto 10% SDS-polyacrylamide gels; one was stained with Coomassie Blue, whereas the other was blotted on Optitran BA-S 83 nitrocellulose membrane (Schleicher & Schüll) and subjected to GST overlay as described above. Internal sequence analysis with the Coomassie Blue-stained bands excised from the SDS-polyacrylamide gel was performed using an electrospray ionization mass spectrometer (LCQ Deca XP, Thermo Electron Corp.). The bands were digested with trypsin, and the resulting peptides were extracted with water, 60% acetonitrile, and 1% trifluoroacetic acid. The fragments were then analyzed by mass spectrometry, and the proteins were identified by analysis of the peptide tandem mass spectrometry data with Turbo SEQUEST (Thermo Electron Corp.) and Mascot (Matrix Science).

Reverse Transcription-PCR Amplifications of TDP-43, hnRNP Factors, and Mutants and Their Expression—The GST-TDP-43 and GST-TDP-43-(101–261) expression plasmids have been described (23). The GST-TDP-43(NR-1) and GST-TDP-43-(101–414) mutants are protein fragments containing residues 1–190 and 101–414 of TDP-43, respectively. The fragments were amplified by PCR using oligonucleotides 571SmaRV (5'-tcccccggggcttctcaaaggctcatcttgg-3') and pGEX5' (5'-gggctggcaagccacgtttggtg-3') for GST-TDP-43(NR-1) and oligonucleotides TDPDNf (5'-cgcggatccagaaaacatccgatttaatagtg-3') and pGEX3' (5'-ccgggagctgcatgtgtcagagg-3') for GST-TDP-43-(101–414). The products were cloned into the pGEX-3X expression vector (Amersham Biosciences) using BamHI and SmaI in the case of GST-TDP-43(NR-1) and BamHI and EcoRI in the case of GST-TDP-43-(101–414). Expression and purification of the mutants were carried out as described previously (23). The GST-TDP-43({Delta}Cter) mutant has been used in a recent study (28). The plasmid expressing His-hnRNP A1 and rabbit antisera directed against this protein were a kind gift of Alexander Ochem and Raffaella Klima (International Centre for Genetic Engineering and Biotechnology). The hnRNP C2 sequence (to obtain GST-hnRNP C2) was amplified by reverse transcription-PCR from total HeLa RNA using primers hnRNPCS (5'-aataaagaattctcatcctccattggcgctgtctctgtcatcctc-3', sense) and hnRNPCAS (5'-tcgactcactgggctggcacaggctgaggggtctctcgcccactatcattaggcccctcgg-3', antisense). The amplified product was then sequenced and inserted in the pGEX-3X plasmid for expression. The same procedure was followed to obtain the GST-hnRNP A2 plasmid using primers hnRNPA2S (5'-aatttaaggatccccatggagagagaaaagga-3', sense) and hnRNPA2AS (5'-aattaagaattctcagtatcggctcctcccaccataa-3', antisense). Mutant hnRNP A2 sequences were produced as follows. GST-hnRNP A2({Delta}Cter) was obtained by amplifying the hnRNP A2 cDNA with primers hnRNPA2S and hnRNPA2/631AS (5'-cctcctctaaagttacttcctggtcc-3', antisense), whereas hnRNP A2({Delta}RRM2) was obtained by a two-round amplification procedure using primers hnRNPA2/{Delta}RRM2S (5'-ctcatgtaactgtggaaatgcaggaagtt-3', sense) and hnRNPA2/{Delta}RRM2AS (5'-aacttcctgcatttccacagttacatgag-3', antisense). All proteins were expressed and purified as described previously (23). Polyclonal antibodies were then obtained by immunizing a 3-month old New Zealand white rabbit according to standard immunization protocols.

GST Pull-down Analysis—Approximately 40 µg of each recombinant protein were bound to 500 µl of glutathione-Sepharose 4B resin and incubated with 0.5 mg of HeLa nuclear protein extract (C4 Biotech) for 2 h at room temperature in PBS and 0.2% Tween 20. As a control, an equal amount of GST protein was bound to a second batch of glutathione-Sepharose 4B resin. After four wash cycles, the proteins bound to the resin were incubated with SDS-PAGE loading buffer and loaded onto a 10% SDS-polyacrylamide gel. Western blotting was performed following standard protocols using rabbit anti-TDP-43 sera at a dilution of 1:2000, and blots were developed by ECL.

Protein/Protein Interaction by Pull-down Analysis—200 µl of nickel-nitrilotriacetic acid (Ni-NTA; for His-TDP-43) or Sepharose 4B (for GST-TDP-43) resin were washed with 1 ml of PBS and incubated for 1 h at 4 °C with 4 µg of dialyzed recombinant protein. The coated beads were then collected by centrifugation at 3000 rpm for 5 min in a Eppendorf minicentrifuge; and after two additional washes with PBS and 0.2% Tween, each batch was incubated for 1 h at room temperature with 2 µg of its respective recombinant protein (GST-hnRNP A2 and GST-hnRNP C2 for the His-TDP-43-coated resin and His-TDP-43 for the GST-TDP-43-coated resin). After four washes with PBS and 0.2% Tween, the beads were collected, and 60 µl of SDS loading buffer were added before loading onto a 10% polyacrylamide gel. Western blotting was performed to check for the presence of the added recombinant protein. As a control, uncoated beads were used in each experiment.

Band Shift Analysis—Commercial (ug)12 and (ug)6 RNA oligonucleotides (200 ng, ~25 pmol; MWG Biotech) were labeled by phosphorylation with [{gamma}-32P]ATP and T4 polynucleotide kinase (Stratagene) according to a standard protocol and resuspended in 400 µl of water. Each binding reaction was performed at room temperature for 15 min by mixing the purified protein with the labeled RNA in a 20-µl final volume. To obtain the (uaggg(u/a))3 RNAs, we cloned, into the SacI-XbaI restriction fragment of Bluescript KS+ sites, oligonucleotides (after annealing) 5'-ctagggatagggttagggat-3' (sense) and 5'-ctagatccctaaccctatccctagagct-3' (antisense); and to obtain the u21 RNAs, oligonucleotides 5'-cttttttttttttttttttttt-3' (sense) and 5'-ctagaaaaaaaaaaaaaaaaaaaaagagct-3' (antisense). The plasmids were linearized with XbaI, and in vitro transcription was performed as described previously (26). Binding reactions were performed in 1x bind shift binding buffer (20 mM HEPES (PH 7.9), 72 MM KCl, 1.5 mM MgCl2, 0.78 mM magnesium acetate, 0.52 mM dithiothreitol, 3.8% glycerol, 0.75 mM ATP, and 1 mM GTP) and 5 µg/µl heparin and electrophoresed on a 5% polyacrylamide gel at 100 V for 1 h in 0.5 x Tris borate/EDTA buffer at 4 °C. The amounts of each protein used in these experiments are reported in the figure legends. The gel were then dried and exposed to autoradiographic XAR film (Eastman Kodak Co.).

