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Journal of Molecular Endocrinology (2006) 37 251-257    DOI: 10.1677/jme.1.01914
© 2006 Society for Endocrinology
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PGC-1{alpha} regulates the isoform mRNA ratio of the alternatively spliced thyroid hormone receptor {alpha} transcript

D C Thijssen-Timmer, M Platvoet-Ter Schiphorst, J Kwakkel, R Emter1, A Kralli1, W M Wiersinga and O Bakker

Department of Endocrinology and Metabolism, Academic Medical Centre, F5-171, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
1 Department of Cell Biology, The Scripps Research Institute, MB111, Maildrop MB-24, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

(Requests for offprints should be addressed to O Bakker; Email: o.bakker{at}amc.uva.nl)


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Transcripts derived from the thyroid hormone receptor {alpha} (TR{alpha}) gene are alternatively spliced resulting in a functional receptor TR{alpha}1 and a non-T3-binding variant TR{alpha}2 that can exert a dominant negative effect on the transactivation functions of other TRs. There is evidence that the ratio of TR{alpha} isoform transcripts can be modulated and here, we investigate whether the PPAR{gamma} co-activator {alpha} (PGC-1{alpha}) has an effect on this splicing process. PGC-1{alpha} was discovered not only as a transcriptional co-activator, but also has certain motifs characteristic of splicing factors. We demonstrate that PGC-1{alpha} alters the ratio of endogenously expressed TR{alpha} isoform transcripts in HepG2 cells, by decreasing TR{alpha}1 mRNA levels twofold. This change in isoform ratio is accompanied by a decrease in 5'-deiodinase expression, whereas no differences were found in TRß1 expression. Deletion of the RNA-processing domain of PGC-1{alpha} abrogated the effect on the TR{alpha} splicing, whereas expression of only the RNA-processing domain favored TR{alpha}1 expression. PGC-1{alpha} showed a similar effect on the splicing of a TR{alpha} minigene containing only the last four exons and introns of the TR{alpha} gene. These data suggest that PGC-1{alpha} is involved in the RNA processing of TR{alpha} transcripts.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Thyroid hormone receptor {alpha} (TR{alpha}) has an important role in the regulation of physiological functions, such as the control of body temperature and heart rate (Wikstrom et al. 1998). The TR{alpha} gene encodes several variants due to alternative promoter usage and the existence of an alternative splice site in exon 9 (Laudet et al. 1991). TR{alpha}1 is a functional receptor and TR{alpha}2 is a non-T3-binding variant that results from the use of the alternative splice site. TR{alpha}2 can still bind to DNA and may have a dominant negative effect on the transactivation of the other TRs. The balance between TR{alpha}1 and TR{alpha}2 (TR{alpha}1:TR{alpha}2 ratio) is likely to be important for the sensitivity to thyroid hormone. Mice that lack TR{alpha}2, and as a result overexpress TR{alpha}1, show certain features of hyperthyroidism, such as increased heart rate, weight loss, and elevated body temperature, as well as some features of hypothyroidism, such as low serum thyroid hormone levels with an inappropriately normal thyroid-stimulating hormone concentration (Salto et al. 2001). The mixed expression of hyper- and hypothyroidism likely results from tissue-specific changes in thyroid hormone-responsiveness, which may reflect the different ratios of TR{alpha}1:TR{alpha}2 in different tissues of wild-type mice, and consequently tissue-specific increases in TR{alpha}1 signaling in the TR{alpha}2–/– mouse. Similarly, mice that lack all TR{alpha} isoforms show increased sensitivity to thyroid hormone, which can be contributed to the abrogation of the silencing effect of TR{alpha}2 in tissues expressing the TRß isoform (Macchia et al. 2001).

Several studies from our lab have provided evidence that the TR{alpha}1:TR{alpha}2 ratio is not constant and can be modulated by changes in the metabolic (or physiologic) state. For instance, when rats are subjected to fasting, the TR{alpha}2 mRNA levels in liver increase threefold, whereas no change in TR{alpha}1 is observed, resulting in a decrease in the TR{alpha}1:TR{alpha}2 ratio (Bakker et al. 1998). Similarly, HepG2 cells that are treated with pharmacological levels of T3 show a decrease in their endogenous TR{alpha}1:TR{alpha}2 ratio (Timmer et al. 2003). No correlation with the expression levels of known splicing factors has been observed and the mechanism behind the possible regulation of the splicing process remains unknown. Interestingly, both fasting and T3 induce the expression of the PPAR{gamma} co-activator {alpha}(PGC-1{alpha}) in liver (Herzig et al. 2001, Yoon et al. 2001, Weitzel et al. 2003). PGC-1{alpha} was originally identified not only as a transcriptional co-activator interacting with the nuclear receptor (NR) PPAR{gamma} but it also enhances the activity of many other NRs, including TR{alpha} (Puigserver et al. 1998, Knutti et al. 2000). In addition to enhancing transcription, PGC-1{alpha} is involved in RNA processing. It has been shown to alter the splicing of a minigene, depends on the presence of a binding site for an NR and on the putative RNA-processing motifs present in the C-terminus of PGC-1{alpha} (Monsalve et al. 2000). These motifs consist of an RNA recognition motif, which is homologous to domains found in known splicing factors, such as the serine-, arginine-rich (SR), and heterogeneous nuclear ribonucleoproteins (hnRNP) protein families, and two regions rich in serine–arginine pairs, which are characteristic for SR proteins. These findings suggest that PGC-1{alpha} can provide a molecular link between transcription and RNA processing. However, there is no evidence yet that PGC-1{alpha} regulates splicing of genes in their natural context.

We hypothesized that PGC-1{alpha} plays a role in the splicing process of TR{alpha} transcripts. To this end, we studied the effect of PGC-1{alpha} on the endogenously expressed TR{alpha} transcripts in HepG2 cells, as well as on a TR{alpha} minigene.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Materials

T3 was obtained from Sigma Chemical Co. and dissolved in 5 mM NaOH to a concentration of 1 mg/ml. It was further diluted in Eagle’s minimum essential medium (E/MEM) (Biowhittaker, Verviers, Belgium) and stored at –20 °C.

Plasmids

TR{alpha} was expressed from a minigene pCMV-erbAm that spans exons 7–10 of the rat TR{alpha} gene under the control of a CMV promoter and was a kind gift of Dr Munroe (Hastings et al. 2000). The expression plasmid for full-length human PGC-1{alpha} containing a hemagglutinin (HA) epitope-encoding sequence (pcDNA3 pBS/ HA-hPGC-1{alpha}) has been described (Knutti et al. 2000). The plasmid pBS/HA-hPGC-1{alpha} was used as a template for the generation of all PGC-1{alpha} deletion variants. To make SR/E/RRM (565–754), primers C8/1713-NdeI (TAC GCC GGT CAT ATG CGC TCT CGT TCA AGG TCC), and C8/2443-a (GAC TGA CTC GAG TTA CTT GCG TCC ACA AAA GTA C) were used to PCR amplify a DNA fragment encoding amino acids 565–754 of PGC-1{alpha}. The PCR-product was digested with NdeI and XhoI and used to replace the NdeI/XhoI fragment of pBS/HA-hPGC-1{alpha}. PGC-1{alpha} variant {Delta}SR/E/RRM was constructed by a two-step PCR method. In the first round, primer C8/1190 was used in combination with -SR/E/RRM-a (GAA AAA TTG CAT CCT TTG GGG TCT TTG) to amplify the 5'-flanking sequences of the sequences to be deleted. Primer T7 was used with primers – SR/E/RRM-s (CAA AGG ATG CAA TTT TTC AAG TCT AAC) to amplify the 3'-flanking sequences of the sequences to be deleted. Corresponding pairs of 5'- and 3'-flanking regions generated in the first round were used as a template for the second PCR-round with primers C8-1190 and T7. The generated PCR products were digested with XbaI and XhoI and used to replace the wild-type XbaI/XhoI fragment of pBS/HA-hPGC-1{alpha}. To confirm the presence of the right deletions and exclude unwanted mutations, all constructs were sequenced. For expression in the mammalian cells, the HA-tagged PGC-1{alpha} variants were subcloned as BamHI/NotI fragments into pcDNA3 to generate pcDNA3/HA-PGC-1{alpha}.{Delta}SR/E/RRM, and pcDNA3/HA-PGC-1{alpha}.SR/E/RRM. A pcDNA3 plasmid (Invitrogen) was used as empty control vector.

Cell culture and transient transfections

The human hepatoma cell line, HepG2, was obtained from the ATCC (#HB 8065, American Type Culture Collection, Rockville, MD, USA). Cells were cultured in Eagle’s medium supplemented with 10 U/ml penicillin/streptomycin/fungizone (p/s/f) and 5% fetal calf serum (all from Biowhittaker, Verviers, Belgium). Cells were seeded in six-well plates 18 h before transfection and reached approximately 60% confluence at the time of transfection. Per treatment (PGC-1{alpha}forms and/or T3) we performed six independent transfections. We used FuGENE (Roche Diagnostics) as transfection reagent at a 3:2 ratio. Standard amount of reporter and expression plasmids per transfection assay were: 1 µg pCMV-erbAm and 2 µg PGC-1{alpha}-wild type (WT) or 0.2 µg PGC-1{alpha} deletion variants or pcDNA3 (with a total amount of 3 µg DNA per cell). When cells were incubated with T3, medium was changed after 24 h to incubation medium with 5% fetal calf serum (FCS) and 10–7 M T3 or the dilutant NaOH in E/MEM.

RNA isolation and RT-PCR

After 48 h transfection, cells were lysed in 200 µl lysis buffer and RNA was isolated using the MagNaPure LC RNA Isolation Kit II (culture cells) in the MagNaPure LC Instrument (Roche Molecular Biochemicals). RNA was reverse transcribed into single-stranded cDNA using the First Strand cDNA synthesis kit with random primers (Roche Molecular Biochemicals).

Real-time PCR

Real-time PCR reactions were performed in a Light-Cycler (Roche Molecular Biochemicals). TR{alpha}1 and TR{alpha}2 were simultaneously detected in the same sample using sequence-specific hybridization probes and a LightCycler-FastStart DNA Master Hybridization Probes kit. Probes, primers, and program were as previously described (Bakker 2001). PGC-1{alpha}, 5'-deiodinase, GAPDH and total TR{alpha} were measured in a total reaction volume of 20 µl with 2µl cDNA using the LightCycler-DNA Master SYBR Green kit. The sequences of the primers of PGC-1{alpha} were as follows: 5'-GCA CCG AAA TTC TCC CTT GTA-3' (exon 9) and 5'-TTT GCT TGG CCC TCT CAG AC-3' (exon 10). The 5'-deiodinase mRNA was detected using the primer sequences 5'-AGC CAC GAC AAC TGG ATA CC-3' (forward) and 5'-ACT CCC AAA TGT TGC ACC TC- 3' (reverse) and the TRß1 mRNA using forward, 5'-AAG TGC CCA GAC CTT CCA AA-3' and reverse 5'-AAA GAA ACC CTT GCA GCC TTC- 3'. Primers for GAPDH were as described (Schreiber et al. 2003). For each mRNA assayed, a sequence-specific standard was generated and analyzed in the range of 0.1–1000 fg/20 µl in parallel to the samples. The crossing points of the standards with the noise band, which is set at the beginning of the log-linear phase, were plotted against the logarithm of the concentration and fitted to a standard curve. The concentration of cDNA of each gene was then calculated from its own standard curve and normalized to the amount of GAPDH in the sample. Minus-RT samples were tested in each experiment for GAPDH amplification to exclude genomic contamination. The individual efficiencies of the samples calculated using the LinReg program (Ramakers et al. 2003) were within 0.05 from the median efficiency of the run.

Protein extraction and analysis

The cells were washed with cold PBS, scraped in 2 ml PBS and centrifuged at 4 °C for 5 min at 3000 r.p.m. The pellet was resuspended in 100 µl 2 x loading buffer (2% SDS, 10% glycerol, 100 mM DTT, 60 mM Tris (pH 6.8) bromophenol blue) and pre-heated at 80 °C. Subsequently, 8 µl lysate was loaded onto a 10% SDS-PAGE gel. After blotting onto a PVDF membrane (Millipore, Brussels, Belgium), PGC-1{alpha} and its variants were detected by blocking for 60 min in PBS containing 1% casein hydrolysate at 37 °C followed by an incubation of 60 min with a mouse monoclonal antibody against the HA epitope (clone 3F10) conjugated with peroxidase in PBS. After washing three times, LumiLight Plus substrate was added and signals were visualized using a LumiImager (all reagents, Roche Molecular Biochemicals).

Statistical analysis

The balance between the mRNA levels of the two splicing variants, TR{alpha}1 and TR{alpha}2, is expressed as the TR{alpha}1:TR{alpha}2 ratio. The mean TR{alpha}1:TR{alpha}2 ratio in the control cells is always set at 1 and the TR{alpha}1:TR{alpha}2 ratio in the treated groups is calculated relative to control. Statistical differences between groups were calculated using a t-test (only when n = 4).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
T3 alters the ratio of endogenous TR{alpha} transcripts

As shown in Fig. 1Go, 10–7 M T3 decreases the ratio of the endogenous TR{alpha} isoform transcripts. This is the result of a decrease in the level of TR{alpha}1 (from 0.63 ± 0.09 to 0.41 ± 0.04, relative to GAPDH, P < 0.001), whereas no effect on the TR{alpha}2 levels was seen. However, T3 has no significant effect on the TR{alpha}1:TR{alpha}2 ratio derived from a transfected TR{alpha} minigene (pCMV-ErbA), which contains only the last four exons and introns of the TR{alpha} gene.


Figure 1
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  		Figure 1 The effect of T3 on the TR{alpha}1:TR{alpha}2 ratio of the endogenously expressed TR{alpha} transcripts in HepG2 cells, and of TR{alpha} transcripts that are derived from the pCMV-ErbA minigene transfected in HepG2 cells. Cells (six wells/group) were incubated for 24 h with 10–7 M T3 (white bars) or vehicle (black bars). Mean values ± S.D. (n = 6) are depicted, and differences between groups are calculated with a t-test.

 
PGC-1{alpha} modulates the ratio of the endogenous alternatively spliced TR{alpha} transcripts

We next investigated whether PGC-1{alpha} has an effect on the splicing direction of the endogenously expressed TR{alpha} transcripts in HepG2 cells. As shown in Fig. 2AGo, the TR{alpha}1:TR{alpha}2 ratio decreased in a dose-dependent manner in cells when PGC-1{alpha} was expressed. The decrease is mainly the result of a decrease in the TR{alpha}1 mRNA level (from 1.0 ± 0.32 finally to 0.42 ± 0.17, P < 0.01). No change in the total level of TR{alpha} mRNA was observed and PGC-1{alpha} mRNA levels rose by 50-fold as a result of the transfection (data not shown). The PGC-1-induced twofold decrease in the endogenous TR{alpha}1 expression levels is accompanied by a decrease in the expression of the 5'-deiodinase mRNA (Fig. 2BGo). No effect was seen on the expression of TRß1 (Fig. 2CGo).


Figure 2
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  		Figure 2 (A) The TR{alpha}1:TR{alpha}2 mRNA ratio derived from the endogenous TR{alpha} gene after transfection of increasing amounts of PGC-1{alpha} in HepG2 cells. Mean values ± S.D. (n = 6) are depicted, and differences between groups are calculated with a t-test. (B) The 5'-deiodinase mRNA expression levels after transfection of increasing amounts of PGC-1{alpha} in HepG2 cells. Mean values ± S.D. (n = 6) are depicted, and differences between groups are calculated with a t-test. (C) The TRß1 mRNA expression levels after transfection of increasing amounts of PGC-1{alpha} in HepG2 cells. Mean values ± S.D. (n = 6) are depicted, and differences between groups are calculated with a t-test.

 
The effect of PGC-1{alpha} deletion variants on the TR{alpha} transcript ratio

To determine if the effect of PGC-1{alpha} on the endogenous TR{alpha} isoform mRNA ratio depends on its putative RNA-processing domains, we studied two PGC-1{alpha} deletion variants (Fig. 3AGo). One variant lacks the putative RNA-processing domain ({Delta}SR/E/RRM) and the other variant only expresses the putative RNA-processing domain (565–754). Expression of PGC-1{alpha}-WT resulted in a decrease (P = 0.03) in the endogenous TR{alpha}1:TR{alpha}2 ratio when compared with the control cells (Fig. 3BGo). Transfection of PGC-1{alpha} {Delta}SR/E/RRM resulted in a TR{alpha}1:TR{alpha}2 ratio which was not statistically different from control. When cells were transfected with 565–754 a significant increase in the TR{alpha}1:TR{alpha}2 ratio (P = 0.01) was observed compared with control. The increase was a result of an increase of TR{alpha}1 (1.2 ± 0.4 to 2.8 ± 0.6, P < 0.05). There was no difference in the total TR{alpha} mRNA expression between groups (data not shown). Next, we studied the effect of PGC-1{alpha}-WT or deletion variants {Delta}SR/E/RRM and 565–754 on the TR{alpha}1:TR{alpha}2 mRNA ratio when transcripts derived from a TR{alpha} minigene. Again, the TR{alpha}1:TR{alpha}2 ratio decreased significantly as a result of co-transfection of PGC-1{alpha}-WT when compared to control (P < 0.01, Fig. 3CGo). Co-transfection of {Delta}SR/ E/RRM resulted in a TR{alpha}1:TR{alpha}2 ratio that was not significantly different from control. However, when 565–754 was co-transfected, a significant decrease compared to control was observed in the TR{alpha}1:TR{alpha}2 ratio. The decrease in the ratio is a result of a decrease in TR{alpha}1 (7.6 ± 0.8 to 4.9 ± 0.3, P < 0.01) and a modest though not significant change in the TR{alpha}2 level was seen. Expression of PGC-1{alpha} or its variants did not result in a difference in the total TR{alpha} mRNA expression compared to control (data not shown). We confirmed the presence of PGC-1{alpha} proteins by western blotting with an anti-HA antibody. The triplet seen in the case of the SR/E/RRM region of PGC-1{alpha} is most probably the result of post-translational modifications.


Figure 3
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  		Figure 3 (A) Schematic representation of the structure of PGC-1{alpha} WT and deletion variants. PGC-1{alpha} {Delta}SR/E/RRM lacks the putative RNA-processing domain, whereas PGC-1{alpha} 565–754 only expresses this domain. (B) The endogenous TR{alpha}1:TR{alpha}2 ratio after transfection of PGC-1{alpha} WT or variants. (C) The TR{alpha}1:TR{alpha}2 ratio transfected with 1 µg pCMV-ErbA and co-transfected with PGC-1{alpha} WT or variants. Below the graph, the variant PGC-1{alpha} proteins are shown in duplicate, run on an SDS-PAGE gel and detected with anti-HA antibodies. Mean values ± S.D. (n = 6–8) are depicted, and differences between groups are calculated with a t-test.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In a previous study, we have shown that pharmacological levels of T3 can change the splicing direction of TR{alpha} transcripts towards TR{alpha}2 (Timmer et al. 2003). Now we show that, in contrast to the effect on the endogenously expressed TR{alpha} transcripts, T3 does not exert a significant effect on the isoform mRNA levels derived from a TR{alpha} minigene. This minigene contains the last four exons and introns of the TR{alpha} gene and thus lacks the natural promoter sequence. This could indicate that the effect of T3 is promoter dependent. Although the TR{alpha} promoter does not appear to contain a known thyroid hormone response element (TRE), it is a target of the estrogen-related receptor {alpha} (ERR{alpha}; Laudet et al. 1993, Vanacker et al. 1998). Interestingly, the expression and activity of ERR{alpha} are regulated by the transcriptional co-activator PGC-1{alpha}, which in turn is induced by T3 (Schreiber et al. 2003, Weitzel et al. 2003). Since PGC-1{alpha} contains putative RNA-processing motifs and has been shown to affect the splicing of a fibronectin minigene (Monsalve et al. 2000), we hypothesized that PGC-1{alpha} might have an effect on the ratio of TR{alpha} isoform transcripts. Indeed, in this study, we provide the first evidence that PGC-1{alpha} has an effect on the mRNA levels of isoforms that result from the alternative splicing of the endogenously expressed TR{alpha} gene, by favoring the production of TR{alpha}2 over TR{alpha}1 transcripts.

We have recently shown that the expression of the TR isoforms in liver is zonal with TRß1 expressed in a narrow layer of cells around the central vein (Zandieh-Doulabi et al. 2002). Similarly, TR{alpha}1 expression is located around the central vein but extends further towards the portal vein (Zandieh-Doulabi et al. 2003). In the first study, it was also shown that 5'-deiodinase is expressed in a zonal fashion overlapping with, but broader, than TRß1 (Zandieh-Doulabi et al. 2002), suggesting a possible involvement of TR{alpha}1 in 5'-deiodinase regulation as well. Indeed, data derived from mice lacking either TRß or TR{alpha}1 indicate that liver 5'-deiodinase expression, although mainly dependent on the predominant isoform TRß, is also influenced by TR{alpha}1 (Amma et al. 2001). We, therefore, selected 5'-deiodinase, which was recently demonstrated to show a marked responsiveness to T3 (Zavacki et al. 2005), to see whether the effects of PGC-1 on TR{alpha}1 levels resulted in an effect on this gene. Our finding that the PGC-1-induced lowering of TR{alpha}1 levels is associated with a decrease in 5'-deiodinase, supports the idea that TR{alpha}1 plays a role in 5'-deiodinase expression.

A PGC-1{alpha} construct that lacks the C-terminal domain containing the RNA-processing motifs has no effect, indicating that the C-terminal domain is necessary. This agrees with the findings of Monsalve et al.(2000), who have shown that the effect of PGC-1{alpha} on the splicing of a fibronectin minigene depends on the RNA-processing domain (Monsalve et al. 2000). Furthermore, Monsalve et al.(2000) have shown that the effect of PGC-1{alpha} on splicing is promoter dependent. We find an effect of PGC-1{alpha} not only on the TR{alpha} isoform ratio transcribed from the endogenous promoter, but also on the TR{alpha} isoform ratio expressed from the minigene, which may indicate that PGC-1{alpha} acts by loading on to the CMV promoter. Another possibility is that PGC-1{alpha} may bind to an alternative promoter, which is present in intron 7 of the TR{alpha} gene and included in the minigene. This promoter has a consensus binding site for the glucocorticoid receptor and thus a potential site for PGC-1{alpha}, and is responsible for the transcription of the N-terminally truncated TR{alpha} transcripts {Delta}TR{alpha}1 and {Delta}TR{alpha}2 that have been detected in several mouse tissues and embryonic stem (ES) cells (Chassande et al. 1997).

When we tested a construct expressing only the PGC-1{alpha} RNA-processing domain (565–754) opposite effects were observed depending on the experimental setup. The RNA-processing domain acted on the endogenously expressed TR{alpha} transcripts by increasing the levels of TR{alpha}1, whereas in the presence of the TR{alpha} minigene, it decreased the TR{alpha}1 levels. A possible explanation for this context-dependent effect could be the RRM motif and the arginine–serine regions located in the RNA-processing domain of PGC-1{alpha} which are contained in the 565–754 construct. These motifs probably mediate the physical association and localization with splicing factors in the so-called nuclear speckles. The PGC-1{alpha} arginine–serine region is reminiscent of that found in the SR proteins, which are required for general splicing, but which can also regulate alternative splicing by promoting the use of the proximal 5'-splice site (Ge & Manley 1990, Krainer et al. 1990). This effect on alternative splicing is counteracted by proteins of the hnRNP family, which promote the use of the distal 5'-splice site instead (Mayeda & Krainer 1992). In the case of the TR{alpha} transcript, use of the proximal 5'-splice site (which is in fact the poly-A addition site) would lead to TR{alpha}1, whereas the use of the distal 5'-splice site leads to TR{alpha}2. If the alternative splicing direction of TR{alpha} pre-mRNA indeed depends on an equilibrium between the levels of pre-mRNA and splicing proteins from the SR and hnRNP families, the direction could change when either one protein family or the pre-mRNA itself is more abundantly expressed. Therefore, in the case of the endogenous gene where there is not much pre-mRNA but a lot of (SR-like) PGC-1{alpha} the proximal site is used (TR{alpha}1). On the other hand, when there is more pre-mRNA (transfected minigene), the protein/RNA ratio changes and the proximal site loses its supremacy resulting in less TR{alpha}1. This does not happen in the case of the wtPGC-1{alpha}; this could be because in this case a promoter-dependent process is involved as well, coupling transcription and RNA processing. Thus, the effects of PGC-1{alpha} on the endogenous TR{alpha} gene could result in recruitment of splice factors by a PGC-1{alpha} domain other than or additionally to the RRM.

In conclusion, PGC-1{alpha} has an effect on the formation of endogenous TR{alpha} transcripts resulting in a decreased TR{alpha}1:TR{alpha}2 ratio. In addition to the studies on the endogenously expressed TR{alpha}s, we have used a TR{alpha} minigene (which does not contain the natural TR{alpha} promoter) and shown that PGC-1{alpha} decreases the TR{alpha}1:TR{alpha}2 ratio of this minigene as well. Therefore, the TR{alpha} minigene is probably a suitable model to help to distinguish promoter-dependent from promoter-independent effects of PGC-1{alpha} on splicing.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received in final form 30 May 2006
Accepted 7 June 2006




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