Advertisement
JBC

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


     

Originally published In Press as doi:10.1074/jbc.M800017200 on May 7, 2008

J. Biol. Chem., Vol. 283, Issue 27, 19077-19084, July 4, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/27/19077    most recent
M800017200v1
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 Izquierdo, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Izquierdo, J. M.
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?

Hu Antigen R (HuR) Functions as an Alternative Pre-mRNA Splicing Regulator of Fas Apoptosis-promoting Receptor on Exon Definition*

José M. Izquierdo1

From the Departamento de Biología Molecular and the Centro de Biología Molecular "Severo Ochoa," C.S.I.C., Universidad Autónoma de Madrid, Cantoblanco 28049, Madrid, Spain

Received for publication, January 2, 2008 , and in revised form, March 27, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exclusion of exon 6 by alternative RNA splicing of the primary transcript of the apoptosis receptor Fas produces a soluble isoform that prevents programmed cell death. I report that antiapoptotic regulator Hu antigen R (HuR, ELAVL1), a member of the embryonic lethal, abnormal vision, Drosophila-like (ELAVL) family, promotes Fas exon 6 skipping by binding to an exonic splicing silencer. HuR inhibits the association of U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 kDa (U2AF65) with the upstream 3' splice site, without decreasing recognition of the downstream 5' splice site by U1 snRNP but by antagonizing the role of TIA-1 (T-cell intracellular antigen 1)/TIAR (TIA-1 related protein) on exon definition. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is partially required for efficient U2AF65 inhibition. Further, the silencing capacity of HuR as splicing regulator resides in the RRM1 and hinge-RRM3 domains. Taken together, these results support a functional link between HuR as repressor of alternative Fas splicing and the molecular mechanisms modulating programmed cell death.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The molecular basis of the regulation of alternative splicing is linked to splice site selection. Thus, authentic splicing sequence elements are often indistinguishable from cryptic sequences hidden in human pre-mRNAs, raising the question of how the spliceosomal machinery can define an authentic splicing motif. Splice site selection depends on balancing of positive and negative sequence elements and on changes in the activity and/or amount of cellular splicing factors under physiological or pathological conditions (1). Although alternative splicing is widespread in human biology (2), few factors involved in regulation of alternative splicing of humans have been identified, and even fewer have been characterized (3).

T-cell intracellular antigen 1 (TIA-1)2 and TIA-1 related protein (TIAR) are both involved in controlling splice site choice. They were first identified as apoptosis-promoting factors (4). TIA-1 and TIAR contain three RNA recognition motifs (RRMs) and a glutamine-rich C-terminal domain (5). Both proteins regulate the alternative splicing of various human and Drosophila pre-mRNAs (FGFR-2 (fibroblast growth factor receptor-2), msl-2, TIAR, CTFR (cystic fibrosis transmembrane conductance regulator), Fas, and COL2A1 (type II procollagen gene) (6-11)) through binding to U-rich stretches, facilitating atypical 5' splice site recognition by U1 snRNP. TIAR can also enhance U6 snRNP assembly on a pseudo-5' splice site followed by uridines in the calcitonin/calcitonin gene-related peptide pre-mRNA (12).

The RNA-binding proteins HuA (HuR, ELAVL1), HuB (Hel-N1, ELAVL2), HuC (ELAVL3), and HuD (ELAVL4) comprise the embryonic lethal, abnormal vision, Drosophila-like (ELAVL) family (13). Expression of HuB, HuC, and HuD proteins is limited to neuronal tissues/cells, whereas HuR is ubiquitously expressed (14). HuB, HuC, and HuD proteins have been recently identified as splicing regulators in neuron-like cells (15). The primary structures of the Hu proteins are highly conserved (70%) and contain three RNA recognition motifs (RRMs). The region between RRMs 2 and 3 defines a basic hinge domain containing a nuclear localization signal, which allows Hu proteins to shuttle between the cytoplasm and the nucleus (16). HuR has been shown to modulate a variety of post-transcriptional decisions (17, 18) in several key cellular processes, including immune response (19), exposure to stressing agents (20, 21), cell survival (22, 23), inflammation (24), cell cycle control (25), differentiation program (26), oncogenesis (27), and replicative senescence (28).

Human gene functions can change by producing multiple functional isoforms, even antagonistic, from a single gene in different tissues, developmental stages, or even in pathological situations (1). One example is the human Fas (CD95/APO-1) pre-mRNA, which encodes a type 1 transmembrane protein that mediates apoptosis on Fas ligand binding (29). Fas is a death receptor that is constitutively expressed by most tissues (29). Alternative splicing of Fas pre-mRNA can regulate the sensitivity of Fas-expressing cells to Fas-induced apoptosis. The protein isoform of the mRNA lacking exon 6 encodes the soluble form of the receptor able to inhibit Fas signaling (30). This process is regulated in a number of physiological situations, including activation-mediated T-cell death, and failure to switch between isoforms underlies autoimmune lymphoproliferative disorders (31).


Figure 1
View larger version (58K):
[in this window]
[in a new window]

  		FIGURE 1.
Nuclear colocalization of HuR and TIA-1/TIAR in HeLa cells. A, from left to right, images show HeLa cells stained with anti-HuR, To-pro-3, anti-TIA-1, or anti-TIAR antibodies and visualized by confocal microscopy. Images on the right (Merged) are the sum of all images. B, HeLa cells were transfected with plasmid expressing either GFP-HuR or GFP-TIA-1b fusion proteins. From left to right, images show fluorescence from HeLa cells transfected with GFP-tagged plasmids or stained with To-pro-3. Images on the right are the sum of GFP and To-pro-3 images (Merged). C, fractions containing total protein (T), nuclei (N), or post-nuclear supernatant (C) from HeLa cells were analyzed with the antibodies indicated. The identities of tested proteins are indicated by closed arrowheads on the right, and molecular mass markers are shown on the left.

 
Despite extensive characterization and the predominant nuclear localization of HuR, the idea that this protein functions as splicing regulator remains unclear because no target sequences of HuR have been identified. I show here that HuR is a novel regulator of alternative Fas splicing.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs and Recombinant Protein Preparations—All the constructs used have been previously described (10, 32, 33). Recombinant MBP, MBP-HuR, His-PTB, GST-TIA-1b, or GST-TIARb fusion proteins were expressed in Escherichia coli and purified as described (10, 33).