In Vitro Splicing Analysis and Separation of Spliceosomal Complexes— The pY7 and pY7(UG12U5) plasmids have been described previously (24). In vitro splicing reactions were performed using capped SP6-transcribed RNAs according to standard protocols. Recombinant proteins (400 ng each) were added to the depleted nuclear extract prior to assembling the remainder of the splicing reaction mixture. Splicing reactions were incubated at 30 °C for 2 h. The RNA was extracted using RNAwiz (Ambion, Inc.); ethanol-precipitated; and analyzed on a denaturing 6% polyacrylamide gel, followed by autoradiography. Spliceosomal complexes at selected time points during the in vitro splicing reactions were separated by electrophoresis on a native 3.5% acrylamide gel (2 ml of 1% bisacrylamide, 3.7 ml of 40% acrylamide, 2 ml of 1 M Tris (pH 10), 2 ml of 1 M glycine (pH 6), 28.5 ml of H2O, 0.28 ml of ammonium persulfate, and 28 µl of Temed) following addition of 1 µl of heparin (50 µg/µl).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
RNA Secondary Structure of CFTR Exon 9 and the (ug)m-repeated Motif—Before analyzing in detail the interactions of TDP-43 with cellular proteins, we decided to address the alternative explanation for the negative role played by (ug)m repeats in CFTR exon 9 splicing proposed recently by Hefferon et al. (30). In this model, the (ug)m repeats have been suggested to fold upon themselves to inhibit CFTR exon 9 recognition by blocking 3'-splice site usage. This mechanism could thus occur without the need to invoke the presence of any trans-acting factor such as TDP-43. Therefore, to address this issue experimentally, we extended a preliminary structural probing that was performed on CFTR exon 9 in a previous study (22). The probed RNA thus comprised the entire CFTR exon 9 sequence carrying a (ug)11u7 repeat and a significant portion of the intervening sequence 8 intron. Structural probing was performed according to a methodology recently described for the EDA exons of the mouse fibronectin gene (45), which integrates limited RNase digestion with mFold structural predictions (46). As shown in Fig. 1, in this extended RNA context, the enzymatic cleavages confirmed the existence of several stem-loop elements (I–IV) that had already been suggested to exist in a previous study (22) and excluded any tendency for the (ug)11 sequence to fold upon itself, even in the absence of interacting proteins. Moreover, mFold analyses performed using this extended context and after varying the number of ug repeats in no instance predicted this sequence as being capable of folding upon itself (data not shown). The discrepancy between these experimental probing data and the conclusions of Hefferon et al. (30) may be explained by the fact that their in silico predictions were performed by taking into account only the ug-repeated sequences and a very limited region surrounding this region (10 nucleotides on either side). Given these limitations, it is not surprising that mFold predictions suggested the possibility that (ug)m repeats might fold upon themselves. Indeed, the length of RNA examined represents a severe limitation in assigning significance to in silico results, which has been reported for the neurofibromin-1 gene, where the RNA window analyzed by even a few nucleotides could yield striking differences in the prediction results (47). Therefore, we decided to investigate further the splicing inhibitory properties of TDP-43.

In Vitro Effects of TDP-43 Mutants on CFTR Exon 9 Splicing and Analysis of Spliceosomal Complex Formation—In a recent study, we highlighted the importance of the TDP-43 C-terminal region in splicing inhibition (28). To confirm and extend these data, we used a pY7 plasmid containing two tropomyosin exons separated by an ~120-nucleotide-long intron sequence that was modified to contain the 3'-splice site of CFTR exon 9 (Fig. 2A, underlined). Detailed in vitro analysis of the splicing pattern of this RNA has previously shown that this construct successfully mimics the splicing inhibitory effect of TDP-43 in the CFTR exon 9 context (24). Therefore, it was of interest to analyze the effects of different TDP-43 mutants (see Fig. 8A for a schematic diagram) in the recovery of splicing inhibition following inactivation of endogenous TDP-43 in the nuclear extract by titration with a (ug)12 synthetic oligonucleotide, as described previously (24). Fig. 2B shows that only wild-type GST-TDP-43 and GST-TDP-43-(101–414) could successfully restore the splicing inhibition when added back to the splicing reaction. On the other hand, neither of the mutants lacking the C-terminal tail (GST-TDP-43({Delta}Cter) and GST-TDP-43(NR-1)) was able to achieve the same effect. This result is consistent with the experimental data obtained by a recent independent study on the inhibitory ability of the mouse homolog of TDP-43; also in this case, the C-terminal tail of mouse TDP-43 was identified as mediating CFTR exon 9 splicing inhibition (41). In addition, it was also of interest to determine at what stage TDP-43 inhibits splicing complex formation. The native gel electrophoresis results shown in Fig. 3 comparing the behavior of pY7(wt) and pY7(UG12U5) RNAs demonstrate that the ug-repeated sequence in pY7(UG12U5) blocked spliceosomal assembly at an early stage. In fact, Fig. 3 shows that the A pre-spliceosomal and B/C late spliceosomal complexes could not be formed by this RNA.

Identification of Potential TDP-43-interacting Proteins—Equal amounts of total nuclear (NE lane) and cytoplasmic (S100 lane) extracts from HeLa cells (Fig. 4, left panel) were blotted onto a Western blot membrane and incubated with recombinant GST-TDP-43 and GST alone as a control (right panel). Fig. 4 (right panel) shows that GST-TDP-43 recognized several nuclear specific protein bands (NE lane), whereas much weaker signals were detected in the cytoplasmic extract (S100 lane), and no background was evident using the GST protein alone. In particular, the strongest protein bands recognized by TDP-43 were clustered in the 32.5–47.5-kDa range.

To provide an identity for these factors, the nuclear extract was fractionated by HPLC, and the GST overlay analysis was again performed on the separate fractions. Fig. 5A shows the HPLC profile of the nuclear extract following fractionation with a C4 reverse phase column. Each fraction was collected and loaded onto two separate SDS-polyacrylamide gels; one was stained with Coomassie Blue (Fig. 5B), whereas the other was blotted and incubated with GST-TDP-43 in an overlay assay (Fig. 5C). As can be judged from the total nuclear extract (Tot lane), the different proteins in the 32.5–47.5-kDa cluster could all be separated from each other in the different fractions. By carefully aligning the Coomassie Blue-stained gel with the overlay signals, each individual band (designated a–e) could be excised from the gel and sequenced by mass spectrometry. The results of the analysis are reported in TABLE ONE. The peptides obtained for each protein band showed that all of these proteins belonged to the hnRNPs involved (among many activities) in splicing regulation (48): hnRNP A1, hnRNP A2/B1, and hnRNP C1/C2. Interestingly, the Coomassie Blue signal for these three proteins closely reflects previously published data showing similar amounts of hnRNP A1 (band a in Fig. 5B), hnRNP A2 (band b), and hnRNP C1 (band c), with a slight predominance of this latter protein (49). Finally, we also identified the human hnRNP A3 protein as a potential binder (band e), a factor involved in cytoplasmic RNA trafficking (50).



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 1.
A, enzymatic determination of the RNA secondary structure of the entire human CFTR exon 9, including 221 nucleotides of intervening sequence 8 and carrying a (ug)11u7 sequence near the 3'-splice site. In vitro transcribed RNA was enzymatically digested with S1 nuclease and T1 and V1 RNases and reverse-transcribed using an antisense primer. The reverse transcription products were separated on a 6% polyacrylamide sequencing gel. A sequencing reaction performed with the same primer was run in parallel to the cleavages to precisely determine the cleavage sites. The positions of all predicted loop regions are highlighted along the sequence to facilitate visualization. To cover the whole exon length, a short (left panel) and a long (right panel) run were performed. Squares, circles, and arrowheads indicate S1 nuclease, T1 RNase, and V1 RNase cleavage sites, respectively. Black, gray, and white symbols indicate high, medium, and low cleavage intensity, respectively. No enzyme was added to the reaction mixture in the C lane. B, schematic diagram of the CFTR exon 9 structure optimized by computer-assisted RNA modeling and based on the cleavages observed by the structural probing. The ug-repeated motif is highlighted to facilitate recognition.

