flag-sam68 purified protein tp300263  (OriGene)

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

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: Domain organization of Sam68 and NMR analysis of the N- and C-terminal regions. ( A ) Sam68 is a 443 amino acid protein. The QUA1 and KH domains are responsible for dimerization and RNA binding. The N-terminal region (residues 1–96), the QUA2 region (267–283) and the C-terminal region (residues 284–443) are predicted to be intrinsically disordered and contain regulatory motifs such as proline-rich motifs (P0–P5), RG-rich motifs (RG), a tyrosine-rich region (YY) and a nuclear localization signal (NLS). The number and percentage of serines and threonines for each region/domain are indicated and their position is indicated by small bars (blue for Ser and purple for Thr). ( B, C ) NMR ( 1 H- 15 N)-HSQC spectra of the Sam68 N-terminus (residues 1–96) (B) and C-terminus (residues 267–368) at 4°C (C). The assignment of the backbone amide resonances is indicated.

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: RNA Binding Assay

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: List of Sam68 post-translational modifications identified by LC-MS/MS

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Methylation, Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: The Sam68 N- and C-termini are phosphorylated at T33 and T317 by Cdk1. ( A, B ) ( 1 H- 15 N)-HSQC spectra overlay of the Sam68 N-terminus (A) and C-terminus (B) before (black) and after 16 h incubation with commercial active Cdk-1/cyclin B (red) at 4°C. T33 and T317 resonance peaks are indicated. ( C, D ) CSP of backbone amides as a function of the N-terminal (C) and C-terminal (D) amino acid sequence upon Cdk1 phosphorylation. For clarity, the CSPs of T33 and T317 are represented as red bars and are not to scale. ( E, F ) Normalized intensity of the phosphorylated T33 (E) and T317 (F) backbone amide peak as a function of time after Cdk1/cyclin B addition. The data were fitted using a Hill function.

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Incubation, Sequencing, Phospho-proteomics

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: The N- and C-terminal regions of Sam68 bind RNA. ( A ) Nucleotide sequence of the G8.5 RNA identified previously as a high-affinity binder of Sam68 . ( B, C ) ( 1 H- 15 N)-HSQC spectra of the Sam68 N-terminus (B) and C-terminus (C) before (black) and after (red) addition of excess G8.5 RNA (protein:RNA molar ratio of 1:2) at 4°C. ( D, E ) Chemical shift perturbation of the Sam68 N- (D) and C-terminal (E) backbone resonances upon RNA interaction as a function of the amino acid sequence. T33 and T317 CSPs are labelled with a green star. CSPs >0.025 (average CSP) are considered significant

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: T33 and T317 phosphorylation reduces the RNA binding ability of the Sam68 N- and C-termini. ( A, B ) ( 1 H- 15 N)-HSQC spectra of the Cdk1-phosphorylated Sam68 N-terminus (A) and C-terminus (B) before (black) and after (red) addition of 2 molar equivalent of G8.5 RNA at 4°C. Resonances of T33 and T317 are indicated. ( C, D ) CSP of the Sam68 N-terminus (C) and C-terminus (D) in their unphosphorylated (black) or Cdk-1 phosphorylated (red) forms upon RNA binding. T33 and T317 CSPs are labelled with a green star. CSPs >0.025 (average CSP) are considered significant. ( E ) RNA pulldown of GFP–Sam68 WT or mutant by biotinylated G8.5 RNA.

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Phospho-proteomics, RNA Binding Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: Cellular localization of Sam68 WT, T33A/T317A and T33E/T317E mutants in HCT116 cells. ( A ) Confocal fluorescence images of HCT116 cells transfected with either GFP-tagged Sam68 WT or mutants. DAPI is shown in red and GFP in green. ( B ) Representative images of three different classes (patterns A–C) of cellular localization of Sam68 WT and mutants in HCT116 cells (top) and percentage of cells displaying Sam68 or mutants localized in each class. Error bars represent the standard deviation of three independent transfection experiments of WT and mutant Sam68. The localization class was assessed using 50–100 GFP-positive cells in each independent experiment (bottom). P -values were calculated using an independent two-sample t -test (* P <0.05).

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Fluorescence, Transfection, Standard Deviation, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: Splicing activity of Sam68 WT and phospho-mimetic mutants on CD44 exon v5 and Bcl-x minigenes in HCT116 cells. Effect of Sam68 WT, T33E, T317E and T33E/T317E transfection on the alternative splicing of CD44 exon v5 ( A ) and Bcl-x ( B ) minigenes. Bottom: agarose gel electrophoresis showing splicing of the minigenes in response to co-transfected proteins. Top: quantification of biological replicates from three independent co-transfection experiments. Bar chart plotting and analysis were performed using GraphPad Prism. Error bars represent the standard deviation of three independent experiments. P -values were calculated using an independent two-sample t -test (* P <0.1, ** P <0.01, **** P <0.0001). Uncropped gels are shown in .

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Activity Assay, Transfection, Alternative Splicing, Agarose Gel Electrophoresis, Cotransfection, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: Effect of Sam68 WT or phospho-mimetic mutants on cell cycle progression, apoptosis and proliferation of HCT116 cells. ( A ) Percentage of cells in sub-G 1 (black), G 1 /G 0 (dark grey), S (white) and G 2 /M (light grey) phases 48 h after transfection with Sam68 WT or mutants (flow cytometry images are presented in ). Controls are untransfected cells. ( B ) Percentage of apoptotic cells 48 h after transfection with Sam68 WT or mutants (flow cytometry images are presented in ). ( C ) Normalized cell proliferation increase between 24 and 96 h after transfection with Sam68 WT or mutants. Controls are untransfected cells. Error bars represent the standard deviation of two independent experiments. P -values were calculated using an independent two-sample t -test (statistical significance shown as: * P <0.05, ** P <0.005 and *** P <0.0005).

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Transfection, Flow Cytometry, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Cdk1-mediated threonine phosphorylation of Sam68 modulates its RNA binding, alternative splicing activity and cellular functions

doi: 10.1093/nar/gkac1181

Figure Lengend Snippet: Model of splicing regulation by Cdk-1 mediated phosphorylation of Sam68/KHDRBS1. In interphase, Cdk-1 is inactive, so T33 and T317 of Sam68 are unphosphorylated. Sam68 would therefore bind (AU)-rich regions of the pre-mRNA specifically through its STAR homodimerization domain with a dissociation constant ( K d ) in the low micromolar range, and the N- and C-terminal regions would anchor the protein to the RNA, increasing the affinity of full-length Sam68 for RNA ( K d in the low nanomolar range). During mitosis or in cancer cells, activation of Cdk-1/cyclin B induces the phosphorylation of T33 and T317, leading to the dissociation of the N- and C-terminal anchoring region from the RNA and, therefore, a weakening in RNA binding by full-length Sam68, making Sam68 less effective at competing with other splicing factors for RNA binding. As a consequence, Sam68 activity in splicing regulation is reduced, leading to a decrease in cell apoptosis and an increase in proliferation.

Article Snippet: Full-length His-tag/Flag-tag Sam68 was cloned into the pLEICS-12 vector, full-length green fluorescent protein (GFP)–Sam68 into the pLEICS-25 vector, His-Tag Sam68 N-terminal region (amino acids 1–96) into the pLEICS-01 vector and glutathione S -transferase (GST)–Sam68 C-terminal region (267–368) into pGEX-6P-2 ( https://www.genscript.com/express-cloning-vector-list.html ).

Techniques: Phospho-proteomics, Activation Assay, RNA Binding Assay, Activity Assay

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: SAM68 binds SMN2 exon 7 and mediates recruitment of hnRNP A1 in vivo. (A) Schematic representation of the human SMN2 gene. Boxes represent exons, black lines represent introns, and the red box indicates the regulated exon 7. Red arrows indicate the oligonucleotide pairs used in the analysis. (B) CLIP assay of SAM68-bound SMN2 pre-mRNA in brain of non-SMA mice ( SMN2Δ7;SMN2;Smn +/+ ). (C) CLIP assay of hnRNP A1 in brain of non-SMA mice that are wild type or knockout for Sam68 . Signals for SAM68 (B) and hnRNP A1 (C) binding was calculated as fold enrichment versus IgGs and expressed as mean ± SD; n = 3. The p-value was determined by two-tailed t test (B) or one-way ANOVA test followed by Bonferroni’s multiple comparison posttest (C). *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant (P > 0.05). (D) Sequence of human SMN2 probe used to synthesize the biotinylated RNA for the streptavidin pull-down experiment. Lowercase letters indicate the intron 6 sequence; uppercase letters indicate the exon 7 sequence. Bold letters and red arrows indicate primers used to synthesize the probe, and red bold letters indicate the exonic splicing silencers created by the C to T transition in SMN2 to which SAM68 binds. (E and F) Western blot analysis of the binding of endogenous U2AF65 to the biotinylated probe (SMN2 Ex7) in streptavidin pull-down assays using brain extracts from non-SMA Sam68 +/+ (wt) or Sam68 −/− (ko) mice (E) or extracts from HEK293T cells transfected (+) or not (−) with FLAG-SAM68 (F). Results are representative of three experiments that yielded similar results. ko, knockout; N.E., nuclear extracts; wt, wild type.

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: In Vivo, Knock-Out, Binding Assay, Two Tailed Test, Sequencing, Western Blot, Transfection

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 expression partially rescues viability, body weight, and motor function of SMAΔ7 mice. (A) Schematic diagram of transgenic mice crossing (top) and PCR analysis of genomic DNA (bottom) from tails of SMN2Δ7;SMN2;Smn +/+ ;Sam68 +/+ (first lane), SMN2Δ7;SMN2;Smn −/− ;Sam68 +/+ (second lane), and SMN2Δ7;SMN2;Smn −/− ;Sam68 −/− (third lane) mice. Bands showing the transgenic and endogenous DNA bands amplified are indicated; the asterisk indicates an undefined band present in SMAΔ7 transgenic mice. (B) qPCR of SMN2Δ7 to quantify the dosage of transgenic SMN2Δ7 in hemizygous and homozygous transgenic animals. The data shown are from a single representative experiment, and values were normalized with Apolipoprotein B mRNA. (C) Micrograph showing representative non-SMA and SMAΔ7 (SMA) mice that are either wild type ( Sam68 +/+ ) or knockout ( Sam68 −/− ) for Sam68 . (D) Kaplan-Meier survival curves of SMAΔ7/ Sam68 +/+ ( n = 50) and SMAΔ7/ Sam68 −/− ( n = 22). Statistical analysis was performed by the log-rank test (P < 0.0001). (E) Weight curves of non-SMA/ Sam68 +/+ , non-SMA/ Sam68 −/− , SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− mice. Plot shows mean ± SD of at least 15 mice for each group for each day analyzed. The p-values were determined by a two-way ANOVA test followed by Bonferroni’s multiple comparison posttest. # represents the p-value of the non-SMA/ Sam68 +/+ versus non-SMA/ Sam68 −/− comparison; § represents the p-value of the non-SMA/ Sam68 −/− versus SMAΔ7/ Sam68 −/− comparison; and * represents the p-value of the SMAΔ7/ Sam68 +/+ versus SMAΔ7/ Sam68 −/− comparison. For all comparisons, P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 are represented by increasing symbols (from one to four). (F) Time required for pups to stand up after being placed on their sides for SMAΔ7/ Sam68 +/+ mice compared with SMAΔ7/ Sam68 −/− mice at 8 and 10 dpp (nd [nondetected] indicates experimental animals that never stood up during the test). Statistical analysis was performed by two-way ANOVA test followed by Bonferroni’s multiple comparison posttest (*, P < 0.05; ***, P < 0.001).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Transgenic Assay, Amplification, Knock-Out

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Sam68 deletion rescues SMN2 splicing and expression in SMAΔ7 tissues. (A) qPCR analysis of exon 7–containing SMN2 transgene transcripts normalized to constant exon 6 in tissues of 10-dpp SMAΔ7/ Sam68 +/+ and SMAΔ7/ Sam68 −/− mice. Bar graph represents mean ± SD. n = 3. The p-value was determined by two-tailed t test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Western blot analysis of SMN protein expression in the indicated tissues of 10-dpp SMAΔ7/ Sam68 +/+ (wt) or SMAΔ7/ Sam68 −/− (ko) mice. Actin was used as a loading control. Quantification of the SMN band is shown at the bottom. Each point value represents the mean ± SD. n = 2. Cereb, cerebellum; ko, knockout; S.cord, spinal cord; wt, wild type.

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Expressing, Two Tailed Test, Western Blot, Knock-Out

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 rescues SMN assembly into nuclear gems in spinal cord motor neurons. (A) Immunodetection of SAM68 (blue) in ChAT-positive motor neurons (red) in the lumbar spinal cord (L1–L5) of 8-dpp non-SMA mice. DAPI was used for staining of nuclei. Motor neurons are indicated by arrows and surrounding cells by arrowheads. (B) Immunodetection of SMN gems (green) in ChAT-positive motor neurons (red) in the spinal cord (L1–L5) of 8-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. DAPI was used for staining of nuclei. Nuclear gems are indicated by arrowheads. Bars: (A and B) 20 µm; (insets) 10 µm. (C) Quantitative analysis of SMN gems in spinal motor neurons. The bar graph (mean ± SD; n = 3) shows the number of gems per 100 nuclei analyzed per sample (top) and the percentage of gem-positive motor neurons (bottom). Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest (*, P < 0.05; ***, P < 0.001).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Immunodetection, Staining

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 rescues motor neuron loss in the spinal cord of SMAΔ7 mice. (A) Schematic representation of a Nissl-stained spinal cord section of the lumbar spinal cord from 8-dpp non-SMA mice (right). The red circle highlights the ventral horn of the spinal cord analyzed. Bar, 250 µm. (B) Higher magnification of sections of the ventral lumbar spinal cord from 8-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Motor neurons are indicated by arrowheads. Bar, 125 µm. (C) Bar graph representing motor neuron counts (mean ± SD; n = 3) in lumbar spinal cord from mice described in B. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest (***, P < 0.001; ****, P < 0.0001).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Staining

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Ablation of Sam68 ameliorates innervation of NMJs. (A) NMJ immunofluorescence images from whole-mount FDB2 of 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Higher magnifications are shown in the bottom panels. Postsynaptic (AChRs stained with α-bungarotoxin; green), presynaptic (stained with α-synaptophysin; red), and motor neuron axons (stained with an antibody against the heavy chain of neurofilament; red) were visualized. Bars, 100 µm. Arrows point to innervated NMJs (yellow staining), and arrowheads point to denervated NMJs (green staining). (B) Bar graph showing the percentage of innervated (left) and denervated (right) NMJs in FDB2 of non-SMA, non-SMA/ Sam68 −/− , SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− mice (for all, n = 3). Quantification of NMJ innervation was performed on at least 100 optical sections for mice, with a step size of 3 µm from the whole FDB2 of each genotype. Data represent mean ± SEM. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s multiple comparison posttest (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant [P > 0.05]).

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: SAM68 is a physiological regulator of SMN2 splicing in spinal muscular atrophy

doi: 10.1083/jcb.201502059

Figure Lengend Snippet: Skeletal muscle atrophy is ameliorated in the SMAΔ7/ Sam68 −/− mice. (A) Hematoxylin and eosin staining of triceps muscle sections from 10-dpp non-SMA, SMAΔ7/ Sam68 +/+ , and SMAΔ7/ Sam68 −/− (SMA) mice. Bar, 100 µm. (B) Frequency distribution of muscle fiber areas (at least 2,000 for each genotype) of the experimental animals described in A. The colored lines and dotted line (non-SMA mice) represent the median value for each genotype: non-SMA mice ( n = 3) median size = 531 µm 2 and fiber size (mean ± SEM) = 568.76 ± 5.8 µm 2 ; non-SMA/ Sam68 −/− mice ( n = 3) median size = 557 µm 2 and fiber size (mean ± SEM) = 586.60 ± 5.7 µm 2 ; SMAΔ7/ Sam68 +/+ median size = 327 µm 2 and fiber size (mean ± SEM) = 342.32 ± 3.1 µm 2 ; and SMAΔ7/ Sam68 −/− median size = 486 µm 2 and fiber size (mean ± SEM) = 503.59 ± 4.6 µm 2 . Statistical analysis of the median values among groups was performed by one-way ANOVA test followed by Dunn’s multiple comparison posttest. (C) qPCR analysis of the expression of atrophy-related genes in quadriceps and lower leg muscles of mice described in B. Values (mean ± SD; n = 4) were normalized with Actin mRNA. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni’s multiple comparison posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant (P > 0.05). r.l., relative level.

Article Snippet: In brief, HEK293T cells were transfected with FLAG-SAM68 vector as indicated using Lipofectamine 2000 (Invitrogen).

Techniques: Staining, Expressing