Article3 December 2010free access TDP-43 regulates its mRNA levels through a negative feedback loop Youhna M Ayala Youhna M Ayala Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Laura De Conti Laura De Conti Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author S Eréndira Avendaño-Vázquez S Eréndira Avendaño-Vázquez Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Ashish Dhir Ashish Dhir Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Maurizio Romano Maurizio Romano Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Department of Life Sciences, University of Trieste, Trieste, Italy Search for more papers by this author Andrea D'Ambrogio Andrea D'Ambrogio Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, ItalyPresent address: Program in Molecular Medicine, University of Massachusetts Medical School Worcester, MA 01605, USA Search for more papers by this author James Tollervey James Tollervey MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jernej Ule Jernej Ule MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Marco Baralle Marco Baralle Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Emanuele Buratti Emanuele Buratti Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Francisco E Baralle Corresponding Author Francisco E Baralle Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Youhna M Ayala Youhna M Ayala Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Laura De Conti Laura De Conti Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author S Eréndira Avendaño-Vázquez S Eréndira Avendaño-Vázquez Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Ashish Dhir Ashish Dhir Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Maurizio Romano Maurizio Romano Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Department of Life Sciences, University of Trieste, Trieste, Italy Search for more papers by this author Andrea D'Ambrogio Andrea D'Ambrogio Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, ItalyPresent address: Program in Molecular Medicine, University of Massachusetts Medical School Worcester, MA 01605, USA Search for more papers by this author James Tollervey James Tollervey MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Jernej Ule Jernej Ule MRC-Laboratory of Molecular Biology, Cambridge, UK Search for more papers by this author Marco Baralle Marco Baralle Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Emanuele Buratti Emanuele Buratti Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Francisco E Baralle Corresponding Author Francisco E Baralle Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy Search for more papers by this author Author Information Youhna M Ayala1,‡, Laura De Conti1,‡, S Eréndira Avendaño-Vázquez1,‡, Ashish Dhir1,‡, Maurizio Romano1,2, Andrea D'Ambrogio1, James Tollervey3, Jernej Ule3, Marco Baralle1, Emanuele Buratti1 and Francisco E Baralle 1 1Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy 2Department of Life Sciences, University of Trieste, Trieste, Italy 3MRC-Laboratory of Molecular Biology, Cambridge, UK ‡These authors contributed equally to this work *Corresponding author. Department of Molecular Pathology, International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste 34149, Italy. Tel.: +39 040 375 7337; Fax: +39 040 375 7361; E-mail: [email protected] The EMBO Journal (2011)30:277-288https://doi.org/10.1038/emboj.2010.310 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TAR DNA-binding protein (TDP-43) is an evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) involved in RNA processing, whose abnormal cellular distribution and post-translational modification are key markers of certain neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We generated human cell lines expressing tagged forms of wild-type and mutant TDP-43 and observed that TDP-43 controls its own expression through a negative feedback loop. The RNA-binding properties of TDP-43 are essential for the autoregulatory activity through binding to 3′ UTR sequences in its own mRNA. Our analysis indicated that the C-terminal region of TDP-43, which mediates TDP-43–hnRNP interactions, is also required for self-regulation. TDP-43 binding to its 3′ UTR does not significantly change the pre-mRNA splicing pattern but promotes RNA instability. Moreover, blocking exosome-mediated degradation partially recovers TDP-43 levels. Our findings demonstrate that cellular TDP-43 levels are under tight control and it is likely that disease-associated TDP-43 aggregates disrupt TDP-43 self-regulation, thus contributing to pathogenesis. Introduction