 


View this table:
[in this window]
[in a new window]
  		TABLE ONE
Protein identity determination by nanoelectrospray mass spectrometry of the five different Coomassie Blue-stained bands (a–e) specifically recognized by GST-TDP-43 in the overlay assay

 
The hnRNP A1, A2, and C1/C2 Proteins Cannot Bind ug-repeated Sequences—Because of their previously described involvement in the splicing process (51), we decided to utilize a recombinant version of the hnRNP A1, A2/B1 and C1/C2 proteins and to further characterize their interaction with TDP-43. Therefore, both hnRNP A2 and C2 cDNAs were amplified from total HeLa RNA and inserted in the pGEX-3X expression plasmid. (The hnRNP A1 protein was already available as a His-tagged fusion protein.) Fig. 6A shows the three purified proteins on an SDS-polyacrylamide gel together with GST-TDP-43. Each protein was then assessed for its ability to bind different RNAs by band shift analysis. The RNA probes were chosen on the basis of high affinity binding sequences already described in the literature for these different proteins: (ug)12 for TDP-43 (26), (uaggg(u/a))3 for hnRNP A1 (52), and u21 for hnRNP C1/C2 (53, 54). The results of the band shift analysis are shown in Fig. 6B. First of all, the high binding specificity of TDP-43 for ug-repeated sequences was confirmed by the observation that this protein appeared capable of binding only this sequence. On the other hand, hnRNP A2 and hnRNP A1 could both bind the (uaggg(u/a))3 sequence, a finding consistent with recent functional data reporting the ability of hnRNP A2 to bind hnRNP A1-specific sequences to compensate for hnRNP A1 deficiency in splicing inhibition (55). Finally, hnRNP C1 confirmed its ability to bind poly(U) sequences (53). Interestingly, none of these proteins appeared capable of binding ug-repeated sequences. Taken together, these results show that these proteins were folded correctly in our expression system and that their tagged region did not interfere with their specific binding activities.



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 2.
Effects of TDP-43 mutants on CFTR exon 9 splicing inhibition. A, schematic diagram of the pY7(wt) and pY7(UG12U5) constructs in the pY7 plasmid in which the original polypyrimidine tract was substituted with the 3'-splice site of CFTR exon 9. nt., nucleotides. B, in vitro splicing pattern of each construct. The processed and unprocessed RNAs are indicated on the left. The samples were run on a denaturing 6% sequencing gel. To inactivate native TDP-43, we added unlabeled (ug)12 RNA to the splicing mixture as a competitor (NE-TDP-43). Add-back experiments were then performed using 400 ng of each mutant to determine whether splicing inhibition could be restored. Only addition of the recombinant proteins carrying the C-terminal tail (wild-type TDP-43 protein and the GST-TDP-43-(101–414) mutant) successfully rescued splicing inhibition. In contrast, no rescue could be observed for the mutants in which the C-terminal tail had been removed. The lower arrows indicate the released lariats from the pY7(wt) and pY7(UG12U5) RNAs as indicated. NE, nuclear extract.

 



View larger version (56K):
[in this window]
[in a new window]
  		FIGURE 3.
Native 3.5% acrylamide gel electrophoresis of the pY7(wt) and pY7(UG12U5) RNAs to detect the formation of the different spliceosomal complexes at different time intervals starting from the nonspecific H complex. The pY7(wt) pre-mRNA could sequentially assemble all spliceosomal complexes. In particular, ATP-dependent spliceosomal complexes A (containing U2 snRNP stably bound to the branch point), B (in which there is the joining of the U4/U6/U5 tri-snRNP particle), and C (which follows an extensive rearrangement of complex B and corresponds to the catalytically active, mature spliceosome containing U2, U5, and U6 snRNPs) could all be observed to assemble at different time points following addition of the nuclear extract. On the other hand, the pY7(UG12U5) RNA appeared to be blocked at the earliest stages of spliceosomal complex assembly.

 



View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 4.
TDP-43 specifically interacts with a set of nuclear proteins. Left panel, Coomassie Blue-stained protein profile of HeLa nuclear (NE) and cytoplasmic (S100) extracts run on a standard 10% SDS-polyacrylamide gel. The molecular masses are indicated. Right panel, results of two GST overlay assays performed on a Western blot containing the same amount of nuclear and cytoplasmic protein extracts with GST-TDP-43 and GST alone as a control. The proteins specifically recognized by GST-TDP-43 were revealed by ECL.

 



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 5.
HPLC purification of putative TDP-43 interactors. An HPLC profile of a total nuclear extract fractionation using a C4 reverse phase column is shown (A). The absorbance was monitored at 218 nm, and fractions 1–11 were collected as indicated. mAU, milli-absorbance units. Each fraction was divided in two, and the aliquots were loaded onto two 10% SDS-polyacrylamide gels. One gel was stained with Coomassie Blue, (B) whereas the other was transferred to a Western blot membrane and subjected to GST overlay using GST-TDP-43 (C). Overlay signals are indicated with a–e according to their relative mobility. By carefully aligning the GST overlay signals (C) with the Coomassie Blue-stained protein bands (B), each protein could be excised from the gel and sequenced by mass spectrometry (see TABLE ONE for results). Briefly, band a corresponds to hnRNP A1, band b to hnRNP A2, band c to hnRNP C1/C2, band d to hnRNP B1, and band e to hnRNP A3. Tot, total nuclear extract.

 
Confirmation by Band Shift and GST Pull-down Analyses of the hnRNP/TDP-43 Interactions—To confirm the observed interactions in GST overlay between TDP-43 and the hnRNPs, we performed a band shift experiment to check the ability of hnRNP A2, hnRNP A1, and hnRNP C2 to "supershift" the (ug)6·TDP-43 complex (Fig. 7A, lane 2). Fig. 7A shows that addition of each of these recombinant proteins to TDP-43 already bound to (ug)6 gave rise to the appearance of supershifted complexes (indicated by the upper arrow in lanes 3–5). As a control, we showed that comparable amounts of a related hnRNP, GST-PTB (hnRNP I), did not produce a supershifted (ug)6 RNA·protein complex (lane 6) and that these proteins alone could not bind (ug)6 RNA (lanes 7–10). Notably, these results also show that, under our binding conditions, the GST tag alone did not affect protein dimerization, a phenomenon that has been reported in some experimental systems (56). In addition, it should be noted that this experiment demonstrated that the hnRNP/TDP-43 interaction was compatible with the binding of TDP-43 to its target RNA, an important functional detail that could not have been tested in the GST overlay experiments (Figs. 4 and 5). In addition, direct interactions between these hnRNPs and the TDP-43 protein was tested by binding this protein to beads and incubating it separately with purified GST-hnRNP A2, His-hnRNP A1, and GST-hnRNP C2. Fig. 7B demonstrates that all three proteins interacted with TDP-43 in the absence of (ug)6 RNA.



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 6.
A, shown is a Coomassie Blue-stained gel of the recombinant proteins used in this study: GST-TDP-43, His-hnRNP A1, GST-hnRNP C2, and GST-hnRNP A2. The molecular masses are indicated. B, these proteins were assayed for the ability to bind their respective RNAs of reference as described in the literature: (ug)12 for TDP-43 (26), (uaggg(u/a))3 for hnRNP A1/A2 (52), and u21 for hnRNP C1/C2 (53). Binding was assayed by band shift analysis on a native 5% polyacrylamide gel at 100 V for 1 h in 0.5x Tris borate/EDTA buffer at 4 °C. For the (ug)12, (uaggg(u/a))3, and u21 RNAs, 50 ng of recombinant proteins were used in each binding assay together with 1 ng of the respective labeled RNAs in a 20-µl final volume.