Cell Cultures, DNA Transfection, and RNA Interference Analysis—HeLa cells were grown and transfected with 20 nM (final concentration) of either a control siRNA (non-silencing siRNA duplex fluorescein labeled 27-6411-02FL from Gene Link) or an siRNA directed to the 3'-untranslated region of HuR (TCTTAAGTTTCGTAAGTTA) as described (10, 32-34). For double transfection, siRNAs were transfected first followed by transfection of plasmid DNA 72 h later. The cells were collected after 24 h.

Protein Analysis, RNA Isolation, and RT-PCR Analysis—Immunoblots were carried out using the following antibodies: anti-U2AF65 (MC3, kindly provided by Dr. J. Valcárcel), anti-TIA-1 (C-20), anti-TIAR (C-18), anti-hnRNPC1/C2, and anti-HuR (3A2) from Santa Cruz Biotechnology; anti-{alpha}-tubulin (B-5-1-2, Sigma); and anti-GFP (JL-8, Clontech). Cytoplasmic RNAs were purified and analyzed as described previously (7, 10).

Fluorescence Microscopy—HeLa cells were grown for 24 h on coverslips and processed as described previously (34). Endogenous HuR/TIA-1/TIAR antibody complexes, To-pro-3, or GFP-fused proteins were viewed by confocal microscopy.

In Vitro Splicing, RNA-RNA, and RNA-Protein Interaction Assays—In vitro transcription and splicing assays were carried out as described previously (7) using capped RNAs synthesized by T7 transcription from PCR templates. UV cross-linking and immunoprecipitation, psoralen-mediated cross-linking, and RNase H-mediated U1 inactivation were performed as described (10).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HuR siRNA-mediated Depletion Promotes Fas Exon 6 Inclusion—HuR is an RNA-binding protein mainly localized in the nucleus of HeLa cells (>90%), with nucleolar exclusion, whereas TIA-1 and TIAR showed a characteristic nuclear-cytoplasmic localization pattern (Fig. 1, A-C). However, both endogenous and ectopic HuR and TIA-1 proteins showed similar nuclear colocalization (Fig. 1, A-C), suggesting that they might have related functions in nuclear events such as, for example, the control of alternative pre-mRNA splicing. To test this idea, I reduced the abundance of endogenous HuR in HeLa cells (80-90%) by RNA interference, resulting in increased levels of Fas exon 6 inclusion (Fig. 2, A and B, compare lanes 2-4 with lanes 1-3, respectively). In these experiments, to detect reliable differences in exon 6 inclusion, I have used a Fas minigene harboring a mutation at the polypyrimidine tract of intron 5 (Fas U-20C) that increases exon 6 skipping (10). To exclude off-target siRNA effects, I used an siRNA-resistant variant of HuR, refractory to the siRNA directed to the 3'-untranslated region of HuR, in line with previous data (33). The results indicate that the resistant protein can provide the full range of their activity even under silencing conditions (Fig. 2, A and B, compare lanes 2, 4, and 5 with lanes 1, 3, and 4, respectively), thus strongly arguing that HuR does promote exon 6 skipping. These results suggest that HuR might function as a Fas exon 6 splicing repressor.


Figure 2
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 2.
HuR represses Fas exon 6 inclusion through the URE6 exonic silencer. A and B, effect of control or HuR siRNA-mediated depletion in HeLa cells on alternative splicing of U-20C Fas minigene. A, protein extracts (5 µg (lane 1) or 25 µg (lanes 2-5)) from post-transfected HeLa cells were analyzed by Western blot using antibodies indicated. HuR* indicates expression of a HuR-resistant to the siRNA used in the assay. B, cytoplasmic RNAs (1.5 µg) from post-transfected HeLa cells in A were analyzed by RT-PCR. The intensities of the different bands were quantified by densitometry. The values of the ratios between 5-6-7 and 5-7 amplification products are means ± S.E. for at least 2-3 independent experiments. Substantial levels of exon 6 inclusion were achieved by transfecting the U-20C Fas mutant minigene in HuR siRNA-transfected cells. In contrast, siRNA-resistant HuR provides significant exon 6 skipping activity under conditions of HuR silencing. C and D, the effects of HuR or TIAR overexpression on alternative splicing of Fas reporter minigenes in HeLa cells. C, Western blot analysis of the expression levels of GFP (lanes 1-3 and 7), GFP-HuR (lanes 4-6) and GFP-TIAR (lanes 8 and 9) proteins in cotransfected HeLa cells using anti-GFP antibody, D, analysis of alternatively spliced products derived from Fas reporter minigenes (10) cotransfected with GFP-tagged plasmids indicated. Cytoplasmic RNAs isolated from post-transfected HeLa cells in C were analyzed by RT-PCR as in B. E, tethering of HuR or PTB to Fas exon 6 through heterologous RNA binding domains promotes exon skipping. The Fas 2xMS2 minigene was cotransfected with plasmids expressing the MS2 fusion proteins indicated. Cytoplasmic RNAs were analyzed as above. Molecular mass markers (MW) for protein (A and C) and DNA (B, D, and E) are indicated on the left. The identities of protein bands (A and C) are indicated on the right by arrowheads. PCR amplification products corresponding to the Fas alternatively spliced isoforms (B, D, and E) are indicated at the right by boxes.

 
An Exonic Splicing Silencer Mediating HuR Repression—Co-transfection of Fas-derived minigenes (10, 34) and plasmids expressing either GFP or GFP-HuR in HeLa cells was used to characterize the sequence through HuR function as a Fas splicing regulator. HuR overexpression over wild type (WT) Fas minigene promoted exon 6 skipping (Fig. 2, C and D, compare lane 1 with lane 4), indicating that HuR down-regulates exon 6 inclusion. Nevertheless, the mutant m0 minigene, with replacement of a uridine-rich sequence on exon 6 (URE6), was fully insensitive to equivalent levels of HuR overexpression (Fig. 2, C and D, compare lane 2 with lane 5). In these studies, TIAR overexpression and the effects on U-20C and mURI6 reporter minigenes were used as positive and negative controls of exon 6 inclusion and skipping, respectively (Fig. 2, C and D, lanes 3 and 6-9). I conclude that HuR recruitment to the URE6 sequence induces Fas exon 6 skipping.