The TAR DNA-binding protein (TDP-43) is a highly conserved heterogeneous nuclear ribonucleoprotein (hnRNP). Like other members of this family, it has been linked to different aspects of RNA processing (reviewed in Buratti and Baralle, 2008). Its function as a regulator of splicing is the one characterized in best detail, wherein TDP-43 recruitment to 3′ splice sites, rich in GU repeats inhibits exon recognition (Buratti et al, 2001). The abnormal cellular distribution and post-translational modification of TDP-43 are key markers for a group of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) (Arai et al, 2006; Neumann et al, 2006), now also referred to as TDP-43 proteinopathies. Accumulation of TDP-43 in ubiquitin-positive insoluble inclusions was subsequently found in a range of neurodegenerative pathologies, such as in some Alzheimer's disease (AD) cases (Amador-Ortiz et al, 2007; Higashi et al, 2007; Uryu et al, 2008) and in additional types of dementia (Higashi et al, 2007; Nakashima-Yasuda et al, 2007; Neumann et al, 2007; Geser et al, 2008). The connection between TDP-43 and disease was further strengthened by the isolation of TDP-43 mutations in ∼5% of patients with inherited and sporadic forms of ALS (Pesiridis et al, 2009). TDP-43 aggregation occurs in the cytoplasm and nuclear compartments of neurons and glial cells, and is often accompanied by nuclear clearance of the protein. TDP-43 isolated from TDP-43 proteinopathies is often modified through extensive ubiquitination, phosphorylation, and aberrant proteolysis (Neumann et al, 2006). The role of TDP-43 in pathogenesis is still unknown, but the lethal and paralytic phenotypes resulting from knockout transgenic mouse and fly models, respectively, highlight the importance of TDP-43 function (Feiguin et al, 2009; Wu et al, 2010). It remains to be seen whether the formation of TDP-43 inclusion bodies is toxic to cells per se, whether the TDP-43 insoluble aggregates sequester the protein resulting in cellular TDP-43 loss of function, or whether both phenomena contribute to pathogenicity. Two RNA-binding domains, RRM1 and RRM2, are present in TDP-43 of which RRM1 is necessary and sufficient for nucleic acid-binding activity (Buratti and Baralle, 2001). The protein binds single-stranded RNA with high specificity for GU-rich sequences, measured in the low nanomolar range (Ayala et al, 2005). Phe 147 and 149 are located in the RNP-1 sequence of RRM1 forming a canonical binding platform for nucleic acid association (NMR structure:pdb2cqg). Substitution of these two key amino acids is sufficient to abolish RNA-binding and TDP-43 splicing regulatory activity (Buratti and Baralle, 2001; D'Ambrogio et al, 2009). The function of RRM2 is still unclear and does not appear to have a significant role in RNA interaction. In fact, its RNA-binding affinity is two orders of magnitude lower than that of RRM1 (Kuo et al, 2009). The splicing activity of TDP-43 also depends on the integrity of the glycine-rich C-terminus. More specifically, residues 321–366 within this domain recruit additional hnRNPs of the hnRNP-A and hnRNP-C families (D'Ambrogio et al, 2009). We now show that TDP-43 controls its own homoeostasis in human cells by downregulating TDP-43 transcript levels. This control requires TDP-43 RNA-binding activity through the association to specific sequences in the 3′ UTR of the TDP-43 transcript as well as the presence of the 321–366 amino acid region. The autoregulatory mechanism seems to at least partly involve an exosome-mediated pathway of mRNA degradation. Results Induced expression of TDP-43 downregulates endogenous levels of the protein We generated a HEK293 human kidney cell line to stably express a single copy of the TDP-43 cDNA on induction of the tetracycline promoter using the Flp-In technology (Invitrogen) as described (Glatter et al, 2009). The protein was tagged at the N-terminus either with a double tag (SH-TDP43) or with a FLAG tag (F-TDP43). Induced expression of the tagged transgenic forms of TDP-43 (TG-TDP43) resulted in a dramatic decrease in the levels of endogenous protein expression as seen by western blot (see Figure 1 WT). The same was not true on expression of TG-GFP in a similarly generated HEK293 cell line used as control (data not shown). After 24 h of tetracycline treatment, the levels of induced protein were similar to endogenous levels in untreated cells, consistent with the single-site integration provided by the Flp-In system. Longer times of induction (72 h) resulted in higher levels of TG-TDP43 and the consequent