 
Finally, pull-down assays using the available recombinant GST/His-hnRNP proteins were used to test whether they were capable of pulling down native TDP-43 from the HeLa nuclear extract. Each recombinant protein was bound to glutathione-Sepharose 48 beads (or Ni-NTA beads for hnRNP A1) and incubated with the HeLa nuclear extract. After prolonged washing, the proteins bound to the beads were then run on an SDS-polyacrylamide gel and analyzed by Western blotting for the presence of native TDP-43. The results show that only the beads coated with hnRNP A2 could successfully pull down native TDP-43 from the nuclear extract (Fig. 7C). Although these results do not fully reflect the results of the supershift experiments, it should be noted that the two assays differ in several respects. In fact, it should be taken into account that, in the GST overlay and supershift assays, there is no competition between the different hnRNPs. On the other hand, in the pull-down assay, all of the native hnRNPs can compete with the recombinant proteins bound to the beads for TDP-43 binding. It is therefore very probable that no signal can be detected in the pull-down assays using hnRNP A1 and hnRNP C2 because their interaction is not strong enough to compete out the presumably predominant interaction between hnRNP A2/B1 and TDP-43 in the nuclear extract. We therefore decided to focus on the interaction of TDP-43 with hnRNP A2 for further studies.

Mapping the Interaction between TDP-43 and the hnRNP A/B Proteins—This was performed by band shift assay using a selected panel of TDP-43 mutants (Fig. 8A). Each mutant contained different portions of this protein with the exception of the first RNA recognition motif (RRM), which is essential to obtain binding of TDP-43 to (ug)6 RNA. The results demonstrate that only GST-TDP-43 and GST-TDP-43-(101–414) were capable of binding recombinant hnRNP A2 (Fig. 8B, lanes 3 and 11), suggesting that the C-terminal region is essential for binding hnRNP A2. Interestingly, the C-terminal tail seemed to be involved with the binding of hnRNP A1 as well. In fact, Fig. 8C shows that this protein could also bind GST-TDP-43 (lane 1) and GST-TDP-43-(101–414) (lane 2), but that deletion of the C-terminal tail in mutant GST-TDP-43({Delta}Cter) (lane 3) completely abolished the binding of hnRNP A1. It follows that the interaction between TDP-43 and hnRNP A2 and hnRNP A1 seems to be mediated by its glycine-rich C-terminal region.

Mapping the Region of hnRNP A2 Responsible for Its Interactions with TDP-43—It was also interesting to map the regions of hnRNP A2 that are involved in the interaction with TDP-43. Fig. 9A shows two hnRNP A2 mutants lacking RRM2 (GST-hnRNP A2({Delta}RRM2)) and the C-terminal tail (GST-hnRNP A2({Delta}Cter)). These mutants were tested first for their ability to bind the (uaggg(u/a))3 sequence (Fig. 9B), and the results show that deletion of RRM2 completely abolished the binding of hnRNP A2 to this sequence, whereas deletion of the C-terminal tail did not appear to have any effect. The same mutants were then tested for binding to TDP-43 in the presence of (ug)6 RNA. As shown in Fig. 9C (lanes 3 and 4), both wild-type hnRNP A2 and the hnRNP A2 mutant lacking RRM2 could successfully supershift GST-TDP-43. However, GST-hnRNP A2({Delta}Cter) had lost the ability to bind TDP-43 (lane 5), identifying the C-terminal region as the probable binding site for TDP-43. The fact that this hnRNP A2 mutant was still capable of binding to the (uaggg(u/a))3 sequence (Fig. 9B) gave us the opportunity to use it as a negative control to test whether the hnRNP A2/TDP-43 interaction was also present when hnRNP A2 was bound to the (uaggg(u/a))3 sequence. However, Fig. 10A shows that GST-TDP-43 did not interact with hnRNP A2 when the latter was bound to its target sequence. In addition, Fig. 10B (left panel) shows that this inability to bind hnRNP A2 was also shared by other TDP-43 mutants such as GST-TDP-43-(101–414), which could still bind hnRNP A2 when bound to the (ug)6 RNA (see Fig. 8B). It should also be noted that TDP-43 mutants such as GST-TDP-43-(101–261) and GST-TDP-43({Delta}Cter) (used as controls) could not bind hnRNP A2 in this type of experiment (Fig. 10B, right panel).



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 7.
A, shown are the results from a supershift assay with the recombinant hnRNP A2, C2, and A1 proteins and GST-PTB (used as a negative control) with labeled (ug)6 RNA and GST-TDP-43 (lower left arrow). In this experiment, 50 ng of GST-TDP-43 were incubated with 50 ng of each of the recombinant proteins together with 1 ng of labeled (ug)6 RNA in a 20-µl final volume. The appearance of a supershifted hnRNP·(ug)6·TDP-43 complex is indicated by the upper left arrow and bracket. Each protein was also incubated with (ug)6 RNA alone as a control. B, direct interaction between these proteins in the absence of (ug)6 RNA was also tested by pull-down analysis using recombinant GST/His-TDP-43-coated beads and incubation with GST-hnRNP A2, His-hnRNP A1, and GST-hnRNP C2 as indicated. Following washes, bound hnRNPs were detected by Western blot analysis with their respective polyclonal antibodies. The results show that all three proteins interacted directly with TDP-43. Input, 50% of the hnRNP protein load during each experiment. C, pull-down analysis using recombinant hnRNPs was performed to determine whether they could pull down native TDP-43 from the HeLa nuclear extract (NE). His-hnRNP A1 was bound to Ni-NTA resin, and GST-hnRNP A2 and GST-hnRNP C2 were bound to glutathione-Sepharose 4B resin and incubated with the nuclear extract. Ni-NTA resin alone or GST protein was used as a parallel control (Cont.). The proteins bound to the resins were loaded onto an SDS-polyacrylamide gel and recognized with anti-TDP-43 antibody. The nuclear extract was loaded alongside each pull-down to show the mobility of native TDP-43.

 
In conclusion, Fig. 10C shows a model depicting the experimental data obtained in Figs. 8, 9, 10. TDP-43 is shown as capable of interacting with the (ug)6 RNA with the first RRM sequence and with the C-terminal tail of hnRNP A2 (left). On the other hand, when hnRNP A2 is bound to the (uaggg(u/a))3 sequence, its C-terminal tail is presumably unavailable for interaction with TDP-43, and binding cannot occur (right).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In general, splicing enhancer and silencer elements (17, 18) can bind a variety of nuclear factors and play a dominant role in the splicing process of particular exons (57). Among the best known family of proteins that recognize the majority of enhancers present in exons is the serine/arginine-rich protein family (58, 59). Binding of these proteins at enhancer elements promotes the formation of a complex network of protein/protein interactions that reinforce the exon recognition by the splicing machinery and participate in controlling the joining of exons in the correct 5' to 3' order (60). Concerning their exact mode of action, it has been recently reported that serine/arginine-rich proteins bound to the exonic splicing enhancer through their RNA-binding domain with the RS domain promote splicing by sequentially contacting both the branch point and the 5'-splice site during spliceosomal assembly (61, 62). Equally well known, but in general with the opposite effect, are several members of the hnRNP protein family (48) such as the hnRNP A/B proteins (63) and PTB (64), which bind to many silencer elements and can antagonize the effects of the serine/arginine-rich proteins (65, 66). This, together with the great variety of potential RNA-binding sequences for each factor, may help to explain their general modulatory ability (67).