To further document HuR function through the URE6 element, this sequence was substituted for two MS2 stem-loop binding sites in tandem for the bacteriophage MS2 protein (Fas 2xMS2 minigene) (10). Cotransfection with a plasmid expressing an MS2-HuR fusion protein led to significant exon 6 skipping (Fig. 2E, lane 4), whereas expression of GFP or MS2 proteins did not (Fig. 2E, lanes 1 and 2). As I described previously (10), expression of an MS2-PTB fusion protein also led to efficient exon 6 skipping (Fig. 2E, lane 3). However, the URE6 substitution rendered the minigene insensitive to PTB or HuR overexpression (Fig. 2E, lanes 5 and 6). The simplest interpretation of all these results is that URE6 sequence silences exon 6 inclusion in a HuR-dependent way.

Fas Splicing Regulation in Vivo and in Vitro by HuR and TIA-1/TIAR Antagonism—Given that antagonism between TIA-1 and HuB/HuC/HuD proteins has been reported previously (15), in vivo splicing experiments in HeLa cells were performed by cotransfection of WT or m0 Fas minigenes and plasmids expressing GFP, HuR alone, or HuR with TIA-1 or TIAR (Fig. 3). Exon 6 skipping by HuR overexpression was rescued by the simple overexpression of equivalent levels of TIA-1 or TIAR proteins (Fig. 3B, compare lanes 2 and 3 with lanes 6 and 7), whereas m0 minigene was refractory to the HuR overexpression (Fig. 3B, lanes 4 and 5). These results point to functional antagonism between TIA-1/TIAR and HuR proteins.

In vitro splicing assays were then done to assess whether the effects of HuR and TIA-1/TIAR observed ex vivo are direct. In vitro-transcribed RNA corresponding to Fas genomic sequences between exons 5 and 6 plus 30 nucleotides of intron 6 were incubated with HeLa nuclear extracts under splicing conditions. ATP-dependent accumulation of the product containing exons 5 and 6 was observed (Fig. 3C, lane 2). In line with transfection data, the addition of purified recombinant HuR abolished accumulation of 5-6 spliced product (Fig. 3C, lanes 3-5), whereas additional supplementation of purified recombinant TIA-1 or TIAR (data not shown) rescued product accumulation (Fig. 3C, lanes 6 and 7). Most of the effects observed previously (Figs. 2, D and E, and 3B) were detectable and recapitulated with the m0 mutant containing mutation of URE6 sequence. For example, this mutant remained insensitive to HuR excess (Fig. 3C, compare lanes 2 and 9 with lane 10). Similarly, the addition of purified recombinant PTB reduced the 5-6 spliced product (Fig. 3C, lane 12), whereas the m0 mutant was insensitive to PTB excess (Fig. 3C, lane 13). Overall, the results recapitulate the effects of cis-acting sequences and trans-acting factors observed ex vivo and indicate that HuR acts directly through the exonic silencer URE6.


Figure 3
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 3.
Regulation of Fas splicing in vivo and in vitro by antagonistic effects of HuR and TIA-1/TIAR. A and B, the effects of HuR, TIA-1, and TIAR overexpression on alternative splicing of WT and mutant m0 Fas minigenes. A, analysis of the expression levels of GFP (lane 1), GFP-HuR (lanes 2-7), GFP-TIA-1 (lane 6), and GFP-TIAR (lane 7) were carried out in cotransfected HeLa cells with WT Fas minigene (0.5 µg) plus plasmids expressing GFP (1.5 µg) (lane 1) or HuR (1.0 and 1.5 µg) (lanes 2 and 3), m0 Fas minigene (0.5 µg), plus plasmid expressing HuR (1.0 and 1.5 µg) (lanes 4 and 5), WT Fas minigene (0.5 µg), plus plasmids expressing HuR (1 µg) and TIA-1 (1 µg) or TIAR (1 µg) (lanes 6 and 7). Protein extracts (20 µg) were purified and analyzed by Western blot with anti-GFP antibody. Molecular mass markers are shown on the left, and the identities of protein bands are indicated by arrowheads on the right. B, cytoplasmic RNAs were purified from post-transfected HeLa cells in A and analyzed by RT-PCR. Molecular mass markers (MW) are indicated on the left. The identities of PCR products are indicated on the right by boxes. C, recapitulation of regulation of Fas splicing in vitro by HuR and TIA-1. In vitro-synthesized RNA corresponding to Fas exons 5 and 6 plus 30 nucleotides of intron 6 was incubated with nuclear extracts under splicing conditions in the absence or presence of ATP. RNAs were isolated and fractionated in denaturing polyacrylamide gels. The patterns of splicing for WT RNA in the absence (lanes 1, 2, and 11) or presence of 10, 25, or 50 ng/µl of HuR (lanes 3-5) or presence of 25 ng/µl of HuR plus 10 or 20 ng/µl of TIA-1 (lanes 6 and 7) together with the patterns of splicing for WT or m0 RNA in the absence (lanes 8 and 9) or presence of 50 ng/µl of HuR (lane 10) or in the absence (lane 11) or presence of 50 ng/µl of PTB (lanes 12 and 13) were analyzed. The expected products of splicing in vitro are schematically indicated on the right.

 
Interaction between HuR and URE6—To confirm that HuR binds to the exonic silencer URE6, RNA binding assays were done. Cross-linking assays with an RNA corresponding to Fas exon 6 (E6 WT) indicated that the addition of purified recombinant HuR supplemented with HeLa nuclear extracts results in a significant increase in HuR cross-linking, without affecting the overall pattern of cross-linking of other proteins (Fig. 4A, compare lanes 2 and 3), whereas mutation of the URE6 sequence (E6 m0) reduced HuR cross-linking 5-10-fold (Fig. 4A, lanes 5 and 6). This preferential binding to the URE6-containing RNA was also observed in UV-cross-linking plus immunoprecipitation assays. In fact, HuR present in HeLa nuclear extracts was cross-linked to the radioactively labeled WT RNA corresponding to the Fas exon 6 followed by HuR immunoprecipitation (Fig. 4B, lane 2), and this cross-linking was significantly reduced by mutation of the URE6 sequence (Fig. 4B, lane 4). These results indicate that HuR binds to the URE6 sequence. Additionally, purified recombinant HuR was incubated with RNAs corresponding to the WT or m0 Fas exon 6 (Fig. 4C). HuR binding was significantly reduced (4-5-fold) by mutation of the URE6 sequence (Fig. 4C, compare lanes 1-6 with lanes 7-12). This result indicates that HuR binds directly to URE6. In line with the function of Hu proteins in other RNAs (35, 36), my results showed cooperative HuR binding to Fas RNA substrates since the fastest migrating HuR-RNA complexes accumulate according to protein concentration, rather than sequentially (Fig. 4C). Taken together, these results suggest that the URE6 sequence can function as a high affinity site facilitating HuR recruitment and favoring multimerization.

Mechanism of Fas Splicing Regulation by HuR—Fas intron 5 splicing is completely dependent on the presence of the 5' splice site of intron 6 and downstream sequences as well as recognition of these sequences by U1 snRNP and TIA-1/TIAR, which facilitate exon definition and splicing of the upstream intron (10). Given that HuR can form multimers and so alter recognition of the sequences at the 5' splice site in intron 6, I analyzed the effect on psoralen-mediated cross-linking between U1 snRNA and the 5' splice site region of Fas exon 6 when adding recombinant purified HuR protein to nuclear extracts. As shown in Fig. 4D, excess HuR did not diminish but rather increased interaction between U1 snRNA and 5' splice site (Fig. 4D, compare lanes 1 and 5 with lanes 4 and 8, respectively). This analysis was repeated with PTB as a positive control (Fig. 4D, compare lanes 1 and 5 with lanes 3 and 7, respectively). However, U1 snRNA binding to the 5' splice site sequence was clearly reduced in extracts with U1 snRNP inactivated by RNase H-mediated digestion of U1 snRNA 5' end (Fig. 4D, lanes 2 and 6). I conclude that HuR stabilizes U1 snRNP recruitment to the 5' splice site of exon 6.

I have previously shown that Fas intron 5 splicing was facilitated by an exon definition phenomenon, probably due to stabilization of U2AF binding mediated by U1 snRNP association with the downstream 5' splice site across the exon (3, 37). Consistent with this, U2AF65 cross-linking was abolished in the absence of a 5' splice site (Fig. 4E, lane 6) and in the presence of excess HuR (Fig. 4E, lane 4) at levels equivalent to those corresponding to affinity chromatography U2AF-depleted extract (Fig. 4E, lane 5). U2AF65 cross-linking was also reduced in the presence of PTB (Fig. 4E, lane 3) as well as in extracts in which U1 snRNP was inactivated by RNase H-mediated digestion of U1 snRNA 5'end (Fig. 4E, lane 2). Furthermore, I demonstrated that inhibition of association of U2AF65 with the 3' splice site by HuR occurs in an URE6-dependent manner since this association is re-established with m0 mutant (Fig. 4F, lanes 1-3). Finally, I tested whether the residual cross-linking of U2AF65 observed in the absence of the 5' splice site of intron 6 (not mediated by exon definition) was inhibited by HuR. In the absence of intron 6 sequences, U2AF65 cross-linking did not depend on the integrity of U1 snRNA (Fig. 4F, lane 5). Remarkably, U2AF65 cross-linking was completely inhibited by HuR (Fig. 4F, lane 7) but not by PTB (Fig. 4F, lane 6; the stronger background is due to the longer exposure required to detect the low levels of U2AF65 cross-linking in the absence of a 5' splice site). Taken together, these results indicate that HuR binding to the URE6 element interferes with U2AF65 association with the 3' splice site region and suggest that HuR inhibition mainly requires the 5' splice site of intron 6 (i.e. interactions across the exon that stabilize U2AF65 binding to the 3' splice site on recognition of the 5' splice site by U1 snRNP) and a direct interaction with the U2AF65 binding site. I conclude that HuR can mediate Fas splicing repression through its capacity to interfere in exon definition.


Figure 4
View larger version (41K):
[in this window]
[in a new window]

  		FIGURE 4.
HuR inhibits U2AF65 binding by interfering with the molecular interactions that lead to exon definition. A and B, HuR cross-links to Fas exon 6 URE6 sequence. A, RNAs uniformly labeled with 32P-uridine corresponding to WT or mutated (m0) Fas exon 6 were incubated with none (lanes 1 and 4) or HeLa nuclear extracts (NE) in the presence of either MBP (lanes 2 and 5) or MBP-HuR (lanes 3 and 6), irradiated with short wave UV light, treated with RNase A, fractionated by electrophoresis on a sodium-dodecyl sulfate gel, dried, and exposed to film. Note that HuR does not affect the cross-linking profile of other proteins in nuclear extracts. B, RNAs corresponding to WT or mutant m0 exon 6 as in A were incubated with HeLa nuclear extracts, cross-linked with UV light, treated with RNase A, and followed by immunoprecipitation with anti-hnRNPC1/C2 (used as a control) or anti-HuR antibodies. The RNA-protein immunocomplexes were analyzed by electrophoresis as in A. The positions of molecular mass markers (MW) and cross-linked HuR in panels A and B are indicated. C, band shift assays using various concentrations of purified recombinant MBP (10-6 M) or MBP-HuR (10-8, 10-7, 3 x 10-7, 6 x 10-7, and 10-6 M) proteins and radioactively labeled RNA sequences corresponding to WT/m0 Fas exon 6. RNA-protein complexes were analyzed on native 6% polyacrylamide gel, dried, and exposed to film. The positions of the free RNAs and RNA-protein complexes are indicated. D, HuR stabilizes U1 cross-linking to the 5' splice site associated with exon 6. Uniformly labeled WT or m0 RNAs were incubated with control (lanes 1, 3-5, 7, and 8), or U1 knock-out (ko) nuclear extracts (lanes 2 and 6) in the presence of psoralen and in the absence (lanes 1 and 5) or the presence of PTB (50 ng/µl) (lanes 3 and 7) or HuR (50 ng/µl) (lanes 4 and 8) were irradiated with long wave UV light, and RNA was isolated and resolved by electrophoresis in denaturing polyacrylamide gel. The positions of uncross-linked RNA and the product of cross-linking to U1 snRNA are schematically indicated. E, HuR inhibits the U1 snRNP-dependent, 5' splicing site-dependent cross-linking of U2AF65. Uniformly labeled RNAs corresponding to exon 6 and neighboring sequences (-68 to +30 or -68 to the 3' end of exon 6) were incubated with normal nuclear extracts in the absence (lanes 1 and 6) or presence of PTB (lane 3) or HuR (lane 4) (50 ng/µl), U1-inactivated nuclear extracts (lane 2), or chromatographically depleted extracts of U2AF ({Delta}U2AF) (lane 5) as indicated. F, HuR does not inhibit U2AF65 cross-linking in the absence of the URE6 (lane 3) but does inhibit it in the absence of 5' splice site sequences (lane 7). Assays were carried out using RNAs with the URE6 sequence mutated (m0) or lacking intron 6 sequences, as indicated.

 
RRM1 and Hinge-RRM3 Domains of HuR Are Necessary and Sufficient to Promote Exon 6 Exclusion—Human HuR protein is made up of two RRMs arranged in tandem near the N terminus followed by a basic hinge domain and a third RRM near the C terminus (14, 38) (Fig. 5A). Structures of peptide-RNA complexes containing RRMs 1 and 2 of HuR protein indicated that these domains are sufficient for specific recognition of AU-rich sequence element (ARE) substrates (36, 39). However, the hinge domain contributes significant binding energy to HuR-ARE complex formation in an ARE length-dependent way, whereas the RRM3 domain is required for cooperative assembly of HuR oligomers on RNA (36). I aimed to establish the domain(s) through HuR functions as a splicing repressor with a tethering approach to test the functionality of each protein domain as fusion protein to MS2. First, I analyzed how overexpression of the RRM1-2 and hinge-RRM3 domains affects the Fas2xMS2 minigene (Fig. 5B). The capacity of both domains to promote exon 6 skipping was similar to full MS2-HuR fusion protein (Fig. 5B, compare lane 2 with lanes 3 and 6). However, on separating and analyzing each domain individually, only RRM1 had sufficient repressor activity to promote exon 6 skipping (Fig. 5B, compare lane 3 with lanes 4 and 5 and lane 6 with lanes 7 and 8). I conclude that the repressor capacity of HuR resides largely in RRM1 and hinge-RRM3 domains.


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 5.
The silencing activity of HuR on Fas splicing resides in RRM1 and hinge-RRM3 domains. A, schematic structure of human HuR protein. The amino acid number is shown. The RRM1-2-3 and basic hinge functional domains are indicated by open boxes. B and C, tethering of HuR domains to Fas exon 6 through heterologous RNA binding domains. B, the Fas 2xMS2 minigene containing two MS2 stem-loop binding sites in tandem by URE6 sequence was co-transfected with plasmids expressing empty vector MS2, MS2-HuR, MS2-RRM12, MS2-RRM1, MS2-RRM2, MS2-hinge-RRM3, MS2-hinge, or MS2-RRM3 as indicated. RNA analyses were performed as described in the legend for Fig. 2 (panel E). Molecular mass markers (MW) are shown on the left. The identities of the alternatively spliced products are indicated on the right by boxes. C, the Fas 2xMS2 minigene was modified by mutagenesis to strengthen the intron 6 5' splice site, intron 5 3' splice site or both, generating three Fas 2xMS2-derived versions identified as U1+, 3'-ss+, and U1++3'-ss+, respectively. The WT, U1+, 3'-ss+, or U1++3'-ss+ Fas 2xMS2 minigenes were cotransfected with plasmids expressing MS2, MS2-HuR, MS2-RRM1, or MS2-hinge-RRM3. RNA analyses were carried out as above. Molecular mass markers and the identities of the alternatively spliced products are indicated. D, effects of GFP-tagged HuR functional domain overexpression on alternative splicing of Fas minigene. HeLa cells were cotransfected with WT Fas minigene (0.3 µg) and empty vector (2.0 µg), empty vector (1.0 µg) plus HuR (1.0 µg), HuR (1.0 µg), plus RRM1 (0.5 and 1.0 µg), HuR (1.0 µg) plus hinge-RRM3 (0.5 and 1.0µg), HuR-{Delta}hingeRRM3 (2.0µg), or HuR-{Delta}RRM1 (2.0µg) plasmids as indicated. Cytoplasmic RNA was purified from post-transfected HeLa cells and analyzed by RT-PCR as above. Molecular mass markers for DNA are indicated on the left. The identities of PCR products are shown on the right by boxes.

 


Figure 6
View larger version (19K):
[in this window]
[in a new window]

  		FIGURE 6.
Model for the regulation of Fas splicing by HuR. Molecular interactions lead to exon 6 skipping in a HuR-dependent manner. HuR acts by multimerizing through the URE6 sequence and through the U2AF65 binding site to inhibit exon definition and, therefore, U2AF (U2AF65-U2AF35 (U2 snRNP auxiliary factor 35 kDa) heterodimer) binding to the upstream 3' splice site, without inhibiting the interactions of TIA-1/TIAR and U1 snRNP to recognize the 5' splice site of exon 6 and downstream sequences.

 
To further clarify the repressor capacity of HuR residing in RRM1/hinge-RRM3 and its dependence on type of RNA substrates, I extended the above tethering analyses using modified Fas 2xMS2 minigenes, which when present strengthened the intron 6 5' splice site, intron 5 3' splice site, or both. In this scenario, the RRM1 domain functioned as a potent repressor of RNA substrates with weak 5' and 3' splice sites and with stronger 5' splice sites but not with stronger 3' splice sites (Fig. 5C, compare lanes 3 and 7 with lanes 11 and 15). Surprisingly, the hinge-RRM3 fusion was a potent repressor in all conditions examined, even when the 5' and 3' splices sites were stronger (Fig. 5C, compare lanes 4 and 8 with lanes 12 and 16). These results point to a dual behavior of the functional domains of HuR splicing repressor and a major role of the hinge-RRM3 domains, where resides the capacity for cooperative assembly of HuR oligomers on RNA, in inhibition of Fas splicing by targeting molecular events that lead to exon definition.

Finally, to demonstrate the functional duality of RRM1 and hinge-RRM3 domains and their decisive contribution to the role exerted by HuR as Fas splicing repressor, I generated these domains as GFP-tagged fusion proteins to test their contribution to the splicing regulation of a WT Fas minigene. These domains were tested as cis- and trans-acting regulatory elements. As shown in Fig. 5D, I did not detect significant differences between the regulatory capacities of domains by themselves, observing dominant-negative effects that might be predicted a priori (Fig. 5D, compare lanes 1 and 2 with lanes 3-6). Overexpression of truncated variants of HuR lacking these domains abolished repressive activity of HuR (Fig. 5D, compare lane 2 with lanes 7 and 8). I conclude that both RRM1 and hinge-RRM3 domains of HuR are necessary to recapitulate the repressor activity of the splicing factor HuR.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The apoptosis-inducing proteins TIA-1/TIAR promote U1 snRNP recruitment to the 5' splice site of intron 6, in turn facilitating exon definition by enhancing U2AF binding to the 3' splice site of intron 5 and, therefore, Fas exon 6 inclusion (10). I show that HuR promotes exon 6 skipping through an exonic splicing silencer, URE6, by inhibiting molecular events leading to exon definition via stabilization of U2AF65 binding (Fig. 6). These findings provide a functional link between HuR, a novel Fas splicing regulator, and molecular events leading to exon definition.

HuR Targets Exon Definition—Hu proteins (HuB, HuC, and HuD) have been recently identified as splicing regulators in neuron-like cells (15). Such proteins can directly block TIA-1 activity on neuron-specific alternative RNA processing of the calcitonin/calcitonin gene-related peptide pre-mRNA (15). Here, I report for the first time that HuR, the only ubiquitously expressed Hu protein in mammalian cells, is a novel splicing regulator with a putative wide range of action. My results show that HuR operates differently to other Hu proteins (15). The repressive effect of HuR on Fas exon 6 splicing does not require 3' of the URE6 element to prevent U1 snRNP and/or TIA-1/TIAR interactions with the 5' splice site of exon 6 and down-stream sequences. HuR-mediated inhibition interferes with the molecular interactions that lead to exon definition (3, 37). I reported previously that recognition of the 3' splice site of Fas exon 6 is strongly dependent on exon definition effects and that PTB/Raver-1 can inhibit the bridging interactions that mediate the events leading to exon definition (10). The results in Fig. 4 strongly suggest that HuR acts by interfering with the molecular bridges between U1 snRNP and U2AF65 that lead to exon definition and by direct competition of U2AF65 binding to the 3' splice site. This double inhibitory effect is exerted through the RRM1 and hinge-RRM3 functional domains of HuR (Fig. 5, B-D). Fialcowitz-White et al. (36) recently demonstrated that the RNA binding affinity of HuR is RRM1-2-dependent, whereas the capacity for cooperative assembly of HuR oligomers on RNA resides in hinge and RRM3 domains (36). I therefore propose that multimerization of HuR, favored by hinge and RRM3 domains, interferes with molecular interactions leading to exon definition and U2AF65 recruitment. Nevertheless, it is still unclear how HuR RRM1 functions as a repressive domain of Fas splicing. It is tempting to suggest that this domain per se may also counteract the bridging interactions leading to exon definition by means of another unknown mechanism. However, consistent with the repressive effects of both domains, RRM1 and hinge-RRM3 fragments of HuR function as trans-acting dominant-negative proteins in an independent manner (Fig. 6). By analogy with the mechanisms operating for other splicing repressors, HuR might partially function like hnRNP A1 (40) or PTB (41). HuR binding to a high affinity site located into URE6 sequence could trigger multimerization on nearby sequences that would prevent or displace the association of factors binding to juxtaposed enhancers and/or splice sites (Fig. 6). My combined results indicate that this model is likely to operate in the regulation of Fas exon 6.

Potential Physiological Consequences—My results support an interesting link between regulation of alternative Fas splicing by HuR and apoptosis in cancer cells. In vivo, HuR overexpression is observed in many human cancers, including brain, breast, colon, lung, and ovary tumors (27). I found that overexpression of HuR in HeLa cells, a cervical carcinoma cell line, leads to Fas exon 6 skipping. The finding that, like TIA-1, TIAR, PTB, FASTK (Fas-activated serine/threonine kinase), and SPF45 (splicing factor 45 kDa) (7, 10, 34, 42), HuR is also involved in the alternative splicing control of Fas exon 6 suggests that the switch in expression between pro- and antiapoptotic Fas isoforms is tightly regulated. I suggest that HuR overexpression might be a strategy for tumor cells to escape apoptosis. This suggestion agrees with the role assigned to HuR as an antiapoptotic and survival factor since this protein can be the master regulator of the coordination of a post-transcriptional antiapoptotic program in human cells (23). The network of HuR interactions might have a more widespread function in the regulation of additional alternative splicing events. Given that about 74% of human pre-mRNAs have alternative isoforms (2), more primary transcripts may be found that can be alternatively spliced through the action of HuR.

HuR is emerging as a key regulator, a new paradigm, of the post-transcriptional regulation in mammals. Given that the same RNA-binding protein can regulate several aspects of gene expression under different environmental conditions, it is interesting to note that a multifunctional protein such as HuR may be involved in coupling different molecular events affecting the post-transcriptional control, including splicing, transport, localization, stability, and translation. HuR is predominantly nuclear under normal growth conditions but shuttles between the nucleus and the cytoplasm through a specialized domain, the shuttling sequence HNS (for HuR nucleocytoplasmic shuttling) (16). HuR regulates RNA metabolism events in the nucleus and cytoplasm. In the nucleus, HuR functions as pre-mRNA splicing regulator, probably as a specific repressor of exon inclusion, like PTB (44), thanks to its capacity to bind to adenosine- and/or uridine-rich tracts located in intronic and exonic sequence elements on the pre-mRNA (this study). Thus, HuR may be regulating other splicing events affecting many relevant genes to control key cellular processes. In the cytoplasm, HuR is involved in the regulation of stability and translation of target mRNA subsets (45). In most systems regulated by HuR, control is achieved through interaction of HuR with multiple HuR binding sites surrounding the target sequence located at the 3'-untranslated regions of the cellular mRNAs (36). Furthermore, HuR post-translational modifications (including phosphorylation by Chk2 (22)) or its cellular compartmentalization could prevent or reverse its regulatory features. In summary, HuR seems to use multiple molecular mechanisms to modulate gene expression at the post-transcriptional level, showing that the control of human genome expression involves complex networks of epigenetic interactions.

Addendum—Interestingly, while this report was under revision, Zhu et al. (43) reported that neuron-specific Hu proteins control the production of the short protein isoform from neurofibromatosis type I (NF1) pre-mRNA by suppressing inclusion of NF1 exon 23a, whereas TIA-1/TIAR proteins promote inclusion of this exon. Mechanistically, they identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In fact, these proteins prevent binding of U1 and U6 snRNPs to the 5' splice site of NF1 exon 23a and decrease U2AF binding to upstream 3' splice site, interfering the molecular events that lead to exon definition. Additional studies of HuR as well as other Hu proteins and their target pre-mRNAs will provide relevant insight into the regulatory mechanisms of alternative splicing.


   FOOTNOTES
 
* This work was supported by a grant from Fondo de Investigaciones Sanitarias (PI051605). 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 To whom correspondence should be addressed: Departamento de Biología Molecular, Centro de Biología Molecular "Severo Ochoa", Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Cantoblanco 28049, Madrid, Spain. Tel.: 34-91-1964530; Fax: 34-91-1964420; E-mail: jmizquierdo{at}cbm.uam.es.

2 The abbreviations used are: TIA-1, T-cell intracellular antigen-1; TIAR, TIA-1 related protein; ELAVL, embryonic lethal, abnormal vision Drosophila-like family; GFP, green fluorescent protein; HuR, Hu antigen R; MBP, maltose-binding protein; PTB, polypyrimidine tract-binding protein; RRM, RNA recognition motif; siRNA, small interference RNA; snRNP, small nuclear ribonucleoprotein; snRNA, small nuclear RNA; U2AF65, U2-snRNP auxiliary factor 65 kDa; URE6, uridine-rich sequence located in Fas exon 6; RT-PCR, reverse transcription-PCR; WT, wild type; ARE, AU-rich sequence element; hnRNP, heterogeneous nuclear ribonucleoprotein. Back


   ACKNOWLEDGMENTS
 
I thank J. Valcárcel and J. M. Sierra for assistance, help, and encouragement. I am grateful to the generosity of M. Gorospe for HuR plasmids and A. Castelló for control siRNA. I also thank J. Alcalde, R. Reyes and J. Barrero for technical assistance. The Centro de Biología Molecular "Severo Ochoa" receives an institutional grant from Fundación Ramón Areces.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wang, G. S., and Cooper, T. A. (2007) Nat. Rev. Genet. 8, 749-761[CrossRef][Medline] [Order article via Infotrieve]
  2. Johnson, J. M., Castle, J., Garret-Engele, P., Kan, Z., Loerch, P. M., Armour, C. D., Santos, R., Schadt, E. E., Stoughton, R., and Shoemaker, D. D. (2003) Science 302, 2141-2144[Abstract/Free Full Text]
  3. Black, D. L. (2003) Annu. Rev. Biochem. 72, 291-336[CrossRef][Medline] [Order article via Infotrieve]
  4. Tian, Q., Streuli, M., Saito, H., Schlossman, S. F., and Anderson, P. (1991) Cell 67, 629-639[CrossRef][Medline] [Order article via Infotrieve]
  5. Kedersha, N., and Anderson, P. (2002) Biochem. Soc. Trans. 30, 963-969[CrossRef][Medline] [Order article via Infotrieve]
  6. Del Gatto-Konczak, F., Bourgeois, C. F., Le Guiner, C., Kister, L., Gesnel, M. C., Stevenin, J., and Breathnach, R. (2000) Mol. Cell. Biol. 20, 6287-6299[Abstract/Free Full Text]
  7. Förch, P., Puig, O., Kedersha, N., Martínez, C., Granneman, S., Seraphin, B., Anderson, P., and Valcárcel, J. (2000) Mol. Cell 6, 1089-1098[CrossRef][Medline] [Order article via Infotrieve]
  8. Le Guiner, C., Lejeune, F., Galiana, D., Kister, L., Breathnach, R., Stevenin, J., and del Gatto-Konczak, F. (2001) J. Biol. Chem. 276, 40638-40646[Abstract/Free Full Text]
  9. Zuccato, E., Buratti, E., Stuani, C., Baralle, F. E., and Pagani, F. (2004) J. Biol. Chem. 279, 16980-16988[Abstract/Free Full Text]
  10. Izquierdo, J. M., Majós, N., Bonnal, S., Martínez, C., Castelo, R., Guigó, R., Bilbao, D., and Valcárcel, J. (2005) Mol. Cell 19, 475-484[CrossRef][Medline] [Order article via Infotrieve]
  11. McAlinden, A., Liang, L., Mukudai, Y., Imamura, T., and Sandell, J. L. (2007) J. Biol. Chem. 282, 24444-24454[Abstract/Free Full Text]
  12. Zhu, H., Hasman, R. A., Young, K. M., Kedersha, N. L., and Lou, H. (2003) Mol. Cell. Biol. 23, 5959-5971[Abstract/Free Full Text]
  13. Antic, D., and Keene, J. D. (1997) Am. J. Hum. Genet. 61, 273-278[Medline] [Order article via Infotrieve]
  14. Ma, W. J., Cheng, S., Campbell, C., Wright, A., and Furneaux, H. (1996) J. Biol. Chem. 271, 8144-8151[Abstract/Free Full Text]
  15. Zhu, H., Hasman, R. A., Barron, V. A., Luo, G., and Lou, H. (2006) Mol. Biol. Cell 17, 5105-5114[Abstract/Free Full Text]
  16. Fan, X. C., and Steitz, J. A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15293-15298[Abstract/Free Full Text]
  17. Peng, S. S., Chen, C. Y., Xu, N., and Shyu, A. B. (1998) EMBO J. 17, 3461-3470[CrossRef][Medline] [Order article via Infotrieve]
  18. Bhattacharyya, S. N., Habermacher, R., Martine, U., Closs, I., and Filipowicz, W. (2006) Cell 125, 1111-1124[CrossRef][Medline] [Order article via Infotrieve]
  19. Atasoy, U., Watson, J., Patel, D., and Keene, J. D. (1998) J. Cell Sci. 111, 3145-3156[Abstract]
  20. Yaman, I., Fernández, J., Sarkar, B., Schneider, R. J., Snider, M. D., Nagy, L. E., and Hatzoglou, M. (2002) J. Biol. Chem. 277, 41539-41546[Abstract/Free Full Text]
  21. Gorospe, M. (2003) Cell Cycle 2, 412-414[Medline] [Order article via Infotrieve]
  22. Abdelmohsen, K., Pullman, R., Jr., Lal, A., Kim, H. H., Galban, S., Yang, X., Blethrow, J. D., Walker, M., Shubert, J., Gillespie, D. A., Furneaux, H., and Gorospe, M. (2007) Mol. Cell 25, 543-557[CrossRef][Medline] [Order article via Infotrieve]
  23. Abdelmohsen, K., Lal, A., Kim, H. H., and Gorospe, M. (2007) Cell Cycle 6, 1288-1292[Medline] [Order article via Infotrieve]
  24. Katsanou, V., Papadaki, O., Millatos, S., Blackshear, P. J., Anderson, P., Kollias, G., and Kontoyiannis, D. L. (2005) Mol. Cell 19, 777-789[CrossRef][Medline] [Order article via Infotrieve]
  25. Wang, W., Furneaux, H., Cheng, H., Caldwell, M. C., Hutter, D., Liu, Y., Holbrook, N., and Gorospe, M. (2000) Mol. Cell. Biol. 20, 760-769[Abstract/Free Full Text]
  26. Figueroa, A., Cuadrado, A., Fan, J., Atasoy, U., Muscat, G. E., Muñoz-Canoves, P., Gorospe, M., and Muñoz, A. (2003) Mol. Cell. Biol. 23, 4991-5004[Abstract/Free Full Text]
  27. López de Silanes, I., Lal, A., and Gorospe, M. (2005) RNA Biol. 2, 11-13[Medline] [Order article via Infotrieve]
  28. Wang, W., Yang, X., Cristofalo, V. J., Holbrook, N. J., and Gorospe, M. (2001) Mol. Cell. Biol. 21, 5889-5898[Abstract/Free Full Text]
  29. Krammer, P. H. (2000) Nature 407, 789-795[CrossRef][Medline] [Order article via Infotrieve]
  30. Cheng, J., Zhou, T., Liu, C., Shapiro, J. P., Brauer, M. J., Kiefer, M. C., Barr, P. J., and Mountz, J. D. (1994) Science 263, 1759-1762[Abstract/Free Full Text]
  31. Bidere, N., Su, H. C., and Lenardo, M. J. (2006) Annu. Rev. Immunol. 24, 321-352[CrossRef][Medline] [Order article via Infotrieve]
  32. Izquierdo, J. M., and Valcárcel, J. (2007) J. Biol. Chem. 282, 19410-19417[Abstract/Free Full Text]
  33. Izquierdo, J. M. (2006) Biochem. Biophys. Res. Commun. 348, 703-711[CrossRef][Medline] [Order article via Infotrieve]
  34. Izquierdo, J. M., and Valcárcel, J. (2007) J. Biol. Chem. 282, 1539-1543[Abstract/Free Full Text]
  35. Gao, F. B., and Keene, J. D. (1996) J. Cell Sci. 109, 579-589[Abstract/Free Full Text]
  36. Fialcowitz-White, E. J., Brewer, B. Y., Ballin, J. D., Willis, C. D., Toth, E. A., and Wilson, G. M. (2007) J. Biol. Chem. 282, 20948-20959[Abstract/Free Full Text]
  37. Berget, S. M. (1995) J. Biol. Chem. 270, 2411-2414[Free Full Text]
  38. Chen, C. Y., Xu, N., and Shyu, A. B. (2002) Mol. Cell. Biol. 22, 7268-7278[Abstract/Free Full Text]
  39. Inoue, M., Muto, Y., Sakamoto, H., and Yokoyama, S. (2000) Nucleic Acids Res. 28, 1743-1750[Abstract/Free Full Text]
  40. Zhu, J., Mayeda, A., and Krainer, A. R. (2001) Mol. Cell 8, 1351-1361[CrossRef][Medline] [Order article via Infotrieve]
  41. Spellman, R., and Smith, C. W. (2006) Trends Biochem. Sci. 31, 73-76[CrossRef][Medline] [Order article via Infotrieve]
  42. Corsini, L., Bonnal, S., Basquin, J., Hothorn, M., Scheffzek, K., Valcárcel, J., and Sattler, M. (2007) Nat. Struct. Mol. Biol. 14, 620-629[CrossRef][Medline] [Order article via Infotrieve]
  43. Zhu, H., Hinman, M. N., Hasman, R. A., Mehta, P., and Lou, H. (2008) Mol. Cell. Biol. 28, 1240-1251[Abstract/Free Full Text]
  44. Wagner, E. J., and García-Blanco, M. A. (2002) Mol. Cell 10, 943-949[CrossRef][Medline] [Order article via Infotrieve]
  45. López de Silanes, I., Zhan, M., Lal, A., Yang, X., and Gorospe, M. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2987-2992[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
 Am. J. Physiol. Renal Physiol.Home page
D. A. Ayupova, M. Singh, E. C. Leonard, D. P. Basile, and B. S. Lee
Expression of the RNA-stabilizing protein HuR in ischemia-reperfusion injury of rat kidney
Am J Physiol Renal Physiol, July 1, 2009; 297(1): F95 - F105.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
O. Papadaki, S. Milatos, S. Grammenoudi, N. Mukherjee, J. D. Keene, and D. L. Kontoyiannis
Control of Thymic T Cell Maturation, Deletion and Egress by the RNA-Binding Protein HuR
J. Immunol., June 1, 2009; 182(11): 6779 - 6788.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Y.-H. Goo and T. A. Cooper
CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA
Nucleic Acids Res., May 14, 2009; (2009) gkp346v1.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
283/27/19077    most recent
M800017200v1
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 Izquierdo, J. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Izquierdo, J. M.
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 © 2008 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement