1. Figure 1. Identification of senataxin-interacting proteins by mass spectrometry. (A) Flow diagram showing the methodology used for identifying and confirming senataxin-interacting proteins. (B) Following SDS–PAGE on a 10% gel, senataxin IPs were silver stained and bands 1–3 were excised, trypsin-digested and analyzed by MS. Excised bands are indicated by the arrows. (C) Regions on senataxin for which nine overlapping GST fragments were generated. The helicase domain of senataxin is shown. (D) Following separation on a 12% SDS–PAGE, senataxin GST pull-downs were stained and multiple bands were excised, trypsin-digested and analyzed by mass spectrometry. Bands (1–13) correspond to proteins involved in RNA metabolism that were identified by mass spectrometry. C2ABR and C3ABR, control lymphoblastoid cells; Ig, pre-immune sheep sera (negative control); Ab-1, senataxin Ab-1.
From: Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation
Hum Mol Genet. 2009;18(18): doi: /hmg/ddp278
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2. Figure 2. Confirmation of senataxin interactions by co-IPs and GST pull-down assays. (A) Confirmation of the interaction between senataxin and SAP155. Senataxin was immunoprecipitated with Ab-1 and binding of SAP155 was determined by immunoblotting using a SAP155 specific antibody. (B) Senataxin interacts with nucleolin. Similarly, senataxin was immunoprecipitated with Ab-1 and binding of nucleolin was determined by immunoblotting with an anti-nucleolin antibody. (C) Mapping the senataxin binding region on nucleolin. Senataxin binds to the N-terminal region of nucleolin (N1 and N2). Coomassie stain shows equivalent loading of the GSTs. (D) Confirmation of the interaction between senataxin and PABP. Binding of PABP to senataxin was demonstrated by immunoblotting a senataxin immunoprecipitation with PABP antibody. (E) Senataxin interacts with RNA polymerase II. RNA polymerase II was co-immunoprecipitated with anti-senataxin antibody (Ab-1). (F) Co-immunoprecipitation of senataxin with the RNA polymerase II large subunit (Rpb1p) using an N-terminal RNA polymerase II antibody. IgH shows equal loading. (G) Co-immunoprecipitation of the mRNA processing factor SMN with senataxin Ab-1. (H) Mapping of the interaction domains on senataxin. Senataxin GST pull-downs were immunoblotted with nucleolin, SAP155, PABP, RNA polymerase II and SMN antibodies, in order to map the domain(s) of interaction on senataxin. C2ABR and C3ABR, control cells; SETX-2RM, AOA2 cells; Ig, pre-immune sheep sera (negative control); WCE, whole cell extract.
From: Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation
Hum Mol Genet. 2009;18(18): doi: /hmg/ddp278
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3. Figure 3. Comparative analysis of RNA polymerase II binding in control and SETX deficient cells. (A) RNA polymerase II ChIP assay in control and AOA2 cells to determine the binding of RNA polymerase II to SOD1, IMPDH2, CYC, RPL36 and CypA in control and AOA2 cells. Shown is the INPUT and OUTPUT for SOD1, IMPDH2, CYC, RPL36 and CypA from control and AOA2 cells run on an agarose gel after PCR. (B) RT–PCR of SOD1, IMPDH2, CYC, RPL36 and CypA from control and AOA2 cells run on an agarose gel. CypA shows the loading. (C) Comparative analysis of RNA polymerase II binding and corresponding endogenous mRNA levels in control and AOA2 cells. There is a significant difference in the relative binding of RNA polymerase II to SOD1, IMPDH2, CYC and RPL36 genomic loci in AOA2 cells relative to controls after normalization against CypA levels (loading control). Relative mRNA levels of SOD1, IMPDH2, CYC and RPL36 in control and AOA2 cells are also shown. (D) Upper panel: schematic representation showing the location of the three SETX RNAi sequences on the SETX coding region. SETX 1, SETX 2 and SETX 3 were targeted against regions 1579–1603, 1450–1474 and 428–452 bp, respectively. Lower panel: RNAi-mediated depletion of senataxin after 48 h in HeLa cells was revealed by immunoblotting with senataxin Ab-1. Anti-Rad50 antibody shows equivalent loading. The expression of SAP155, nucleolin, RNA polymerase II and SMN are also shown. (E) RNA polymerase II ChIP assay in senataxin RNAi knocked-down cells. INPUT and OUTPUT of an RNA polymerase II ChIP assay in cells knocked-down with control and SETX RNAi is shown to determine the binding of RNA polymerase II to SOD1, IMPDH2 and CYC. CypA shows equivalent loading. (F) RT–PCR of SOD1, IMPDH2, CYC and CypA from control and RNAi-depleted cells run on an agarose gel. CypA shows the loading. (G) Comparative analysis of RNA polymerase II binding and corresponding endogenous mRNA levels of SOD1 in cells with control and SETX RNAi. There is significantly less RNA polymerase II binding to SOD1 in cells with SETX RNAi knock-down (SETX 1, SETX 2 and SETX 3) compared with cells with control RNAi. Relative mRNA levels are also shown. Error bars indicate SD for three independent experiments. C3ABR, control cells; SETX-2RM, AOA2 cells; NS antibody, non-specific antibody; WCE, whole cell extract.
From: Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation
Hum Mol Genet. 2009;18(18): doi: /hmg/ddp278
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4. Figure 4. Transcription read-through in SETX deficient cells
Figure 4. Transcription read-through in SETX deficient cells. (A) Schematic illustration of the in vivo read-through construct. The read-through reporter construct (CSAG-1) contains a CMV promoter and was generated by the insertion of a sequence specific region containing a poly(A) site to allow the termination of mRNA during transcription. Read-through is measured by EGFP expression, located downstream of the poly(A) site. Primers pairs used for RT–PCR are shown. (B) Upper panel: RT–PCR run on an agarose gel with the reporter construct on control and SETX RNAi knocked-down HeLa cells using primer pairs F1/R1 and F2/R2. Lower panel: RT–PCR run on an agarose gel with control/SETX RNAi HeLa cells using β-2M as an internal standard to show equal loading. (C) Percentage relative read-through in SETX-depleted cells compared with cells with control RNAi. Read-through in cells transfected with SETX RNAi was observed compared with those transfected with control RNAi. Error bars indicate SD for three independent experiments.
From: Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation
Hum Mol Genet. 2009;18(18): doi: /hmg/ddp278
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5. Figure 5. Reduced splicing efficiency in SETX deficient cells
Figure 5. Reduced splicing efficiency in SETX deficient cells. (A) Schematic of the in vivo splicing assay (TN24). The reporter genes encoding β-gal and luciferase are fused in-frame via a recombinant fragment of the adenovirus and human άs-tropomyosin genes, respectively, containing three in-frame translation stop signals (XXX). Transfection gives rise to two different types of mRNA. Inefficient splicing—RNA contains the stop codons and only β-gal is expressed. Efficient splicing—removes the stops codons and β-gal-luc fusion protein is expressed (22). (B) No detection of TN24 reporter splicing construct expression by RT–PCR in AOA2 (SETX-2RM) lymphoblastoid cells compared with HeLa and control (C3ABR) lymphoblastoid cells, indicating very poor transfection efficiency of AOA2 cells. β-2M was used to show loading for HeLa, C3ABR and SETX-2RM. (C) Percentage relative ratio of luc to β-gal (luciferase/β-galactosidase) in control and senataxin-depleted HeLa cells transfected with TN24. A significant decrease in splicing in cells with reduced SETX levels was observed. (D) The splicing efficiency in U-251 cells depleted of senataxin was also measured. A significant reduction in splicing efficiency in cells with reduced SETX expression levels is shown. (E) A significant reduction in splicing efficiency in A549 cells with reduced SETX expression levels was seen. Error bars indicate SD for three independent experiments.
From: Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation
Hum Mol Genet. 2009;18(18): doi: /hmg/ddp278
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6. Figure 6. Loss of senataxin affects alternative splice-site selection
Figure 6. Loss of senataxin affects alternative splice-site selection. (A) Structure of the Tra2β1 minigene (24). Dotted lines indicate alternative splicing patterns. RT–PCR run on an agarose gel with the Tra2β1 minigene with control and SETX 1 on HeLa cells is also shown. (B) Quantitation of the percentage of exon 2 inclusion with the reporter and endogenous Tra2β1 in control and senataxin-depleted cells. (C) Quantitation of percentage of exon 2 inclusion with Tra2β1 minigene in U-251 cells transfected with control/SETX RNAi. (D) Structure of SRp20 minigene (24). Dotted lines indicate alternative splicing patterns. Lower panel: RT–PCR run on an agarose gel with the SRp20 minigene with control and SETX 1 on HeLa cells. (E) Quantitation of percentage of exon 4 inclusion with reporter and endogenous SRp20. (F) Quantitation of percentage of exon 4 inclusion with SRp20 minigene in U-251 cells transfected with control/SETX RNAi. Error bars indicate SD for three independent experiments.
From: Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation
Hum Mol Genet. 2009;18(18): doi: /hmg/ddp278
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7. Figure 7. Neurodegeneration and the novel roles of senataxin
Figure 7. Neurodegeneration and the novel roles of senataxin. Lack of senataxin in both AOA2 and senataxin-depleted cells influences the binding of RNA polymerase II to specific gene loci and subsequently affects mRNA transcript levels, suggesting a regulatory role for senataxin during transcription initiation and/or elongation. Furthermore, a reduction in splicing and alternative splicing efficiency and increased transcriptional read-through was also observed. It is not surprising that multiple defects are observed in several aspects of RNA transactions given that both transcription and mRNA processing are intimately coupled and co-regulated (21). Thus, sensitivity of AOA2 cells to H₂O₂, elevated levels of oxidative DNA damage in senataxin deficient cells and the protective function of senataxin in the repair of H₂O₂-induced DNA double strand breaks, combined with the defects in transcription and mRNA processing observed in senataxin deficient cells may all act as compounding factors for the neurodegeneration observed in AOA2.
From: Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation
Hum Mol Genet. 2009;18(18): doi: /hmg/ddp278
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