The cellular fate of mutant α1(S326fs328X) subunits was studied using a minigene strategy
The minigene strategy has been used to study transcript alteration and NMD in a number of diseases, including cystic fibrosis, since it contains introns that support mRNA editing and splicing. We thus used this approach to study the intracellular processing of human wild-type and mutant α1 subunits. The α1 subunit mutation, 975delC, S326fs328X, is located in the middle of TM3, and the PTC produced by the deletion would result in a loss of the C-terminal 141 amino acids, thus producing a truncated α1(S326fs328X) subunit (). The human α1 subunit gene, GABRA1, has 9 exons and 8 introns. Minigenes were constructed by including the entire GABRA1 intron 8 (1242 bp) between exon 8 and 9 of the wild-type and mutant α1 subunit cDNA constructs (). The stop codon for the wild-type α1 subunit minigene is located at the end of exon 9 (), and the PTC for the mutant α1 subunit minigene α1(S326fs328X) subunit minigene is located in exon 8, which is 128 bp downstream from intron 7 and 74 bp upstream from intron 8 (). We also generated an α1(A322D) subunit minigene as a control (). The α1 subunit mutation, A322D, associated with juvenile myoclonic epilepsy, is a missense mutation four residues upstream of the 975delC, S326fs328X frameshift mutation and should not elicit NMD. In addition, we generated an artificial α1 subunit nonsense mutation, Y368X, in the middle of the last exon (exon 9) as an NMD-insensitive control ().
A minigene strategy was used to study the α1 subunit mutation, 975delC, S326fs328X
In HEK 293T cells, wild-type and mutant α1(S326fs328X) subunit minigenes were cotransfected with β2 and γ2 subunit cDNAs. In this and all other experiments, the Calcium transfection technique was used unless otherwise noted. Total RNAs were extracted 48 hrs after transfection, and equal amounts of total RNA were transcribed to cDNA. With flanking primers in exon 8 and 9, equal amounts of cDNAs from wild-type and mutant α1 subunit minigenes were used for a template in the PCR amplification, and both minigenes displayed a band at 260 bp, suggesting correct splicing in the reverse transcribed cDNA products from α1 and α1(S326fs328X) subunit minigene mRNAs (). The mutant α1 subunit minigene band integrated density values (IDVs), however, were only about ~20% of the wild-type α1 subunit minigene band IDVs, suggesting reduction of mutant transcript levels.
Expression of the NMD competent α1(S326fs328X) subunit minigenes in heterologous cells and rat cortical neurons produced reduced mutant protein
It is unknown how robust the endogenous NMD machinery is in different cell types, and if NMD in a given cell type can degrade mRNAs equally when transfection methods with different efficiencies are employed. We thus first used the less efficient Calcium transfection technique and then the more efficient Fugene transfection technique (see Methods). With Calcium transfection, we cotransfected HEK 293T cells with β2 and γ2S subunit and wild-type and/or mutant α1 subunit cDNAs (; left) or minigenes (; middle) at a cDNA ratio of 1:1:1 for wild-type α1 subunits and mutant α1(S326fs328X) subunits and at a cDNA ratio of 1:1:0.5:0.5 for mixed α1/α1(S326fs328X) subunits. Wild-type α1 subunits should migrate at about 50 KDa, and mutant α1(S326fs328X) subunits should migrate at about 37 KDa (; left, green arrows). Our data are consistent with the original report that mutant α1(S326fs328X) subunit protein was reduced in both mixed and mutant conditions, suggesting that the mutant protein was degraded (; left) (0.328 ± 0.063 mutant vs 0.765 ± 0.082 wt; p = 0.002). By flow cytometry, the total amount of mutant protein was less than 3% of wild-type protein (Supplementary Figure 1
). Interestingly, the ratio of wild-type to mutant protein with mixed expression was larger than the ratio of wild-type to mutant protein with wt and mut expression, suggesting that the presence of the wild-type α1 subunit or a failure to compete with the wild-type subunit during assembly somehow facilitated degradation of the mutant protein.
Expression of the NMD competent α1(S326fs328X) subunit minigene produced minimal mutant protein
Second, HEK 293T cells were cotransfected with β2 and γ2S subunit cDNAs and wildtype and mutant α1 subunit minigenes (wt, cDNA ratio of 1:1:1), mixed α1/α1(S326fs328X) subunit minigenes (mix, cDNA ratio of 1;1:0.5:0.5 or mutant α1(S326fs328X) subunit minigene (cDNA ratio of 1:1:1) (; middle). The wild-type α1 subunit minigene displayed a robust 50 KDa band in both wild-type and mixed conditions. With mixed or mutant expression of the minigene constructs, however, mutant protein (37 KDa) was substantially reduced (; middle, green arrows) (; middle), suggesting an additional mechanism for loss of mutant protein.
Finally, HEK 293T cells were cotransfected with β2 and γ2S subunit cDNAs and the wild-type α1 subunit minigene (wt, cDNA ratio of 1:1:1), mixed α1/α1(Y368X) subunit minigenes (mix, cDNA ratio of 1:1:0.5:0.5) or mutant α1(Y368X) subunit minigene (cDNA ratio of 1:1:1) minigenes (; right). Expression of the NMD-insensitive α1(Y368X) subunit minigene in both mixed and mutant conditions produced truncated proteins but at reduced levels that migrated at the predicted molecular mass of 43 KDa (; right).
We next extended our study to include other cell types including HeLa cells and rat cortical neurons. Since these cells are less efficiently transfected, we employed the more efficient Fugene transfection technique, and included Fugene transfection of HEK 293T cells for comparison. In HEK 293T cells with Fugene transfection of wild-type and/or mutant α1 subunit minigenes, mutant α1(S326fs328X) subunit protein was present, although substantially reduced compared to wild-type subunit protein in both mixed and mutant conditions (; left), despite being almost absent with Calcium transfection (; middle). However, the mutant protein was not detectable with Fugene transfection in either HeLa cells (; middle) or rat cortical neurons (; right), despite robust expression of wild-type α1 subunit protein.
Consistent with the biochemical data, similar data were obtained with immunocytochemistry and confocal microscopy for total human α1 subunit protein expression (). In rat cortical neurons expressing wild-type α1β2γ2S subunits six days following transfection of β2 and γ2S subunit cDNAs and wild-type α1 subunit minigenes (; left), there was robust signal in both the cell bodies and dendrites, but in neurons expressing α1(S326fs328X)β2γ2S subunits, there was only a minimal fluorescent signal in the soma (; middle). In contrast, a significant amount of fluorescence was detected in the cell somata of neurons expressing α1(Y368X)β2γ2S subunits (; right). The levels of mutant relative to wild-type subunit protein seemed to vary somewhat with different transfection reagents, different cell types and the presence or absence of the β2 and γ2 subunits (Supplementary Figure 1
). It is likely that the more efficient transfection technique (Fugene) and use of efficiently transfected cell types (HEK 293T cells) were able to partially overwhelm the NMD machinery and ER quality control mechanisms due to high minigene copy number. Nonetheless, reduction of mutant protein following minigene expression occurred with all transfection techniques and in all cell types, suggesting common and consistent pathways for loss of mutant protein, likely NMD and ERAD.
Peak current amplitudes and surface α1 subunit levels were reduced with mixed expression of α1/α1(S326fs328X)β2γ2S subunits
We recorded currents evoked by 6 () or 28 () sec applications of 1 mM GABA from HEK 293T cells cotransfected with β2 and γ2S subunit cDNAs and the wild-type α1 subunit minigene (1:1:1 cDNA ratio; wt, black), half the amount of wild-type α1 subunit minigene as a haploinsufficiency control (1:1:0.5 cDNA ratio; hc, silver), mixed α1/α1(S326fs328X) subunit minigenes (1:1:0.5:0.5 cDNA ratio; mix, green) or mutant α1(S326fs328X) subunit minigene (1:1:1 cDNA ratio; mut, gray). Peak haploinsufficiency control and mixed receptor currents were smaller than wild-type currents, and there was no significant difference between them (p = 0.65) (). No currents were recorded from cells following mutant subunit expression. Compared with wild-type type peak currents (2490 ± 189.3 pA,), peak amplitudes of both haploinsufficiency control (1461 ± 294.7 pA; p = 0.01) and mixed receptor currents (1299 ± 158.2 pA; p = 0.0004) were significantly reduced () without change in macroscopic kinetic properties. When normalized to the peak wild-type receptor current, there was no difference in the steady state/peak current ratio between wild-type and haploinsufficiency control or wild-type and the receptor from mixed expression of wild-type and mutant receptors (), suggesting that the functional receptors on the cell surface were likely wild-type receptors.
Mixed α1/α1(S326fs328X)β2γ2S receptors had reduced peak current amplitudes and reduced α1 subunit surface expression
Results from cell surface receptor biotinylation and immunoblotting were consistent with the electrophysiological results. HEK 293T cells were cotransfected with β2 and γ2S subunit cDNAs and the wild-type α1 subunit minigene (1:1:1 cDNA ratio; wt), half the amount of wild-type α1 subunit minigene as a haploinsufficiency control (1:1:0.5 cDNA ratio; hc), mixed α1/α1(S326fs328X) subunit minigenes (1:1:0.5:0.5 cDNA ratio; mix) or mutant α1(S326fs328X) subunit minigene (1:1:1 cDNA ratio; mut) (). Compared with transfection of wild-type subunits, transfection of haploinsufficiency control or mixed subunits resulted in reduced wild-type α1 subunit surface levels. There was no mutant α1(S326fs328X) subunit on the surface with either mixed or mutant expression. When normalized to wild-type α1 subunit surface levels, α1 subunit levels were reduced with expression of either haploinsufficiency control (0.64 ± 0.04, p<0.0001) or mixed (0.62 ± 0.03, p<0.0001) subunits, but there was no significant difference in α1 subunit surface levels between the haploinsufficiency control and mixed subunits (p = 0.67) (). A similar pattern of surface α1 subunit protein expression was obtained by a more quantitative flow cytometry (Supplementary Figure 2A, B
Mutant α1(S326fs328X) subunits were not significantly “rescued” by proteasome inhibition, and mutant protein expressed was subject to ERAD
The mutant α1(S326fs328X) subunit that was detected was likely retained in the ER and degraded. To confirm this, following coexpression of β2 and γ2S subunits with either wild-type α1 subunit minigenes or mutant α1(S326fs328X) subunit cDNAs, total lysates from HEK 293T cells were either undigested (U) or digested with endoglycosidase H (Endo-H) () or peptide-N-glycosidase F (PNGase-F) (). Since only small amounts of protein were produced with the mutant minigene expression, the mutant cDNA construct was used for this experiment. Endo-H removes N-linked high-mannose oligosaccharides attached in the ER, but not complex oligosaccharides attached in the trans-Golgi region. In contrast, PNGase-F removes N-linked oligosaccharides attached in both ER and trans-Golgi region. Therefore, protein retained inside the ER should have similar molecular mass with either Endo-H or PNGase-F digestion. Wild-type α1 subunits migrated at a higher molecular mass after Endo-H digestion and migrated at a lower molecular mass after PNGase-F digestion as we previously described (Gallagher et al., 2005
). The mutant protein, however, migrated at a similar molecular mass after either Endo-H or PNGase-F digestion. The different shifts in molecular mass of wild-type and mutant α1 subunits after Endo-H or PNGase-F digestion suggested that the majority of the wild-type α1 subunits were trafficked beyond the ER, and that the mutant subunits were retained in the ER.
The truncated mutant α1(S326fs328X) subunit protein was subject to ERAD
Loss of mutant protein could have been due to rapid degradation after protein synthesis, NMD of the PTC-containing transcripts before protein synthesis or both. If the mutant protein was mainly subject to ERAD after synthesis, proteasomal inhibition should “rescue” the mutant protein. Therefore, we applied lactacystin (Lac, 10 μM) and MG-132 (3 μM) (data not shown), two structurally different proteasome inhibitors, for 6 hours to HEK 293T cells cotransfected with β2 and γ2S subunit cDNAs and wild-type, haploinsufficiency control, mixed or mutant α1/α1(S326fs328X) subunit minigenes (). With either Lac or MG-132 (data not shown) treatment, the total mutant α1(S326fs328X) subunit proteins from both less efficient Calcium or more efficient Fugene transfections were increased, but still minimal, compared to the wild-type subunit (). Relative to the wild-type α1 subunits, the magnitude of the total protein reduction in the haploinsufficiency control, mixed or mutant condition was greater with Calcium transfection ( top) than with Fugene transfection ( bottom) but was significantly reduced with both transfection techniques (). The mutant α1(S326fs328X) subunit protein, but not wild-type, haploinsufficiency control or mixed α1 subunit proteins, were increased by lactacystin treatment when normalized to the subunits with lactacystin treatment, suggesting enhanced degradation of the mutant subunit (). There was no difference between the ratio of the total protein of the mixed and wild-type subunit proteins before or after lactacystin, probably because the mutant protein was minimal compared to the wild-type protein in the mixed condition ( Fugene; ), thus obscuring any increase of the mutant subunit. The NMD-insensitive control α1(Y368X) subunit proteins were all significantly increased by both proteasomal inhibitors (Supplementary Figure 3
). The data suggest that the loss of the mutant α1(S326fs328X) subunit was mainly due to NMD while the loss of α1(Y368X) subunit was mainly due to ERAD. The mutant α1(S326fs328X) subunit, however, was partially rescued by blocking protein degradation, thus suggesting that any mutant subunit produced would be subject to degradation by ERAD.
We then used intronless cDNA constructs, which would not be subject to NMD, and Fugene transfection to produce more mutant protein (). Mutant α1(S326fs328X) subunit levels in both HEK 293T and HeLa cells were reduced compared to wild-typeα1 subunit levels but were increased by proteasomal inhibition to a greater extent than wild-typeα1 subunits (1.24 ± 0.12 vs 1.99 ± 0.11, p = 0.0007 for HEK 293T cells; 1.66 ± 0.20 vs 2.36 ± 0.23%, p = 0.046 for HeLa cells) (). The increased production of mutant α1(S326fs328X) subunit protein with transfection of intronless cDNA constructs and with Fugene transfection of minigene constructs () also suggested that any mutant protein produced would be subject to rapid degradation by ERAD through the ubiquitin-proteasome system.
Mutant α1(S326fs328X) subunits were degraded faster than the wild-type α1 subunits
To further investigate the cellular fate of the mutant protein, we performed 35S methionine radio-labeling pulse-chase experiment to determine if the mutant protein had an altered rate of degradation. We used HEK 293T cells since they had highly efficient protein expression, which produced higher levels of mutant subunit. The cells were transfected with either wild-typeα1 or mutant α1(S326fs328X) subunits alone with Fugene and then used for study after 48 hours. After radionuclide labeling for 20 min, the cells were lysed immediately and analyzed as time 0 or as “chased” for 30, 60, 90, 120, 180 and 240 min (). Compared with the amount of protein at time 0, the remaining amounts of wild-type and mutant subunit protein were unchanged at 30 min, but the mutant subunit degraded faster than wild-type subunit from 60 min to 240 min. From 60 min to 240 min of chase, the mutant subunit was significantly reduced compared to wild-type α1 subunit. The half life of the mutant subunit was 64 min, and the half life of wild-typeα1 subunits was 194 min, which was more than 3 fold higher than that of the mutant subunit. To exclude the possibility that the increased mutant protein after proteasomal inhibition was due to increased synthesis, we applied cycloheximide (CHX) (100 μg/ml) to block protein synthesis (data not shown) and found that the mutant protein was degraded more rapidly either with or without CHX treatment.
Mutant α1(S326fs328X) subunit protein had a reduced half life
The reduced mutant protein could also be due to the reduced synthesis. To determine the rate of synthesis of wild-type and mutant subunits, we pulse-labeled cells transfected with α1FLAG or α1(S326fs328X)FLAG subunits with 35S methionine for 0, 5, 10, 15 and 20 min. We used intronless α1 subunit cDNA constructs since they were used for degradation pulse chase experiments above. Normalized to the total wild-type or mutant protein labeled for 20 min, there were no significant differences in the amount of wild-type or mutant α1 subunits, demonstrating no difference in the rate of synthesis of these subunits.
Mutant α1(S326fs328X) subunits had an increased association with the chaperone Bip
Molecular chaperones are a group of proteins that mediate protein folding, assembly, trafficking and degradation (Hartl, 1996
). Our data above suggested that the mutant protein was subject to ERAD, which is known to be mediated by chaperones. We thus tested the association of the mutant protein with Bip, which is an ER luminal chaperone that binds misfolded or unfolded, unassembled secretory proteins and ensures proper maturation and movement of proteins from the ER to the Golgi apparatus (Connolly et al., 1996
). The cells were transfected with either wild-type α1FLAG
or mutant α1(S326fs328X)FLAG
subunits alone with Fugene and studied after 48 hours. The total lysates were purified with FLAG antibody conjugated beads and probed with either anti-FLAG antibody for α1FLAG
subunits or anti-Bip antibody (). The ratio of Bip to α1FLAG
subunit IDV was then determined. Consistent with the accelerated degradation of the mutant subunit, there was less mutant than wild-type subunit protein. Also, more Bip protein was pulled down with the mutant subunit compared to the wild-type subunit. Thus the ratio of Bip to mutant subunit was enhanced substantially (7 fold) compared to the wild-type subunit (wt 0.12 ± 0.011 vs mut 0.8430 ± 0.053) (P<0.001, n = 4), consistent with an increased association of Bip with the mutant subunit.
Mutant α1(S326fs328X) subunit protein had increased association with the ER resident chaperone Bip
Mutant α1(S326fs328X) subunit expression was increased by ribosomal or hUPF-1 inhibition, suggesting that loss of mutant protein was due also to NMD
Was loss of mutant α1(S326fs328X) subunit due also to loss of mRNA triggered by NMD? To determine this, we first quantified wild-type and mutant α1 subunit mRNAs in HeLa and HEK 293T cells and rat cortical neurons transfected with wild-type α1 and mutant α1(S326fs328X) subunit minigenes. Total RNAs were extracted 2–4 days after transfection, and mRNAs were analyzed by Real-time PCR. In HEK 293T and HeLa cells and in neurons, mutant α1(S326fs328X) transcripts were substantially reduced (). When normalized to wild-type α1 subunit mRNA levels, mutant α1(S326fs328X) subunit mRNA levels were reduced to 39.1 ± 7.3% (p = 0.0001) in HEK 293T cells, to 16.6 ± 4% (p = 0.006) in rat cortical neurons and to 23.8 ± 3.6% (p < 0.0001) in HeLa cells.
Mutant α1(S326fs328X) subunit mRNA was reduced and reversed by ribosome inhibition or by silencing the NMD essential factor hUpf-1
Treatment with the protein synthesis inhibitor cycloheximide (CHX) or silencing of the core component of the NMD machinery, Upf-1 (also known as Rent1), are widely used to determine if transcripts are subject to NMD (Montfort et al., 2006
). CHX is an indirect NMD inhibitor since NMD activation is post-transcriptional, but translation-dependent, and ribosomal association is required during the pioneer round of translation. Upf-1 silencing is believed to produce direct suppression of NMD. The RNA helicase, Upf-1, is a central effector of NMD that links the translation-termination event to the assembly of a surveillance complex. Upf2 and Upf3 are believed to recruit and activate Upf1 on NMD substrates (Lykke-Andersen et al., 2000
). Although the discrimination of PTCs from normal termination codons and the molecular links that trigger NMD are still unclear, it is well established that the core components of the NMD machinery are the conserved proteins, Upf-1, Upf-2 and Upf-3. PTC-containing mRNAs marked by the Upf complex are finally degraded by decay factors (Conti and Izaurralde, 2005
). A recent study reported that Upf-1 phosphorylation triggers translation repression, which is a key step preceding mRNA decay (Isken et al., 2008
). With CHX (100 μg/ml) treatment for 6 hrs, mutant α1(S326fs328X) subunit mRNA transcripts were increased in HeLa cells from 27.4% ± 5.2% to 84.5% ± 8.5% of wild-type α1 subunit mRNA after CHX treatment (). Addition of CHX reversed the down-regulation of the mutant transcripts, having no discernible effect on wild-type mRNA transcripts ().
We then investigated the role of Upf-1 in reducing α1(S326fs328X) subunit levels. We transfected cells with synthetic short-interfering RNA (siRNA) duplexes targeting the human Upf-1 (hUpf-1) gene (Chan et al., 2007
;Mendell et al., 2002
) and harvested the cells 48 hrs after transfection. Western blotting of whole cell lysates demonstrated significant loss of hUpf-1 protein in cells treated with hUpf-1 siRNA in HEK 293T (from 50 to 100 nM) and HeLa cells (from 10 to 100 nM) (). Treatment with 100 nM siRNA for either 2 or 4 days significantly reduced hUpf-1 protein levels in both HEK 293T and HeLa cells, but the siRNA appeared to be more effective in HeLa cells at both time points (). We thus used HeLa cells in these experiments. A similar effect, but to a lesser magnitude, was observed with HEK 293T cells (Supplementary Figure 4
Wild-type or mutant α1 subunit minigene constructs were transfected into HeLa cells treated 48 hr earlier with siRNA. The cells were harvested after an additional 48 hr for protein () and transcript () assays. All results were normalized to the cells treated only with siRNA delivery reagents (mock). Cells treated with siRNA (100 nM) against hUpf-1 (Upf) had increased expression of the mutant α1(S326fs328X) subunit compared with cells treated with the negative control (neg) siRNA () in both HEK 293T cells (Supplementary Figure 4
) and HeLa cells (0.80 ± 0.35 vs 2.48 ± 0.45; p = 0.006). Cells treated with lactacystin, in addition to the hUpf-1 specific siRNA, had increased expression of the mutant protein compared to cells treated with lactacystin alone in both cell lines (3.93 ± 0.83 (Upf + lac) vs 1.46 ± 0.20 (lac) (p = 0.013). There was no significant change in expression of wild-typeα1 subunit proteins () with hUpf-1 specific siRNAs treatment (100 nM). In contrast, the mutant α1(A322D) subunit protein was similarly increased when treated with lactacystin in addition to the hUpf-1 specific siRNA or with lactacystin alone (). The data suggest that the α1(S326fs328X) subunit protein was reduced mainly due to mRNA degradation, and that the α1(A322D) subunit protein was reduced mainly due to protein degradation by ERAD (Gallagher et al., 2007
Similarly, normalized to mRNA levels in cells expressing wild-typeα1 subunits with mock treatment, expression of mutant α1(S326fs328X) subunit transcripts was increased in cells treated with siRNA against hUpf-1 () compared to expression of mutantα1(S326fs328X) subunit transcripts in cells treated with negative control siRNA (11.3 ± 5 vs 91.4 ± 12; p = 0.002) in HeLa cells. There was no significant change in the wild-type (data not shown) or α1(A322D) (p = 0.28) subunit transcripts after hUpf-1 siRNA treatment (). Taken together, our data suggest that elimination of the mutant α1(S326fs328X) subunit was due primarily to loss of transcripts due to mRNA surveillance (NMD), but that ERAD also played a role in reducing mutant protein levels.
Differential molecular mechanisms underlying two GABRA1 epilepsy mutations, 975delC, S326fs328X) and A322D
Although both GABRA1 epilepsy mutations, A322D and 975delC, S326fs328X, are located in the same domain of the α1 subunit (TM3) and both mutations produced haploinsufficiency, the molecular bases for the functional consequences are different. The α1(A322D) subunit mutation is a missense mutation, and the mutant mRNAs are translated and produce an unstable mutant protein, which is subject to rapid ERAD. The α1 subunit mutation, 975delC, S326fs328X, is a PTC-generating mutation and activated NMD. However, NMD is not complete; it degrades the majority of the mutant mRNA, but a small portion of mutant mRNA escapes NMD. The surviving mutant mRNA would result in translation of a mutant, truncated protein, which is not stable and would be subject to ERAD like the other mutant α1(A322D) subunit. The loss of mutant message and protein from both molecular pathways would directly contribute to the loss of the functional α1 subunit-containing GABAA receptors, and thus, the loss of inhibition that would result in epilepsy ().
Different molecular pathways contribute to epileptogenesis of a missense and a frameshift PTC-generating epilepsy GABRA1 gene mutation