View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 8.
A, left panel, schematic diagram of the GST-TDP-43 mutants used to map the protein regions of TDP-43 involved in its binding to hnRNP A2. The white and black boxes represent the RRM1 and RRM2 regions of TDP-43, respectively. Right panel, Coomassie Blue-stained gel of GST-TDP-43 and each mutant. B, wild-type GST-TDP-43 and each mutant shown in A (25 ng) were used in a band shift assay with 50 ng of recombinant hnRNP A2 and 1 ng of labeled (ug)6 oligonucleotide in a 20-µl final volume to check for the formation of supershifted complexes. The (ug)6·TDP-43 protein/mutant binary complexes are indicated by the lower arrow and bracket. The upper arrow indicates the supershifted complexes. C, similar experiment performed using His-hnRNP A1 (50 ng) incubated with 25 ng of GST-TDP-43 (lane 1), GST-TDP-43-(101–414) (lane 2), or GST-TDP-43({Delta}Cter) (lane 3). The lower arrow shows the (ug)6·TDP-43 protein/mutant binary complexes, whereas the upper arrow indicates the supershifted complex that occurred only with the wild-type TDP-43 protein.

 
Besides these two main families of splicing regulators, there is also growing evidence that the eukaryotic nucleus contains many potential positive and negative splicing factors such as the CELF protein family (68), cyclin L (69), ZNF265 (70), Y box-binding protein-1 (71, 72), Tra2 proteins (73), hnRNP L (39), hnRNP H (74), and Nova1 (75). Although somewhat less represented in the general regulation of splicing, these novel factors are by no means less significant. Among these types of factors, TIA-1 (72, 76) and TDP-43 (23, 26) are the ones that have been found to be involved in CFTR exon 9 splicing regulation with opposite effects. Indeed, their relative expression levels may be of vital importance in promoting exon inclusion/exclusion in selected tissues or developmental stages.

RNA Structure and ug-mediated Splicing Inhibition—The role of TDP-43 binding to the (ug)m repeats at the 3'-splice site of CFTR exon 9 has been recently questioned, and an alternative explanation based on RNA secondary structure has been offered (30). In this work, we have addressed this possibility by structural probing, and our results show that this explanation is not supported by experimental testing. Moreover, it should also be noted that recent data confirm the involvement of TDP-43 in the inhibition mediated by (ug)m repeats. For example, the fact that exon inhibition mediated by (ug)12u5 in an in vitro system sequence can be successfully rescued following addition of a competitor (ug)12 oligonucleotide and restored by adding back recombinant TDP-43 (24) suggests that trans-acting factors (rather than cis-acting effects) are responsible for the inhibitory action of ug-repeated sequences. In addition, a recent study investigating the degree of CFTR exon 9 exclusion in a panel of human cell lines has shown that endogenous TDP-43 concentration correlates well with the degree of splicing inhibition (29), which would not be expected if the inhibition had been mediated by a cis-acting factor. In conclusion, the experimental evidences confirm and extend our initial findings that identify TDP-43 as a negative regulator of CFTR exon 9 splicing (23).



View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 9.
A, shown is a schematic diagram of the hnRNP A2 mutants used to map the regions involved in binding to TDP-43. B, shown are a Coomassie Blue-stained gel of each mutant (left panel) and results from a band shift experiment performed with 50 ng of each hnRNP A2 mutant with a labeled (uaggg(u/a))3 oligonucleotide to determine the effect of each mutation on the RNA-binding activity (right panel). C, the same amount of each mutant was then used in a band shift assay with 50 ng of recombinant GST-TDP-43 and 1 ng of labeled (ug)6 oligonucleotide in a 20-µl final volume to determine the formation of supershifted complexes. The (ug)6·TDP-43 binary complex is indicated by the lower arrow. The upper arrow indicates the supershifted hnRNP A2·(ug)6·TDP-43 complexes.

 
TDP-43 Interactions with Other Splicing Regulators—Interestingly, many splicing factors have been found to frequently interact with each other. For example, hnRNP L has been described to interact with PTB (77), another well known splicing regulator (64), whereas Y box-binding protein-1 has been found to interact with SRp30c (78). Recently, we showed that the glycine-rich C-terminal part of TDP-43 is conserved in Drosophila (but not in Caenorhabditis) and that this correlates with the splicing inhibitory function (28). Therefore, to understand the mechanism through which TDP-43 can play this inhibitory role, we studied the possible interactors of this protein. In this work, we identified several proteins that can interact directly with TDP-43, in particular hnRNP A1 and hnRNP A2/B1. These proteins have been described to act in a variety of systems as splicing repressors (55, 66, 79–81); and in this respect, it is interesting to note that hnRNP A1 has been previously reported to inhibit CFTR exon 9 splicing (82). One of the characteristics of the family of hnRNP A/B proteins is a glycine-rich region that they use to interact with other proteins of this family. Interestingly, the observation that both the hnRNP A1 and A2 proteins interact with the C-terminal region of TDP-43 is in agreement with previous data identifying the C-terminal region of the mouse and Drosophila TDP-43 proteins as the region responsible for mediating CFTR exon 9 splicing inhibition (28, 41).



View larger version (45K):
[in this window]
[in a new window]
  		FIGURE 10.
A, a supershift assay was performed using increasing concentrations of GST-TDP-43 (50, 100, and 200 ng) in the presence of 50 ng of GST-hnRNP A2 (left panel) or GST-hnRNP A2({Delta}Cter) (right panel) with 1 ng of labeled (uaggg(u/a))3 oligonucleotide in a 20-µl final volume. The arrow indicates the formation of hnRNP·(uaggg(u/a))3 complexes. Even in the presence of increasing GST-TDP-43 concentrations, no supershifted complex was detected with either GST-hnRNP A2 or GST-hnRNP A2({Delta}Cter). B, no supershift could be detected with increasing concentrations of GST-TDP-43-(101–414) (50, 100, and 200 ng) (left panel) or, as an additional control, with 100 and 200 ng of GST-TDP-43-(101–261) or GST-TDP-43({Delta}Cter) (right panel), two proteins that cannot normally bind hnRNP A2 when bound to (ug)6 RNA. C, shown is a schematic diagram summarizing the results from this mapping analysis. Left, when TDP-43 is bound to the (ug)6 repeat by RRM1, complex formation with hnRNP A2 can be achieved through the interaction between the C-terminal tails of hnRNP A2 and TDP-43. Right, when hnRNP A2 is bound to the (uaggg(u/a))3 sequence, its C-terminal tail is unavailable for recognition of TDP-43, and no supershifted complex can be detected.

 
Finally, from a protein/protein interaction point of view, probably one of the most interesting results of this work is the observation that TDP-43 could bind hnRNP A2 while interacting with (ug)m-repeated sequences, but TDP-43 could not bind hnRNP A2 when the latter was bound to its high affinity RNA target sequence (Fig. 10). This observation suggests that the functionality of this interaction is tightly regulated according to which of the two proteins happens to bind to the RNA. At the moment, it is difficult to speculate on the potential consequences of this behavior, as the real biological function of TDP-43 is still unknown. However, the fact that this interaction seems to be tightly regulated suggests that TDP-43 may play a role in multiple biological processes, which is quite consistent with its proposed involvement in bridging the various nuclear bodies (43); in the response following Rous sarcoma virus infection (44); and as a component of one of the two multiprotein complexes that contain human Drosha, an RNase III enzyme that mediates the genesis of micro RNAs (83).

Mechanism of TDP-43-mediated Splicing Inhibition—It is worth noting, however, that the exact role played in TDP-43 splicing-repressive action by each of the hnRNPs identified in this study remains to be evaluated. Recent reports have shown that hnRNP factors may act through the functional disruption of interactions by essential splicing factors near the exon splice sites, such as U2/U1 small nuclear ribonucleoprotein (snRNP) (84, 85) and U2AF65 (86). The observation that pY7(UG12U5) cannot proceed to A complex formation (the first ATP-dependent spliceosomal complex in which U2 snRNP binds stably to the branch point) suggests that TDP-43 blocks the binding of this factor. Depletion experiments that we performed using antisera against these single hnRNPs yielded scant results, probably because of the functional redundancy of these proteins that compensate for each other, as has been observed to occur in other systems (55). Indeed, our attempts to deplete all hnRNP A1/A2/B1 and C1/C2 proteins from the splicing mixture invariably resulted in inactivation of the splicing pathway (data not shown), probably because of aspecific depletion of essential splicing factors. Work is currently in progress to define the contribution of each interactor to the splicing repression mediated by TDP-43. In conclusion, TDP-43 potentially represents a multifunctional nuclear protein acting also in splicing regulation, and further studies will be aimed at elucidating its biological role in the different nuclear processes.


