pgex 6p3 sam68 flag  (GE Healthcare)

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: In vivo association of SAM68 with U1 snRNP. ( A ) Schematic representation of a portion of mTor pre-mRNA spanning from exon4 to exon6 (upper panel), with a close-up of the 5′ splice site and the subsequent SAM68-binding site (SB-1), as well as the cryptic polyadenylation signal that harbor SAM68-binding site (SB-A). ( B ) Co-immunoprecipitation of U1 snRNP with Flag-hSAM68. HEK-293T cells depleted of endogenous SAM68 (sh SAM68 HEK-293T) were transiently transfected with Flag-hSAM68 or Flag-YFP (yellow-fluorescent protein), the latter serving as negative control. Flag-tagged proteins were immunoprecipitated using anti-Flag M2 agarose beads and immunoprecipitated proteins were detected with antibodies specific to U1–70K, U1A and U1C. β-Actin was used as negative control. Portion of the Flag-immunoprecipitates was used for RNA isolation and RT-PCR using U1 snRNA specific primers. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) RNA was used as negative control of the RT-PCR made from the RNA immunoprecipitation. ( C ) Co-immunoprecipitation of endogenous hSAM68 with U1–70K. Immunoprecipitated proteins were detected with antibodies directed against SAM68 and U1–70K. β-Actin was used as negative control of immunoprecipitated proteins. ( D ) Co-immunoprecipitation of endogenous hSAM68 with U1A. Immunoprecipitated proteins were detected with antibodies directed against SAM68 and U1A. β-Actin was used as negative control of immunoprecipitated proteins. ( E ) Coomassie staining of purified human SAM68 and U1A. ( F ) RNA binding assay with purified SAM68 and labeled U1snRNA. Reactions contained 10 nM γ-p32 labeled U1snRNA in buffer with no protein (lane 1) or with purified SAM68 (lanes 2–5). Bottom panel: quantification from three independent binding experiments. Error bars represent the corresponding standard error. Unpaired two-tailed t -tests were used to compare the different concentrations of purified protein to the RNA only control. SAM68 P -values are 0.0014, 0.0005, <0.0001, <0.0001 in increasing order of SAM68 concentration. ( G ) RNA binding assay with purified U1A and labeled U1snRNA. Reactions contained 10 nM γ-p32 labeled U1snRNA in buffer with no protein (lane 1) or with purified U1A (lanes 2–5). Bottom panel: U1snRNA P -values = 0.0008, <0.0001, <0.0001, <0.0001 in increasing order of U1A concentration. ** P -value < 0.005, *** P -value < 0.001.

Article Snippet: pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag were constructed by inserting full-length human and mouse SAM68 coding sequence (cds) into pGEX-6P3 (GE Healthcare) with N-terminal GST tag, a PreScission protease cleavage site (see below ‘Protein purification and GST pulldown’ section) between GST and SAM68.

Techniques: In Vivo, Binding Assay, Immunoprecipitation, Transfection, Negative Control, Isolation, Reverse Transcription Polymerase Chain Reaction, Staining, Purification, RNA Binding Assay, Labeling, Two Tailed Test, Concentration Assay

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: In vitro Purified SAM68 associated with U1 snRNP in an RNA-independent manner. ( A ) In vitro purified hSAM68-Flag was added to sh SAM68 HEK-293T cell lysates for 1 h at 4°C, in the presence or absence of 50 μg/ml RNaseA. hSAM68-Flag and associated proteins were immunoprecipitated using Flag-M2 affinity beads and treated further with RNaseA at 37°C for 30 min. Bound proteins were eluted with Laemmli and immunoblotted with antibodies specific to U1–70K, U1A and U1C. To assess RNaseA treatment efficiency, total RNA from sh SAM68 HEK-293T was treated with either Mock or RNaseA for 30 min at 37°C, and the remaining total RNA was assessed on agarose gel. ( B ) RNA-binding defective mutant hSAM68 I184N interacts with U1 snRNP. sh SAM68 HEK-293T were transiently transfected with Flag-hSAM68, Flag-hSAM68 I184N and Flag-YFP (negative control). The Flag-tagged proteins were immunoprecipitated using anti-Flag M2 agarose beads and immunoblotted with antibodies directed against U1–70K, U1A and U1C. ( C ) Association of hSAM68-Flag with U1 snRNP withstands high salt washes. Purified in vitro produced hSAM68-Flag was added to cell lysates of sh SAM68 HEK-293T for 1 h at 4°C. Flag-M2 affinity beads were added to the reaction and left for 1 h at 4°C. The washes were done, by increasing salt concentration, from 150 to 500 mM of NaCl. Bound proteins were eluted with Laemmli and immunoblotted with antibodies directed against U1–70K, U1A and U1C. ( D ) SAM68 interacts with U1A in vitro . About 300 ng of purified hSAM68-Flag was incubated with 100 ng of glutathione-agarose bound GST-U170k-His, GST-U1A-His, GST-U1C-His and GST-His. Following washes, the beads were washed five times in binding buffer and the bound proteins eluted with Laemmli and immunoblotted using anti-Flag or anti-His antibodies.

Article Snippet: pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag were constructed by inserting full-length human and mouse SAM68 coding sequence (cds) into pGEX-6P3 (GE Healthcare) with N-terminal GST tag, a PreScission protease cleavage site (see below ‘Protein purification and GST pulldown’ section) between GST and SAM68.

Techniques: In Vitro, Purification, Immunoprecipitation, Agarose Gel Electrophoresis, RNA Binding Assay, Mutagenesis, Transfection, Negative Control, Produced, Concentration Assay, Incubation, Binding Assay

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: SAM68 interaction with U1A is mediated through its C-terminal portion. ( A ) Schematic representation of C-terminus (aa. 1–280) and N-terminus (aa.281–443) deletion domains of hSAM68 fused to flag. ( B ) sh SAM68 HEK-293T cells were transiently transfected with Flag- SAM68 (N-term), Flag- SAM68 (C-term), Flag- SAM68 (FL) and flag-YFP (negative control). Forty-eight hours post transfection, the flag-tagged proteins were immunoprecipitated using anti-flag M2 agarose beads and immunoblotted with antibodies specific to U1–70K, U1A and U1C. ( C ) Schematic representation of full-length SAM68, C-terminus deleted SAM68 (NT, aa. 1–280), C-terminus truncated to proline rich C1 (aa. 269–364) and tyrosine rich C2 (aa. 365–443), C3 (aa. 370–443), C4 (aa. 385–443), C5 (aa. 340–443) and NLS (aa. 430–443). Fragments were fused to GFP tag at their N-terminus and all fragments had SAM68 NLS at their C-terminus. ( D ) GFP-Trap-A pulldown of GFP-tagged proteins. sh SAM68 HEK-293T cells were transiently transfected with GFP, GFP-SAM68 (FL), GFP - SAM68 (NT), GFP - SAM68 (C1) and GFP-SAM68 (C2). Forty-eight hours post transfection, cells were lysed and GFP-Trap-A beads were used to pull down GFP-tagged proteins, and their association with U1A was validated by western blot using specific antibodies. ( E ) Primary amino acid sequence of the various deletion constructs of SAM68 YY domain (GFP-hSAM68 C2 to C5). Underlined indicates YXXY motifs in the YY domain. Also highlighted is the minimal ARM-binding region. ( F ) GFP-Trap-A pulldown of GFP-tagged proteins. shSAM68 HEK-293T cells were transiently transfected with GFP, GFP-SAM68 (C2), GFP-SAM68 (C3), GFP-SAM68 (C4), GFP-SAM68 (C5) and GFP-SAM68 (NLS). Forty-eight hours post transfection, cells were lysed and GFP-Trap-A beads were used to pull down GFP-tagged proteins, and their association with U1A was validated by western blot using specific antibodies. ( G ) U1A binds preferentially to the minimal ARM motif (YEGYEGY) within the YY domain of SAM68. Flag-hSAM68(FL) and Flag-hSAM68(ΔARM) were transiently transfected in sh SAM68 HEK-293T cells. Forty-eight hours post transfection, cells were lysed and Flag-tagged proteins were immunoprecipitated using anti-flag M2 agarose beads, and U1A association was assessed using U1A antibody. ★: denotes an unspecific band.

Article Snippet: pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag were constructed by inserting full-length human and mouse SAM68 coding sequence (cds) into pGEX-6P3 (GE Healthcare) with N-terminal GST tag, a PreScission protease cleavage site (see below ‘Protein purification and GST pulldown’ section) between GST and SAM68.

Techniques: Transfection, Negative Control, Immunoprecipitation, Western Blot, Sequencing, Construct, Binding Assay

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: Tyrosine-rich (YY) domain of SAM68 mediates the interaction with U1 snRNP via YXXY repeated motif. ( A ) Overlay of the 2D 15 N- 1 H HSQC spectra of GB1-hSAM68 (C2) recorded before and after the addition of unlabeled GB1-U1A. The spectra are colored according to the molar ratio hSAM68 (C2):U1A (1:0; 1:0.6 and 1:1.4 are colored in blue, red and black, respectively). Strongly perturbed signals are marked by red arrows and their assignment is indicated. ( B ) Overlay of the 2D 15 N- 1 H HSQC spectra of U1A RRM1 recorded before and after the addition of unlabeled GB1-hSAM68 (C2). The spectra are colored according to the molar ratio U1A RRM1:hSAM68 (C2) (1:0; 1:0.6 and 1:1.4 are colored in blue, red and black, respectively). ( C ) Plot of the normalized chemical shift perturbations observed in panel (B) in function of the sequence of U1A RRM1. The chemical shift perturbations are then plotted onto the surface representation of the structure of the free form of the RRM1 of U1A . Amino acids that experienced chemical shift perturbation between 0.03 and 0.05 are colored in orange while the CSP higher than 0.05 are colored in red.

Article Snippet: pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag were constructed by inserting full-length human and mouse SAM68 coding sequence (cds) into pGEX-6P3 (GE Healthcare) with N-terminal GST tag, a PreScission protease cleavage site (see below ‘Protein purification and GST pulldown’ section) between GST and SAM68.

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: Both SAM68 and intronic enhancer sequences in mTor intron 5 are required for U1A recruitment to 5′SS in vitro . ( A ) Schematic representation of the various in vitro transcribed mTor minigene baits with the 5′ splice site. As shown, the baits span from last 7 nucleotides of exon5 to the poly-adenylation signal in intron 5. WT refers to the wild-type intronic SAM68-binding sequences of SB-1 (UUUUAU) and SB-A (UAAAA), the latter is embedded in the cryptic poly-adenylation signal (AAUAAA). The ‘mut’ denotes the combined mutations of SB-1 (UUUUAU to UUUCAU) and SB-A (AAUAAAA to AAUAACC). ( B ) SAM68 recruits U1A to 5′ splice site in vitro . Recombinant in vitro purified hSAM68-Flag was tested for its ability to recruit U1A to mTor intron 5 baits with either WT or mutated SAM68-binding sites. GST-Flag was used as negative control. ( C ) Schematic representation of the various in vitro transcribed mTor minigene baits that are deleted for the 5′ splice site. As shown, the baits span 18 nucleotides downstream of the 5′ splice site to the poly-adenylation signal of intron 5. WT refers to the wild-type intronic SAM68-binding sequences, SB-1 (UUUUAU) and SB-A (UAAAA). The ‘mut’ denotes the combined mutations of SB-1 (UUUUAU to UUUCAU) and SB-A (AAUAAAA to AAUAACC). ( D ) SAM68 recruits U1A in the absence of 5′ splice site in vitro . Recombinant in vitro purified hSAM68-Flag was tested for its ability to recruit U1A to mTor intron 5 baits lacking 5′SSs with either WT or mutated SAM68-binding sites. GST-Flag was used as negative control. ( E ) Schematic representation of the in vitro transcribed mTor minigene bait and the primers used for the RppH/Xrn1 protection assays. ( F ) Assessment of the processivity of RppH and Xrn1 enzyme on the naked mRNA bait, showing that RppH treatment is necessary for Xrn1-mediated degradation of the mRNA bait. ( G ) RppH and Xrn1 protection assays in vitro produced mRNA bait incubated with either WT MEFs cell lysate (lane 1), Sam68 −/− MEFs cell lysate (lane 2), in vitro produced mSAM68(WT) + Sam68 −/− MEFs cell lysate (lane 3) or in vitro produced mSAM68(WT) + Sam68 −/− MEFs cell lysate + U1 nRNAs antisense oligo (lane 4). U1snRNP components (U1A, U1C) and mSAM68 levels were assessed by western blot, while U1 snRNA levels was assessed by RT-PCR. GAPDH served as loading control for the western blot. ( H ) SAM68 protects the mTor RNA bait from Xrn1 degradation. Biotinylated RNA baits were incubated with buffer (lane 1), 100 ng of GST-Flag (lane 2) or 100 ng of mSAM68-Flag (lane 3) for 30 min on ice. Sam68 levels were assessed by western blotting using anti-Flag, while baits levels were measured by semi-quantitative RT-PCR using FSS-RSB primers for the full-length RNA and FSB-RSB for the SAM68 protected fragment.

Article Snippet: pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag were constructed by inserting full-length human and mouse SAM68 coding sequence (cds) into pGEX-6P3 (GE Healthcare) with N-terminal GST tag, a PreScission protease cleavage site (see below ‘Protein purification and GST pulldown’ section) between GST and SAM68.

Techniques: In Vitro, Binding Assay, Recombinant, Purification, Negative Control, Produced, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: U1snRNP is recruited in a SAM68-dependent manner at the exon5–intron 5 junction (ei5) in mTor pre-mRNA. ( A ) RNA immunoprecipitation (RIP) assay of mSAM68 on mTor pre-mRNA. (Top) Schematic representation of mTor pre-mRNA showing location of amplicon used to detect U1 snRNP binding by RIP (ei4 in red, ei5 in green and ei37 in black). (Below) U1A-RIP was done from WT MEFs or Sam68 −/− MEFs using anti-U1A or control IgG antibodies. Bound RNA was analyzed by RT-qPCR using the highlighted primers. Mean values are expressed as fold enrichment over input and normalized to WT signal. Error bars represent ± standard deviations of the means. ( B ) U1snRNP recruitment is restored at mTor EI5, in Sam68 −/− MEFs expressing mSam68(WT) but not with mSam68(ΔArm) . U1A-RIP was done using anti-U1A or control IgG antibodies in Sam68 −/- MEFs, Sam68 −/- MEFs rescued with mSam68(WT) or mSam68(ΔArm) . Bound RNA was analyzed in triplicates by RT-qPCR using the highlighted primers. Mean values are expressed as fold enrichment over input and normalized to WT signals. Error bars represent ± standard deviations of the means.

Article Snippet: pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag were constructed by inserting full-length human and mouse SAM68 coding sequence (cds) into pGEX-6P3 (GE Healthcare) with N-terminal GST tag, a PreScission protease cleavage site (see below ‘Protein purification and GST pulldown’ section) between GST and SAM68.

Techniques: Immunoprecipitation, Amplification, Binding Assay, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: SAM68 interaction with U1A modulates U1 snRNP recruitment and regulates mTor pre-mRNA splicing

doi: 10.1093/nar/gkz099

Figure Lengend Snippet: SAM68 deletion of ‘ARM binding region’ shows decrease in U1A binding. ( A ) Schematic of the pcDNA mTor 4–6 minigene, comprising the mTor genomic fragment from exon4 to exon6. ( B ) (Left panel) Sam68 −/- MEFs cells were infected with Sam68 (WT) or Sam68 (ΔARM) and compared to uninfected Sam68 −/- MEFs or WT MEFs. Total RNA was extracted in each cell lines and semi-quantitative RT-PCRs were performed using endogenous mTor specific primers. Forward (Fe4) and Reverse (Re6) were used to quantify mTor transcripts that were spliced normally (mTor 4–6 ), while Forward (Fe4) and Reverse (Ri5) were used to quantify intron 5 including mTor transcripts ( mTor i5 ). Gapdh was used to normalize the values obtained. Total protein was also extracted and run on 10% SDS-PAGE and blotted with SAM68, U1A and GAPDH antibodies. (Right panel) Quantification of intron 5-induced termination over normally spliced mRNA based on three independent experiments using endogenous mTor specific primers. ** P -value ≤ 0.05 and # = non-significant (two-tailed t -test).

Article Snippet: pGEX-6P3- SAM68 -Flag and pGEX-6P3- Sam68 -Flag were constructed by inserting full-length human and mouse SAM68 coding sequence (cds) into pGEX-6P3 (GE Healthcare) with N-terminal GST tag, a PreScission protease cleavage site (see below ‘Protein purification and GST pulldown’ section) between GST and SAM68.

Techniques: Binding Assay, Infection, SDS Page, Two Tailed Test