total depletion of endogenous TDP-43. Figure 1.Induced expression of wild-type and mutant TDP-43 in HEK293 cells. Tagged forms of TDP-43 (F-TDP43) were expressed for 24 or 72 h on tetracycline (Tet) induction (1 μg/ml). The diagrams on the left are a schematic representation of the different TDP-43 protein domains, numbering denotes amino acid residues. The N-terminal domain tag is represented by the hashed box and the RNA recognition motifs 1 and 2 are shown as black and grey boxes, respectively. Deletion of the C-terminal region in mutant Δ321–366 is depicted as a white box. The right panels are immunoblot analyses of the protein extracts with an antibody against TDP-43 that detects both endogenous (end) and tagged (TG-TDP43) protein. Tubulin was used as loading control. Download figure Download PowerPoint The RRM1 and C-terminal domain of TDP-43 are necessary for autoregulation To begin addressing the mechanism of self-regulation by TDP-43, we examined the importance of key TDP-43 regions (Figure 1). The variants were expressed as FLAG-tagged TDP-43 in the HEK293 Flp-In system. RRM1 and RRM2 were disrupted by double-site mutations F147/149L and F229/231L, respectively, and both RNA-binding domains were simultaneously targeted with the mutations F147/149/229/231L (F4L). In addition, the 321–366 region was deleted from the C-terminal domain (Δ321–366). Western blot analysis revealed that the RRM1 mutant failed to inhibit endogenous TDP-43 expression even after 72 h of induction (Figure 1 F147/149L), as did the mutant wherein both RRM1 and RRM2 were disrupted (F4L). The TDP-43 carrying mutations in RRM2 only (F229/231L), however, had no effect on the inhibition of endogenous TDP-43. Finally, in the case of the C-terminal deletion, the expression of variant Δ321–366 did not change endogenous TDP-43 levels after 72 h and failed to do so even after 120 h of transgene induction (Figure 1 and data not shown). These results indicate that autoregulation is mediated by an RNA–TDP-43 interaction and requires the recruitment of additional factors through the 321–366 C-terminal region. TDP-43 self-regulation occurs at the transcript level We next investigated whether TDP-43 overexpression similarly repressed endogenous transcript levels through northern blot analysis. Our experiments, using a probe specific for endogenous TDP-43 (indicated by arrows in Figure 2A), identified two main isoforms that correspond to the differential use of polyadenylation sites. Bioinformatic analysis of the 3′ UTR of TDP-43 predicted four polyadenylation signals located in the second half of the 3′ UTR (Figure 2A). Analysis of the TDP-43 ESTs reported in the database (UC Santa Cruz Genome Browser), however, indicated the near exclusive usage of polyadenylation sites labelled as pA1 and pA4 with predicted molecular weights of 2.8 and 4.2 Kb, respectively. Also found in the EST database is a minor form, we refer to as V2, that has a smaller molecular weight because of intron activation at the 3′ end of the gene. The two main isoforms detected in our northern blot assays (V1) are compatible with the use of pA1 and pA4 (Figure 2B, first lane from left, WT, Tet −). Figure 2.TDP-43 negative autogenous regulation occurs at the transcript level. (A) Schematic representation of the TDP-43 gene with coding exons and untranslated regions shown in black and grey, respectively. The various predicted polyadenylation signals are indicated, where (*) denotes the experimentally validated sites. The two principally expressed endogenous isoforms (V1pA1 and V1pA4) are shown including the transgenic TG-TDP43 transcript containing the coding sequences only. The V2 form corresponds to a minor isoform discussed in Supplementary Figure 2. (B) Northern blot analysis of control and after 24 and 72 h of induction. V1pA1 and V1pA4 correspond to the 2.8 and 4.2 Kb bands, respectively. Endogenous TDP-43 transcripts were detected by probing the region indicated by the arrows in (A), while TG-TDP43 was identified with a probe spanning exons 2 and 3. GAPDH detection was used as loading control. Download figure Download PowerPoint A comparison of the endogenous TDP-43 transcript levels as a function TG-TDP43 expression by northern blot analysis showed a striking reduction of TDP-43 mRNA isoforms on tetracycline induction (Figure 2B second lane, WT, Tet +). Mutations in RRM1 (F147/149L and F4L) and the Δ321–366 deletion prevented significant regulation of endogenous TDP-43 mRNA after 24 and 72 h of TG-TDP43 expression (Figure 2B). Again, the RRM2 mutant was not