   FOOTNOTES
 
* This work was supported by Grant GGP02453 from the Telethon Onlus Foundation (Italy) and Grant RBNE01W9PM from Fondo per Gli Investimenti della Recerca di Base. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed: International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy. Tel.: 39-040-375-7337; Fax: 39-040-375-7361; E-mail: baralle{at}icgeb.org.

3 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; hnRNP, heterogeneous nuclear ribonucleoprotein; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; Ni-NTA, nickel-nitrilotriacetic acid; Temed, N,N,N',N'-tetramethylethylenediamine; wt, wild-type; PTB, polypyrimidine tract-binding protein; RRM, RNA recognition motif; snRNP, small nuclear ribonucleoprotein. Back


   ACKNOWLEDGMENTS
 
We thank Alexander Ochem and Raffaella Klima for the His-hnRNP A1 expression plasmid and the antibodies against this protein.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Krainer, A. R. (1997) in Frontiers in Molecular Biology: Eukaryotic mRNA processing (Hames, B. D., and Glover, D. M., eds) Oxford University Press, New York
  2. Adams, M. D., Rudner, D. Z., and Rio, D. C. (1996) Curr. Opin. Cell Biol. 8, 331–339[CrossRef][Medline] [Order article via Infotrieve]
  3. Burge, C. B., Tuschl, T., and Sharp, P. A. (1999) in The RNA World (Gesteland, R. R., Cech, T. R., and Atkins, J. F., eds) 2nd Ed., pp. 525–560, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  4. Modrek, B., and Lee, C. J. (2003) Nat. Genet. 34, 177–180[CrossRef][Medline] [Order article via Infotrieve]
  5. Lareau, L. F., Green, R. E., Bhatnagar, R. S., and Brenner, S. E. (2004) Curr. Opin. Struct. Biol. 14, 273–282[CrossRef][Medline] [Order article via Infotrieve]
  6. Boue, S., Letunic, I., and Bork, P. (2003) BioEssays 25, 1031–1034[CrossRef][Medline] [Order article via Infotrieve]
  7. Pagani, F., and Baralle, F. E. (2004) Nat. Rev. Genet 5, 389–396[CrossRef][Medline] [Order article via Infotrieve]
  8. Faustino, N. A., and Cooper, T. A. (2003) Genes Dev. 17, 419–437[Free Full Text]
  9. Caceres, J. F., and Kornblihtt, A. R. (2002) Trends Genet 18, 186–193[CrossRef][Medline] [Order article via Infotrieve]
  10. Cartegni, L., Chew, S. L., and Krainer, A. R. (2002) Nat. Rev. Genet 3, 285–298[CrossRef][Medline] [Order article via Infotrieve]
  11. Nissim-Rafinia, M., and Kerem, B. (2002) Trends Genet 18, 123–127[CrossRef][Medline] [Order article via Infotrieve]
  12. Garcia-Blanco, M. A., Baraniak, A. P., and Lasda, E. L. (2004) Nat. Biotechnol. 22, 535–546[CrossRef][Medline] [Order article via Infotrieve]
  13. Nilsen, T. W. (2003) BioEssays 25, 1147–1149[CrossRef][Medline] [Order article via Infotrieve]
  14. Zhou, Z., Licklider, L. J., Gygi, S. P., and Reed, R. (2002) Nature 419, 182–185[CrossRef][Medline] [Order article via Infotrieve]
  15. Buratti, E., and Baralle, F. E. (2004) Mol. Cell. Biol. 24, 10505–10514[Free Full Text]
  16. Kornblihtt, A. R., De La Mata, M., Fededa, J. P., Munoz, M. J., and Nogues, G. (2004) RNA (N. Y.) 10, 1489–1498
  17. Smith, C. W., and Valcarcel, J. (2000) Trends Biochem. Sci. 25, 381–388[CrossRef][Medline] [Order article via Infotrieve]
  18. Blencowe, B. J. (2000) Trends Biochem. Sci. 25, 106–110[CrossRef][Medline] [Order article via Infotrieve]
  19. Stamm, S. (2002) Hum. Mol. Genet. 11, 2409–2416[Abstract/Free Full Text]
  20. Shin, C., and Manley, J. L. (2004) Nat. Rev. Mol. Cell. Biol. 5, 727–738[CrossRef][Medline] [Order article via Infotrieve]
  21. Kreahling, J., and Graveley, B. R. (2004) Trends Genet. 20, 1–4[CrossRef][Medline] [Order article via Infotrieve]
  22. Niksic, M., Romano, M., Buratti, E., Pagani, F., and Baralle, F. E. (1999) Hum. Mol. Genet. 8, 2339–2349[Abstract/Free Full Text]
  23. Buratti, E., Dork, T., Zuccato, E., Pagani, F., Romano, M., and Baralle, F. E. (2001) EMBO J. 20, 1774–1784[CrossRef][Medline] [Order article via Infotrieve]
  24. Buratti, E., Brindisi, A., Pagani, F., and Baralle, F. E. (2004) Am. J. Hum. Genet. 74, 1322–1325[CrossRef][Medline] [Order article via Infotrieve]
  25. Groman, J. D., Hefferon, T. W., Casals, T., Bassas, L., Estivill, X., Des Georges, M., Guittard, C., Koudova, M., Fallin, M. D., Nemeth, K., Fekete, G., Kadasi, L., Friedman, K., Schwarz, M., Bombieri, C., Pignatti, P. F., Kanavakis, E., Tzetis, M., Schwartz, M., Novelli, G., D'Apice, M. R., Sobczynska-Tomaszewska, A., Bal, J., Stuhrmann, M., Macek, M., Jr., Claustres, M., and Cutting, G. R. (2004) Am. J. Hum. Genet. 74, 176–179[CrossRef][Medline] [Order article via Infotrieve]
  26. Buratti, E., and Baralle, F. E. (2001) J. Biol. Chem. 276, 36337–36343[Abstract/Free Full Text]
  27. Ou, S. H., Wu, F., Harrich, D., Garcia-Martinez, L. F., and Gaynor, R. B. (1995) J. Virol. 69, 3584–3596[Abstract]
  28. Ayala, Y. M., Pantano, S., D'Ambrogio, A., Buratti, E., Brindisi, A., Marchetti, C., Romano, M., and Baralle, F. E. (2005) J. Mol. Biol. 348, 575–588[CrossRef][Medline] [Order article via Infotrieve]
  29. Disset, A., Michot, C., Harris, A., Buratti, E., Claustres, M., and Tuffery-Giraud, S. (2005) Hum. Mutat. 25, 72–81[CrossRef][Medline] [Order article via Infotrieve]
  30. Hefferon, T. W., Groman, J. D., Yurk, C. E., and Cutting, G. R. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 3504–3509[Abstract/Free Full Text]
  31. Arrisi-Mercado, P., Romano, M., Muro, A. F., and Baralle, F. E. (2004) J. Biol. Chem. 279, 39331–39339[Abstract/Free Full Text]
  32. Gabellini, N. (2001) Eur. J. Biochem. 268, 1076–1083[Medline] [Order article via Infotrieve]
  33. Yeo, G., Hoon, S., Venkatesh, B., and Burge, C. B. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 15700–15705[Abstract/Free Full Text]
  34. Zhao, B., Schneid, C., Iliev, D., Schmidt, E. M., Wagner, V., Wollnik, F., and Mittag, M. (2004) Eukaryot. Cell 3, 815–825[Abstract/Free Full Text]
  35. Anyanful, A., Ono, K., Johnsen, R. C., Ly, H., Jensen, V., Baillie, D. L., and Ono, S. (2004) J. Cell Biol. 167, 639–647[Abstract/Free Full Text]
  36. Takahashi, N., Sasagawa, N., Suzuki, K., and Ishiura, S. (2000) Biochem. Biophys. Res. Commun. 277, 518–523[CrossRef][Medline] [Order article via Infotrieve]
  37. Faustino, N. A., and Cooper, T. A. (2005) Mol. Cell. Biol. 25, 879–887[Abstract/Free Full Text]
  38. Perez Canadillas, J. M., and Varani, G. (2003) EMBO J. 22, 2821–2830[CrossRef][Medline] [Order article via Infotrieve]
  39. Hui, J., Stangl, K., Lane, W. S., and Bindereif, A. (2003) Nat. Struct. Biol. 10, 33–37[CrossRef][Medline] [Order article via Infotrieve]
  40. Bilbao, D., and Valcarcel, J. (2003) Nat. Struct. Biol. 10, 6–7[CrossRef][Medline] [Order article via Infotrieve]
  41. Wang, H. Y., Wang, I. F., Bose, J., and Shen, C. K. (2004) Genomics 83, 130–139[CrossRef][Medline] [Order article via Infotrieve]
  42. Georgi, A. B., Stukenberg, P. T., and Kirschner, M. W. (2002) Curr. Biol. 12, 105–114[CrossRef][Medline] [Order article via Infotrieve]
  43. Wang, I. F., Reddy, N. M., and Shen, C. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 13583–13588[Abstract/Free Full Text]
  44. Brasier, A. R., Spratt, H., Wu, Z., Boldogh, I., Zhang, Y., Garofalo, R. P., Casola, A., Pashmi, J., Haag, A., Luxon, B., and Kurosky, A. (2004) J. Virol. 78, 11461–11476[Abstract/Free Full Text]
  45. Buratti, E., Muro, A. F., Giombi, M., Gherbassi, D., Iaconcig, A., and Baralle, F. E. (2004) Mol. Cell. Biol. 24, 1387–1400[Abstract/Free Full Text]
  46. Zuker, M. (2003) Nucleic Acids Res. 31, 3406–3415[Abstract/Free Full Text]
  47. Vandenbroucke, I., Callens, T., De Paepe, A., and Messiaen, L. (2002) BMC Genomics http://www.biomedcentral.com/1471–2164/3/13
  48. Krecic, A. M., and Swanson, M. S. (1999) Curr. Opin. Cell Biol. 11, 363–371[CrossRef][Medline] [Order article via Infotrieve]
  49. Huang, M., Rech, J. E., Northington, S. J., Flicker, P. F., Mayeda, A., Krainer, A. R., and LeStourgeon, W. M. (1994) Mol. Cell. Biol. 14, 518–533[Abstract/Free Full Text]
  50. Ma, A. S., Moran-Jones, K., Shan, J., Munro, T. P., Snee, M. J., Hoek, K. S., and Smith, R. (2002) J. Biol. Chem. 277, 18010–18020[Abstract/Free Full Text]
  51. Chabot, B., LeBel, C., Hutchison, S., Nasim, F. H., and Simard, M. J. (2003) in Progress in Molecular and Subcellular Biology: Regulation of Alternative Splicing (Jeanteur, P., ed) Vol. 31, pp. 59–82, Springer-Verlag New York Inc., New York
  52. Burd, C. G., and Dreyfuss, G. (1994) EMBO J. 13, 1197–1204[Medline] [Order article via Infotrieve]
  53. Gorlach, M., Burd, C. G., and Dreyfuss, G. (1994) J. Biol. Chem. 269, 23074–23078[Abstract/Free Full Text]
  54. Swanson, M. S., and Dreyfuss, G. (1988) Mol. Cell. Biol. 8, 2237–2241[Abstract/Free Full Text]
  55. Hutchison, S., LeBel, C., Blanchette, M., and Chabot, B. (2002) J. Biol. Chem. 277, 29745–29752[Abstract/Free Full Text]
  56. Niedziela-Majka, A., Rymarczyk, G., Kochman, M., and Ozyhar, A. (1998) Protein Expression Purif. 14, 208–220[CrossRef][Medline] [Order article via Infotrieve]
  57. Zheng, Z. M. (2004) J. Biomed. Sci. 11, 278–294[Medline] [Order article via Infotrieve]
  58. Graveley, B. R. (2000) RNA (N. Y.) 6, 1197–1211[CrossRef]
  59. Bourgeois, C. F., Lejeune, F., and Stevenin, J. (2004) Prog. Nucleic Acid Res. Mol. Biol. 78, 37–88[Medline] [Order article via Infotrieve]
  60. Ibrahim, E. C., Schaal, T. D., Hertel, K. J., Reed, R., and Maniatis, T. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 5002–5007[Abstract/Free Full Text]
  61. Shen, H., and Green, M. R. (2004) Mol. Cell 16, 363–373[CrossRef][Medline] [Order article via Infotrieve]
  62. Shen, H., Kan, J. L., and Green, M. R. (2004) Mol. Cell 13, 367–376[CrossRef][Medline] [Order article via Infotrieve]
  63. Mayeda, A., Munroe, S. H., Caceres, J. F., and Krainer, A. R. (1994) EMBO J. 13, 5483–5495[Medline] [Order article via Infotrieve]
  64. Wagner, E. J., and Garcia-Blanco, M. A. (2001) Mol. Cell. Biol. 21, 3281–3288[Free Full Text]
  65. Caceres, J. F., Stamm, S., Helfman, D. M., and Krainer, A. R. (1994) Science 265, 1706–1709[Abstract/Free Full Text]
  66. Zhu, J., Mayeda, A., and Krainer, A. R. (2001) Mol. Cell 8, 1351–1361[CrossRef][Medline] [Order article via Infotrieve]
  67. Reed, R. (2000) Curr. Opin. Cell Biol. 12, 340–345[CrossRef][Medline] [Order article via Infotrieve]
  68. Ladd, A. N., Charlet, N., and Cooper, T. A. (2001) Mol. Cell. Biol. 21, 1285–1296[Abstract/Free Full Text]
  69. Dickinson, L. A., Edgar, A. J., Ehley, J., and Gottesfeld, J. M. (2002) J. Biol. Chem. 277, 25465–25473[Abstract/Free Full Text]
  70. Adams, D. J., van der Weyden, L., Mayeda, A., Stamm, S., Morris, B. J., and Rasko, J. E. (2001) J. Cell Biol. 154, 25–32[Abstract/Free Full Text]
  71. Stickeler, E., Fraser, S. D., Honig, A., Chen, A. L., Berget, S. M., and Cooper, T. A. (2001) EMBO J. 20, 3821–3830[CrossRef][Medline] [Order article via Infotrieve]
  72. Zuccato, E., Buratti, E., Stuani, C., Baralle, F. E., and Pagani, F. (2004) J. Biol. Chem. 279, 16980–16988[Abstract/Free Full Text]
  73. Tacke, R., Tohyama, M., Ogawa, S., and Manley, J. L. (1998) Cell 93, 139–148[CrossRef][Medline] [Order article via Infotrieve]
  74. Buratti, E., Baralle, M., De Conti, L., Baralle, D., Romano, M., Ayala, Y. M., and Baralle, F. E. (2004) Nucleic Acids Res. 32, 4224–4236[Abstract/Free Full Text]
  75. Jensen, K. B., Dredge, B. K., Stefani, G., Zhong, R., Buckanovich, R. J., Okano, H. J., Yang, Y. Y., and Darnell, R. B. (2000) Neuron 25, 359–371[CrossRef][Medline] [Order article via Infotrieve]
  76. Forch, P., Puig, O., Kedersha, N., Martinez, C., Granneman, S., Seraphin, B., Anderson, P., and Valcarcel, J. (2000) Mol. Cell 6, 1089–1098[CrossRef][Medline] [Order article via Infotrieve]
  77. Hahm, B., Cho, O. H., Kim, J. E., Kim, Y. K., Kim, J. H., Oh, Y. L., and Jang, S. K. (1998) FEBS Lett. 425, 401–406[CrossRef][Medline] [Order article via Infotrieve]
  78. Raffetseder, U., Frye, B., Rauen, T., Jurchott, K., Royer, H. D., Jansen, P. L., and Mertens, P. R. (2003) J. Biol. Chem. 278, 18241–18248[Abstract/Free Full Text]
  79. Caputi, M., Mayeda, A., Krainer, A. R., and Zahler, A. M. (1999) EMBO J. 18, 4060–4067[CrossRef][Medline] [Order article via Infotrieve]
  80. Nasim, F. U., Hutchison, S., Cordeau, M., and Chabot, B. (2002) RNA (N. Y.) 8, 1078–1089
  81. Del Gatto-Konczak, F., Olive, M., Gesnel, M. C., and Breathnach, R. (1999) Mol. Cell. Biol. 19, 251–260[Abstract/Free Full Text]
  82. Nissim-Rafinia, M., Chiba-Falek, O., Sharon, G., Boss, A., and Kerem, B. (2000) Hum. Mol. Genet. 9, 1771–1778[Abstract/Free Full Text]
  83. Gregory, R. I., Yan, K. P., Amuthan, G., Chendrimada, T., Doratotaj, B., Cooch, N., and Shiekhattar, R. (2004) Nature 432, 235–240[CrossRef][Medline] [Order article via Infotrieve]
  84. Tange, T. O., Damgaard, C. K., Guth, S., Valcarcel, J., and Kjems, J. (2001) EMBO J. 20, 5748–5758[CrossRef][Medline] [Order article via Infotrieve]
  85. Eperon, I. C., Makarova, O. V., Mayeda, A., Munroe, S. H., Caceres, J. F., Hayward, D. G., and Krainer, A. R. (2000) Mol. Cell. Biol. 20, 8303–8318[Abstract/Free Full Text]
  86. Domsic, J. K., Wang, Y., Mayeda, A., Krainer, A. R., and Stoltzfus, C. M. (2003) Mol. Cell. Biol. 23, 8762–8772[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
A. D'Ambrogio, E. Buratti, C. Stuani, C. Guarnaccia, M. Romano, Y. M. Ayala, and F. E. Baralle
Functional mapping of the interaction between TDP-43 and hnRNP A2 in vivo
Nucleic Acids Res., May 8, 2009; (2009) gkp342v1.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y.-J. Zhang, Y.-F. Xu, C. Cook, T. F. Gendron, P. Roettges, C. D. Link, W.-L. Lin, J. Tong, M. Castanedes-Casey, P. Ash, et al.
Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity
PNAS, May 5, 2009; 106(18): 7607 - 7612.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
P.-H. Kuo, L. G. Doudeva, Y.-T. Wang, C.-K. J. Shen, and H. S. Yuan
Structural insights into TDP-43 in nucleic-acid binding and domain interactions
Nucleic Acids Res., April 1, 2009; 37(6): 1799 - 1808.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. M. Igaz, L. K. Kwong, A. Chen-Plotkin, M. J. Winton, T. L. Unger, Y. Xu, M. Neumann, J. Q. Trojanowski, and V. M.-Y. Lee
Expression of TDP-43 C-terminal Fragments in Vitro Recapitulates Pathological Features of TDP-43 Proteinopathies
J. Biol. Chem., March 27, 2009; 284(13): 8516 - 8524.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
H Daoud, P N Valdmanis, E Kabashi, P Dion, N Dupre, W Camu, V Meininger, and G A Rouleau
Contribution of TARDBP mutations to sporadic amyotrophic lateral sclerosis
J. Med. Genet., February 1, 2009; 46(2): 112 - 114.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
Y. M. Ayala, P. Zago, A. D'Ambrogio, Y.-F. Xu, L. Petrucelli, E. Buratti, and F. E. Baralle
Structural determinants of the cellular localization and shuttling of TDP-43
J. Cell Sci., November 15, 2008; 121(22): 3778 - 3785.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. K. Bose, I.-F. Wang, L. Hung, W.-Y. Tarn, and C.-K. J. Shen
TDP-43 Overexpression Enhances Exon 7 Inclusion during the Survival of Motor Neuron Pre-mRNA Splicing
J. Biol. Chem., October 24, 2008; 283(43): 28852 - 28859.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
A. S. Chen-Plotkin, F. Geser, J. B. Plotkin, C. M. Clark, L. K. Kwong, W. Yuan, M. Grossman, V. M. Van Deerlin, J. Q. Trojanowski, and V. M.-Y. Lee
Variations in the progranulin gene affect global gene expression in frontotemporal lobar degeneration
Hum. Mol. Genet., May 15, 2008; 17(10): 1349 - 1362.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
J. Sreedharan, I. P. Blair, V. B. Tripathi, X. Hu, C. Vance, B. Rogelj, S. Ackerley, J. C. Durnall, K. L. Williams, E. Buratti, et al.
TDP-43 Mutations in Familial and Sporadic Amyotrophic Lateral Sclerosis
Science, March 21, 2008; 319(5870): 1668 - 1672.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
Y.-J. Zhang, Y.-f. Xu, C. A. Dickey, E. Buratti, F. Baralle, R. Bailey, S. Pickering-Brown, D. Dickson, and L. Petrucelli
Progranulin Mediates Caspase-Dependent Cleavage of TAR DNA Binding Protein-43
J. Neurosci., September 26, 2007; 27(39): 10530 - 10534.
[Abstract] [Full Text] [PDF]

Home page
 AM J ALZHEIMERS DIS OTHER DEMENHome page
D. Irwin, C. F. Lippa, and J.M. Swearer
Cognition and Amyotrophic Lateral Sclerosis (ALS)
American Journal of Alzheimer's Disease and Other Dementias, September 1, 2007; 22(4): 300 - 312.
[Abstract] [PDF]

Home page
 Nucleic Acids ResHome page
E. Buratti, C. Stuani, G. De Prato, and F. E. Baralle
SR protein-mediated inhibition of CFTR exon 9 inclusion: molecular characterization of the intronic splicing silencer
Nucleic Acids Res., July 26, 2007; 35(13): 4359 - 4368.
[Abstract] [Full Text] [PDF]

Home page
 BrainHome page
M. Hasegawa, T. Arai, H. Akiyama, T. Nonaka, H. Mori, T. Hashimoto, M. Yamazaki, and K. Oyanagi
TDP-43 is deposited in the Guam parkinsonism-dementia complex brains
Brain, May 1, 2007; 130(5): 1386 - 1394.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
E. Buratti, M. Baralle, and F. E. Baralle
Defective splicing, disease and therapy: searching for master checkpoints in exon definition
Nucleic Acids Res., July 19, 2006; 34(12): 3494 - 3510.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
280/45/37572    most recent
M505557200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Buratti, E.
Right arrow Articles by Baralle, F. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Buratti, E.
Right arrow Articles by Baralle, F. E.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2005 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement