Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Endogenous TERRA is bound to hnRNPA1. ( A ) Immunoprecipitation of hnRNPA1-myc in 293T nuclear extract. TERRA and centromeric RNA were detected by dot blot hybridization. Immunoprecipitation efficiency was determined by western blotting. Twenty-five percent of the RNA extracted from the input (I) and all RNA from the immunoprecipitates were spotted on the membrane and quantified. Bars represent the means ± SD of three independent experiments. ( B ) RNA-ChIP of TERRA associated with endogenous hnRNPA1 in 293T cells untreated (−) or transfected with siRNA against GFP or hnRNPA1. RNA was detected by dot blot hybridization. One percent of the input RNA or supernatant and half of the RNA from the immunoprecipitates was loaded. The same samples were treated with RNase A before loading (+ RNase). Bars represent the means ± SD of three independent experiments. ( C ) RNA-ChIP of TERRA associated with hnRNPA1-ZZ in HT1080 cells. RNA was detected as in (B). One percent of the input RNA or supernatant and half of the RNA from the immunoprecipitates was loaded. *** P = 0.0002. Bars represent the means ± SD of six independent experiments.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Immunoprecipitation, Dot Blot, Hybridization, Western Blot, Transfection

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Purification and characterization of recombinant GST-hnRNPA1. ( A ) GST and GST-hnRNPA1 fractions used for in vitro assays after the second purification step on the Mono Q sepharose column. The left SDS polyacrylamide gel was stained with Coomassie brilliant blue. Lane 1: GST, lane 2: GST-hnRNPA1, lanes 3 and 4: 200 and 400 ng of BSA. On the right are the corresponding western blots. Western blots were probed with α-GST antibody and α-hnRNPA1 antibody as indicated. ( B ) Recombinant GST-hnRNPA1 but not GST binds (UUAGGG) 3 . Electromobility shift assay and supershift of [ 32 P] 5′ end-labeled (UUAGGG) 3 (2 nM) with purified GST or GST-hnRNPA1 (180 nM) with antibody (Ab) or without (−)200 ng α-hnRNPA1 antibody. Lane 1: (UUAGGG) 3 alone. Lane 2: (UUAGGG) 3 incubated with GST. Lane 3: (UUAGGG) 3 incubated with GST and α-hnRNPA1 Ab. Lane 4: (UUAGGG) 3 incubated with GST-hnRNPA1. Lane 5: (UUAGGG) 3 incubated with GST-hnRNPA1 and α-hnRNPA1 Ab. GST-hnRNPA1- (UUAGGG) 3 and Ab-GST-hnRNPA1- (UUAGGG) 3 complexes (supershift) are indicated. ( C ) GST-hnRNPA1 interacts with TERRA RNA and telomeric DNA oligonucleotides but not with the TS primer. EMSA analysis of [ 32 P] 5′ end-labeled (UUAGGG) 3 , (TTAGGG) 3 or TS (2 nM) with purified GST-hnRNPA1 (35, 105 or 315 nM) or 315 nM GST. ( D ) GST-hnRNPA1 has similar affinity for TERRA and telomeric DNA oligonucleotides. EMSA analysis of [ 32 P] 5′ end-labeled (UUAGGG) 3 or (TTAGGG) 3 (2 nM) with purified GST-hnRNPA1 (12.5, 25, 37.5, 62.5, 75, 87.5, 112.5 or 137.5 nM). Kds are given and were determined from four independent experiments. ( E ) EMSA competition experiments to assess specificity of hnRNPA1 for TERRA and telomeric DNA oligonucleotides. On the left gel, GST-hnRNPA1 (60 nM) was incubated with [ 32 P] 5′ end-labeled (TTAGGG) 3 (2 nM) mixed with H 2 O or 17.5, 35, 175 or 350 nM of non-labeled dA 18 ; 175 or 350 nM TS; 17.5, 35, 175 or 350 nM of (TTAGGG) 3 ; 17.5, 35, 175 or 350 nM of rA 18 ; 17.5, 35, 175 or 350 nM of (UUAGGG) 3 . On the right gel, GST-hnRNPA1 (60 nM) was incubated with [ 32 P] 5′ end-labeled (UUAGGG) 3 (2 nM) mixed with H 2 O or 17.5, 35, 175 or 350 nM of rA 18 ; 17.5, 35, 175 or 350 nM of (UUAGGG) 3 ; 17.5, 35, 175 or 350 nM of non-labeled dA 18 ; 175 or 350 nM TS; 17.5, 35, 175 or 350 nM of (TTAGGG) 3 . For both gels, in lane 1, the radiolabeled oligonucleotide was incubated with the protein buffer only. The vertical line between lanes indicates juxtaposition of separate parts of the same gel.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Purification, Recombinant, In Vitro, Staining, Western Blot, Electro Mobility Shift Assay, Labeling, Incubation

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Purification of overexpressed telomerase and inhibiting effects of hnRNPA1 on telomerase activity by primer binding. ( A ) The enriched telomerase fraction used for direct telomerase assays does not contain detectable endogenous hnRNPA1 protein. Load, flow through and eluate from TEV cleavage were analyzed by western blotting using the indicated antibodies. ( B ) hnRNPA1 inhibits telomerase activity by binding to the primer. Direct telomerase assay with telomerase substrates B-(TTAGGG) 3 (left) and B-TS (right). The 3′ end biotinylated 10-mer was [ 32 P] 5′ end-labeled and used as a loading control (added during the purification step of the elongated products on streptavidin beads). The numbers on the left of the gels indicate the number of nucleotides added to the substrates. For both gels, lane 1: DTA performed in presence of the protein buffer, lanes 2, 3 and 4: DTA performed in presence of 17.5, 50 and 150 nM GST-hnRNPA1, lane 5: DTA performed with 150 nM GST.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Purification, Activity Assay, Binding Assay, Western Blot, Telomerase Assay, Labeling

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: Alleviation of telomerase inhibition by TERRA with hnRNPA1. ( A ) Rescue of telomerase activity by GST-hnRNPA1 when using the TS primer. Lanes 1–4: DTA performed in the presence of protein buffer and water, 175 nM rA 18 , 17.5 nM (UUAGGG) 3 or 35 nM (UUAGGG) 3 . Lanes 5–7: DTA performed in presence of 17.5 nM GST-hnRNPA1 and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 or 35 nM (UUAGGG) 3 . Lanes 8–10: DTA performed in the presence of 50 nM GST-hnRNPA1 and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 or 35 nM (UUAGGG) 3 . Lanes 11–14: DTA performed in presence of 150 nM GST-hnRNPA1 and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 , 35 nM (UUAGGG) 3 or 175 nM (UUAGGG) 3 . Lanes 15–18: DTA performed in presence of 150 nM GST and 175 nM rA 18 , 17.5 nM (UUAGGG) 3 , 35 nM (UUAGGG) 3 or 175 nM (UUAGGG) 3 . Lane 19: DTA performed in the presence of 25 mM EDTA. 3′ end biotinylated 10-mer was [ 32 P] 5′ end-labeled and used as a recovery and loading control. The numbers on the left of the gels indicate the number of nucleotides added to the substrates. ( B ) Quantification of (A) from two independent experiments. ( C ) and ( D ) same as (A) and (B), but the assays were performed with the (TTAGGG) 3 primer.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Inhibition, Activity Assay, Labeling

Journal: Nucleic Acids Research

Article Title: A three-state model for the regulation of telomerase by TERRA and hnRNPA1

doi: 10.1093/nar/gkt695

Figure Lengend Snippet: ( A ) hnRNPA1 levels do not vary throughout the cell cycle. Left panel: hnRNPA1 protein level in synchronized HeLa cells after release from a double thymidine block. AS: asynchronous cells, 0 h: before release. Cells were collected every 2 h (2, 4, 6, 8, 10 and 12 h). Protein content was analyzed by western blotting using the indicated antibodies. CyclinE and CyclinB1 were used as markers for cell synchronization. Right panel: hnRNPA1 levels were quantified and normalized to tubulin levels and to the 0 h sample. Error bars correspond to standard deviation of two independent experiments. ( B ) Model: telomerase is switched on or off depending on the local concentrations of telomerase, hnRNPA1, TERRA and free 3′ telomeric single-stranded overhang. (1) TERRA < hnRNPA1: only a subset of hnRNPA1 is bound by TERRA and the remainder is bound to the 3′ telomeric single-stranded overhang, inhibiting the access for telomerase. (2) TERRA = hnRNPA1: TERRA and hnRNPA1 form an inert RNP complex. Telomerase can act on the telomeric single-stranded 3′ overhang. (3) TERRA > hnRNPA1: hnRNPA1-free TERRA will bind telomerase and inhibit its activity.

Article Snippet: For bacterial expression of hnRNPA1, pGEX-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pcDNA6-hnRNPA1 using BamH1-A1_F and A1-EcorRI_R primers ( Supplementary Table S1 ) and subcloned into the BamHI and EcoRI sites of pGEX-6P-1 vector (GE Healthcare). pcDNA6-hnRNPA1 was generated by PCR amplification of the hnRNPA1 open reading frame from pCMV6-XL5-hnRNPA1 (Origene, clone NM_002136) using HindIII-A1_F and EcoRI-A1_R primers. cDNAs were subcloned into retroviral-based pCL vectors for expression of ZZ or hnRNPA1-ZZ proteins in HT1080 cells.

Techniques: Blocking Assay, Western Blot, Standard Deviation, Activity Assay