different from wild-type. These findings are in agreement with the immunoblot results showing the requirement of RRM1 and the 321–366 region for the negative self-regulation of TDP-43. TDP-43 autoregulation involves 3′ UTR-binding elements The accumulation of TG-TDP43 over time observed in our western blot analyses (Figure 1) suggested that the inducible TDP-43, unlike endogenous TDP-43, was not subject to self-regulation through a negative feedback loop. A significant structural difference between endogenous and transgenic TDP-43 transcripts is the lack of the corresponding 3′ UTR in TG-TDP43 (Figure 2A), implying that this region may contain important regulatory elements. Of course, additional important differences exist between the wild-type transgene and the endogenous TDP-43 gene (i.e., presence of introns and different promoters), however, we first focused on the 3′ UTR region for several reasons. First of all, regarding the absence of introns, it must be taken into account that the 3′ UTR region of TDP-43 is highly conserved among different species while none of the intronic regions share an even similar degree of conservation (see Supplementary Figure 1). We also know that evolutionary conservation is probably very important for autoregulation as three heterozygous transgenic knockout mouse models have shown that +/− mice were observed to produce the same amount of TDP-43 protein and mRNA as the wild-type +/+ mice (Kraemer et al, 2010; Sephton et al, 2010; Wu et al, 2010). Second, regarding the use of different promoters, it should be considered that autoregulation is still observed in a heterologous reporter construct that uses a different promoter, which will be discussed later. Finally, and as a side note, the use of two different systems to produce stable cell lines also rules out an artifactual effect mediated by the protein tag sequences attached to the transgenic TDP-43 as these differed from each other in either system: FLAG and streptavidin-binding peptide fused to a hemagglutinin epitope tag. Regarding the 3′ UTR, recent cross-linking immunoprecipitation (CLIP) analysis performed in HEK293 cells determined that TDP-43 associated with several RNA sequences within the 3′ UTR of its own transcript in vivo (Figure 3) in a region that we called TDP-43-binding region (TDPBR, Figure 3A). To confirm TDP-43 binding to these sequences, we first of all performed electromobility shift assays (EMSAs) using fragments (fragment (frg.) 1–4) across this entire region. As shown in Figure 3B, right panel, fragments 2 and 3 that contain most CLIP sequences/clusters can efficiently bind TDP-43 while no binding could be observed for fragments 1 and 4 that contained only one or none (respectively) extended CLIP sequences/clusters. Figure 3.TDP-43 specifically binds its 3′ UTR region. (A) Schematic representation of the TDP-43 gene depicting the location and sequence of the TDPBR (B, fragments 2 and 3) within the 3′ UTR. Coding exons and untranslated regions are shown in black and grey, respectively. (B) In the left panel, the 3′ UTR region of TDP-43 with the different regions tested for TDP-43 binding (frgs. 1–4) is reported. In this panel, all the CLIP sequences/clusters reported from different experiments (J Ule and J Tollervey unpublished results) are highlighted in bold. The major 34-nt CLIP sequence seen in HEK 293 cells and used in comparative-binding analysis (C–E) is boxed. On the right, band-shift analysis using recombinant TDP-43 using these different fragments. (C) GST-TDP43 mutants were analyzed for binding to the major 34-nt CLIP sequence (boxed) and (GU)6 RNA as control. (D, E) Competition assays using constant levels of wild-type protein to bind radiolabelled (GU)6 as a function of increasing concentrations of unlabelled 34-nt CLIP sequence (D), or binding to radiolabelled 34-nt CLIP sequence as a function of increasing concentrations unlabelled (GU)6. Protein was absent from the first lane in each panel. ∼0.5 μg of the different recombinant proteins were used with 1 ng of the various 5′-labeled single-stranded RNA oligonucleotides. Competition analyses were carried out with 2.5, 3.75, 5, and 10 ng of unlabelled 34-nt CLIP sequence and (GU)6. Download figure Download PowerPoint The 34-nucleotide boxed sequence (34 nt) in Figure 3B (frg. 2) was the one with the higher density of hits in the CLIP assay for TDP-43 in HEK 293 cells. However, as it was not a canonical (GU)n repetitive sequence that is known to be the preferred target of this protein, we considered necessary to study the 34 nt-binding efficiency compared with (GU)6. Figure 3C shows that wild-type TDP-43 and the RRM2 mutant (F229/231L) specifically bind the 34 nt sequence while the RRM1 mutant (F147/149L) did not produce a shift, as expected. These results coincide with our in vivo assays showing the lack of regulatory control on disruption of RRM1, and no apparent role of RRM2 (Figures 1 and 2). To compare the binding efficiency of TDP-43 with the 34 nt and (UG)6 RNAs, we then performed EMSA in the presence of competing amounts of unlabelled RNA. TDP-43 binding to the 34 nt was efficiently competed with increasing amounts of (UG)6 RNA, while higher concentrations of TDPBR RNA were necessary to see a reduction in TDP-43 association with (UG)6 (Figure 3D and E, respectively). Collectively, our data suggest that TDP-43 association with these sequences is specific, although it seems to occur with lower efficiency compared with UG repeat binding. A lower affinity may be desirable for regulatory sequences spread thorough a large region of the 3′ UTR that require multiple molecules of TDP 43 to induce a response. Assessing the importance of the TDPBR region in a heterologous context To test the self-regulatory function of the TDP-43-binding sites found in the TDP-43 mRNA 3′ UTR, we used a recombinant construct (GFP-3′ UTR wt) that contained exons 5 and 6, together with the 3′ UTR sequence of TDP-43, fused to the C-terminal end of a GFP reporter sequence (schematically summarized in Figure 4A). This construct provided the advantage to test the autoregulatory mechanism in a heterologous context and was also easier to manipulate than the endogenous gene. When transfected into our inducible HEK293 cell line, the GFP expression levels from the GFP-3′ UTR wt construct decreased following Tet induction (Figure 4B, upper panel), a result that was consistent with the conclusion that the downregulatory effect of TDP-43 did not involve additional TDP-43 exons or introns beside the sequence cloned in this system. Furthermore, no downregulation was observed on a transfected GFP wild-type protein used to normalize transfection efficiencies (Figure 4B, lower panel). We then engineered heterologous constructs wherein we progressively deleted the 3′ UTR across the region of the TDP-43-binding sites. Two constructs (GFP-3′ UTR Δ369 and Δ669, Figure 4A) clearly showed partial and complete loss of self-regulation when transfected into the inducible HEK293 cell line. In fact, it can be seen in Figure 4B that the downregulation of GFP protein expression levels following Tet induction was much reduced in the Δ369 construct compared with GFP-3′ UTR wt and eventually abolished in the Δ669 construct. Interestingly, our western blot analysis also highlighted the production of a smaller GFP-containing protein (Δ1812) of approximately 40 kDa in the lanes belonging to the GFP-3′ UTR Δ369 and Δ669 constructs (see below). Figure 4.A region in the 3′ UTR of TDP-43 mediates autoregulation (A) a schematic diagram of the GFP-3′ UTR wt and Δ369 and Δ669 constructs. Wild-type TDP-43 coding sequences are shown in grey. The lower panel shows the splicing events that lead to the Δ1812 mRNA and protein isoforms. (B) GFP protein expression when the GFP-3′ UTR wt and Δ369 and Δ669 constructs were transfected into the HEK293 stable cell lines following induction (+) of the TG-TDP43 transgene. Transfection efficiencies were normalized by co-transfecting a GFP expression vector (lower panel). In the GFP-3′ UTR Δ369 and Δ669 (−) and (+) lanes, the western blot also contains an additional 40 kDa protein band that was called Δ1812. The different proteins were detected with anti-GFP (C) shows a northern blot analysis of the transcripts derived from the GFP-3′ UTR wt, Δ369, and Δ669 constructs in the HEK293 stable cell lines before (−) and after Tet induction (+). All constructs produce two major mRNA species because of the usage of different polyadenylation sites. In addition to these species, the GFP-3′ UTR Δ369 and Δ669 constructs also produce an aberrantly spliced mRNA isoform (Δ1812) that accounts for the production of the aberrant protein present in the western blots (B). Download figure Download PowerPoint In parallel, we performed northern blot analysis to confirm that autoregulation did not occur in the case of the Δ369 and Δ669 constructs at the mRNA level as expected. First of all, it should be noted that for all three constructs (wt, Δ369, and Δ669) we observed the presence of two major bands, which correctly mimic the endogenous mRNA species shown in Figures 2 and 5. In keeping with the results obtained with the endogenous TDP-43 transcript, Figure 4C shows that the two signals corresponding to the GFP-3′ UTR wt construct completely disappeared following Tet induction (this did not occur in the case of the control GFP used to normalize, see bottom panels). On the other hand, all the corresponding signals for the GFP-3′ UTR Δ369 and Δ669 constructs were progressively unaffected by Tet induction (note that these signals migrate lower than the GFP-3′ UTR wt transcript because of the substantial deletions in the TDPBR). In the deleted constructs, however, a new mRNA species is also visible (Δ1812). RT–PCR and sequencing analyses of this band found that it belongs to mRNA species resulting from alternative splicing of the last exon, similar to what occurs in the endogenous TDP-43 V2 form described in Figure 2A and Supplementary Figure 2. In the form Δ1812, exon and intron 7 were spliced out because of a deletion of the 5′ splice site of exon 7 in both Δ369 and Δ669 constructs (see lower scheme in Figure 4A). It is this band, labelled Δ1812 in Figure 4B, which accounted for the production of the short aberrant protein (the lower scheme in Figure 4A also shows the new coding frame). Figure 5.TDP-43 autoregulation is independent of NMD. (A) The existence of alternative splicing that activates the removal of two downstream introns (V2) was seen in the EST database. The shorter form V2 was seen to undergo NMD (Supplementary Figure 2), while northern blot analysis (B) of control and Tet-induced samples in the presence and absence of CHX treatment (50 μg/ml, for 3 h) did not detect the presence of V2. V2 should have a MW of 1.2 or 2.5 Kb depending on the poly A site usage. The probe was generated with the primers shown in (A) followed by purification of the product corresponding to V2. Download figure Download PowerPoint Collectively, these results show that the sequence important to downregulate the TDP-43 mRNA is made up by a collection of closely localized-binding sites in the TDPBR region. Next, we decided to also investigate the molecular mechanisms involved in this self-regulatory process. Nonsense-mediated decay On the basis of previous autoregulatory mechanisms described for other hnRNPs (Wollerton et al, 2004; Rossbach et al, 2009), we first considered alternative splicing modifications coupled to nonsense-mediated decay (NMD) as a possible mechanism to explain TDP-43 self-regulation. PCR did not show changes in the pattern of TDP-43 mRNAs after Tet induction (Supplementary Figure 2). These analyses, however, did indicate the existence of a lower molecular weight form referred to as V2 in both control and induced cells (Figure 5A and Supplementary Figure 2). Cycloheximide (CHX) treatment showed that only V2 would be subject to NMD. However, from a functional point of view, it should be noted that the V2 mRNA is present at very low levels, and is clearly visible only with PCR (see Supplementary Figure 2B) while not on northern blot analysis (Figure 5B). At the same time, the corresponding protein derived from this isoform is not found in significant amounts after inhibition of the NMD process in the presence of TG-TDP43 induction (data not shown). Thus, although we cannot rule out a role of V2 and NMD in the regulation of TDP-43 through alternative pathways or in different systems wherein this isoform may be expressed at higher levels, we conclude that it is unlikely to mediate TDP-43 self-regulation in our cell lines. Overexpression of TDP-43 promotes instability of its own transcript As a second possibility, we examined the stability of the endogenous TDP-43 transcript as a function of TG-TDP43 expression in the HEK293 cell line. We first established the minimum time of Tet induction that would allow us to adequately measure endogenous transcript levels (data not shown). A 6-h period of induction led to a 20–30% decrease in the levels of endogenous TDP-43 by quantitative PCR (qPCR). Cells were then treated with actinomycin D (ActD) at 6 h after Tet induction to block transcription and were collected at different time points, along with the control-treated cells. As shown in Figure 6A, TG-TDP43 expression led to a decrease in the half-life of the endogenous transcript as measured by qPCR. We confirmed that TG-TDP43 transcript levels also decreased on Act D treatment (data not shown) by measuring TG-TDP43 levels with specific primers at the different time points, confirming the block of transcription under our experimental conditions. More importantly, this control in
