Journal: Molecular Cell

Article Title: Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination

doi: 10.1016/j.molcel.2016.11.039

Figure Lengend Snippet:

Article Snippet: GST-UBE1 (human) , Boston Biochem , Cat. #E-306.

Techniques: Recombinant, Protease Inhibitor, Methylation, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Sequencing, Negative Control, Software

Journal: Brain

Article Title: UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

doi: 10.1093/brain/awy237

Figure Lengend Snippet: Rescue of sensory-motor connectivity defects in the spinal cord of SMA mice following systemic restoration of UBA1. ( A – C ) Lumbar motor neurons from control, SMA and SMA+AAV9-UBA1 mice labelled with ChAT (magenta), VGLUT1 (green) and DAPI. Scale bar = 10 µm. ( D ) Quantification of the number of VGLUT1 synapses per ChAT-positive motor neuron soma. n = 3 mice per condition, n > 20 motor neurons analysed per mouse. ns = not significant, * P < 0.05.

Article Snippet: HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples.

Techniques:

Journal: Brain

Article Title: UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

doi: 10.1093/brain/awy237

Figure Lengend Snippet: Label-free proteomics analysis identifies tRNA synthetases as UBA1-dependent proteins. ( A ) UBA1 expression in HEK293 cells used for proteomics screen showing UBA1 overexpression (OE) and knockdown (KD) compared to control. CoxIV and H3 = loading control. ( B and C ) Quantification of UBA1 overexpression ( B ) and knockdown ( C ). Ctrl = control; ** P < 0.01, *** P < 0.001. ( D ) BioLayout clustering 3D representation of proteomic expression across UBA1 overexpression, control and UBA1 knockdown. Nodes represent proteins and edges represent similarity of expression between proteins and clusters of proteins with similar expression profiles are indicated by different colours. Cluster numbers are indicated. ( E and F ) Expression profile means ± SEM in log scale for the two UBA1-dependent clusters: cluster 1 ( E ) and cluster 4 ( F ). ( G and H ) IPA analysis showing the top network for UBA1 overexpression/control ( G ) and UBA1 knockdown/control ( H ), proteins highlighted in magenta are upregulated, proteins highlighted in green are downregulated. See also – and .

Article Snippet: HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples.

Techniques: Expressing, Over Expression

Journal: Brain

Article Title: UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

doi: 10.1093/brain/awy237

Figure Lengend Snippet: Gene ontology term enrichment of proteins changed following modulation of UBA1 expression

Article Snippet: HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples.

Techniques: Activity Assay, Binding Assay, Conjugation Assay

Journal: Brain

Article Title: UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

doi: 10.1093/brain/awy237

Figure Lengend Snippet: UBA1 influences GARS expression through a non-canonical function. ( A and B ) HEK293 cells were transfected with ubiquitin-HA (Ub-HA) and UBA1 ( A ) or UBA1 siRNA ( B ); western blot of UBA1 and ubiquitin (immunoblotted for HA tag), showing polyubiquitylated substrate proteins (Poly-Ub), and free triubiquitin (Tri-Ub), diubiquitin (Di-Ub) and monoubiquitin (Mono-Ub). ( C and D ) HEK293 cells were transfected with GARS-GFP or GFP along with Ub-HA and UBA1 ( C ) or UBA1 siRNA ( D ). Input control samples were immunoblotted for UBA1 and GARS; immunoprecipitation (IP) with GFP; IP samples were immunoblotted for GFP and HA. IgG bands and polyubiquitylation (Poly-Ub) smears are indicated. High intensity immunoblots show polyubiquitylation smears imaged at increased laser power. See also .

Article Snippet: HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples.

Techniques: Expressing, Transfection, Western Blot, Immunoprecipitation

Journal: Brain

Article Title: UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

doi: 10.1093/brain/awy237

Figure Lengend Snippet: GARS is dysregulated in neuronal tissue from SMA mice. ( A and B ) Western blot ( A ) and quantification ( B ) of GARS protein levels in spinal cord from late-symptomatic SMA mice and control littermates. α-Tubulin (α-Tub) = loading control. ( C and D ) Western blot ( C ) and quantification ( D ) of SMN, UBA1 and GARS protein levels in DRG from late-symptomatic SMA mice and control littermates. α-Tubulin (α-Tub), total protein (Ponceau): loading controls. n = 3 mice per condition. See also and .

Article Snippet: HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples.

Techniques: Western Blot

Journal: Brain

Article Title: UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

doi: 10.1093/brain/awy237

Figure Lengend Snippet: Dysregulation of UBA1 and GARS in dorsal root ganglia sensory neurons from SMA mice. ( A and C ) Spinal column sections from lumbar segments 1 and 2 of late-symptomatic SMA and control mice labelled with UBA1a ( A ) or GARS ( C ) (green), SMI32 (magenta) and DAPI. DRG are outlined in overview panels, boxes indicate the area in lower panels. Arrows in bottom panel indicate sensory neurons with altered UBA1 ( A ) or GARS ( C ) expression. N = NF200-positive neurons; P = peripherin-positive neurons. Scale bars = 100 µm ( overview ), 10 µm ( lower panels). ( B and D ) Late-symptomatic SMA DRG sensory neurons show a reduction in the nuclear to cytoplasmic ratio of UBA1 ( B ) and an increase in the nuclear to cytoplasmic ratio of GARS ( D ) compared to control mice. n = 3 mice per condition, n = 4 DRGs per mouse (14 sensory neurons were analysed per DRG); * P < 0.05, *** P ≤ 0.001.

Article Snippet: HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples.

Techniques: Expressing

Journal: Brain

Article Title: UBA1/GARS-dependent pathways drive sensory-motor connectivity defects in spinal muscular atrophy

doi: 10.1093/brain/awy237

Figure Lengend Snippet: Restoration of UBA1 in SMA mice reverses GARS dysregulation and rescues sensory neuron cell fate phenotypes. ( A and B ) Representative fluorescent western blot ( A ) and quantification ( B ) of SMN, UBA1 and GARS in dorsal root ganglia from late-symptomatic SMA mice and SMA mice injected with AAV9-UBA1 (SMA+AAV9-UBA1). α-Tubulin (α-Tub): loading control. n = 3 mice per condition. ( C ) Spinal column sections from lumbar segments 1 and 2 of late-symptomatic SMA and SMA+AAV9-UBA1 mice labelled with UBA1a (green), SMI32 (magenta) and DAPI. ( D ) Spinal column sections from lumbar segments 1 and 2 of late-symptomatic SMA and SMA+AAV9-UBA1 mice labelled with NF200 (magenta) and peripherin (green). ( C and D ) Dorsal root ganglia (DRG) are outlined. ( E and F ) Quantification of the percentage of NF200-positive (NF200 + ) ( E ) and peripherin-positive (peripherin + ) ( F ) sensory neurons. Data from control mice is shown as reference. n = 3 mice per condition, n = 4 DRGs per mouse. ( G and H ) Quantification of the area of NF200 + ( G ) and peripherin + ( H ) sensory neurons. n = 3 mice per condition, n = 2 DRGs per mouse (seven NF200 + and seven peripherin + neurons per DRG were analysed); ns = not significant. * P < 0.05, ** P < 0.01. See also and .

Article Snippet: HEK293 cells were transfected with RNAiMAX (Invitrogen) and 2.5 µM Silencer® Select Validated UBA1 siRNA (s601, targeted against exons 24 and 25; Life Technologies), or with Lipofectamine™ (Invitrogen) and 3.5 µg pCMV6-XL4-UBA1 plasmid (Origene) for proteomics samples.

Techniques: Western Blot, Injection

Journal:

Article Title: Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery

doi: 10.1021/cb9000348

Figure Lengend Snippet: Ub/Ubl activating enzymes (E1s), conjugating enzymes (E2s) and ligases (E3s) identified from (a) mouse lymphoma (EL4) and (b) human mammary epithelial cell lysates (HMLE).

Article Snippet: Autoubiquitination activity was tested in a 20 μL reaction by incubating ARF-BP1 HECT domain (10 μg) with 100 ng human E1 (Ube1, Boston Biochem), 1 μg UbcH7, and 10 μg [ 32 P]-Ub (see Supporting Information for plasmid construction, protein expression and purification conditions, and [ 32 P]-labeling) with an ATP regenerating system (50 mM Tris [pH 7.6], 5 mM MgCl 2 , 5 mM ATP, 10 mM creatine phosphate, 3.5 U/mL creatine kinase) for 10 min at room temperature.

Techniques: Activity Assay

Journal:

Article Title: Ubiquitin C-terminal electrophiles are activity-based probes for identification and mechanistic study of ubiquitin conjugating machinery

doi: 10.1021/cb9000348

Figure Lengend Snippet: Detection of Ub-thioesters in ARF-BP1 HECT domain. (a) Ub-thioester assay with the indicated ARF-BP1 HECT domain proteins. Purified ARF-BP1 HECT domain proteins were incubated with recombinant E1 and E2 (UbcH7), an ATP regenerating system, and Ub. Reactions were stopped with 4 M urea and either reducing (left panel) or non-reducing (right panel) SDS-PAGE sample buffer, separated by SDS-PAGE, and analyzed by immunoblot with anti-Ub antibody. (b) Ub thioester assay with the indicated ARF-BP1 truncation proteins was performed with all necessary components; without UBE1; or without UbcH7. (c) Substrate ubiquitination assay using the indicated ARF-BP1 HECT domain proteins and recombinant Mcl-1 as substrate. Reactions contained recombinant UBE1, UbcH7, Ub, an ATP regenerating system, and 1 μg recombinant Flag-labeled Mcl-1. Reaction mixtures were quenched as above, separated on SDS-PAGE and analyzed by anti-Flag immunoblot. (d) Substrate ubiquitination assay using the indicated ARF-BP1 HECT domain with all components; no UBE1; and no UbcH7.

Article Snippet: Autoubiquitination activity was tested in a 20 μL reaction by incubating ARF-BP1 HECT domain (10 μg) with 100 ng human E1 (Ube1, Boston Biochem), 1 μg UbcH7, and 10 μg [ 32 P]-Ub (see Supporting Information for plasmid construction, protein expression and purification conditions, and [ 32 P]-labeling) with an ATP regenerating system (50 mM Tris [pH 7.6], 5 mM MgCl 2 , 5 mM ATP, 10 mM creatine phosphate, 3.5 U/mL creatine kinase) for 10 min at room temperature.

Techniques: Purification, Incubation, Recombinant, SDS Page, Western Blot, Ubiquitin Proteomics, Labeling

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Sequencing traces obtained with Uba1 DNA (prepared by reverse transcription of pools of mRNA or from genomic DNA) from wild-type CHO-K1 and mutant tsTM3 cells. The wild type contains a G at nucleotide 768 of Uba1 , whereas the mutant contains an A. The G-to-A transition at nucleotide 768 converts Met to Ile at amino acid 256. (B) Structure of mammalian Uba1. Colored boxes represent four functional domains, adenylation domains (IAD and AAD), catalytic cysteine half-domains (FCCH and SCCH), four-helix bundle domain (4HB), and C-terminal ubiquitin-fold domain (UFD). Locations of the mutation found in ts mutant ts20 and in X-linked spinal muscular atrophy (XL-SMA) are shown above the diagram. Alignments were made with CLUSTAL W relative to positions 203–301 of hamster Uba1 (Accession No. AB661372) with sequences from Homo sapiens (H. s.; NCBI Gene ID: 7317), Mus musculus (M. m.; ID: 22201), and Rattus norvegicus (R. n.; ID: 314432). Asterisks indicate identical residues.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Sequencing, Mutagenesis, Functional Assay

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Western blot analysis of Uba1 in tsTM3 cells. Wild-type CHO-K1 or ts mutant tsTM3 cells were grown at 34°C or incubated at 39°C for the times shown in the figure and lysed. The proteins resolved on acrylamide gels and were detected by immunoblotting with an antibody directed against Uba1 (rabbit polyclonal antibody supplied from Rockland). Uba1 exists as two isoforms: Uba1A (∼117 kDa), localized predominantly in the nucleus and Uba1B (∼110 kDa), localized in the cytoplasm. Similar results were obtained from another antibody (rabbit polyclonal from Calbiochem). Only relevant parts of the blot are shown. SMC3 was included as a loading control. Temperature-dependent reduction in the amount of Uba1 was found in tsTM3 cells. (B, C) Quantitative analyses of the amounts of Uba1 in CHO-K1 and tsTM3 cells. Band intensities, like those shown in panel (A), were measured and expressed relative to that of each at 34°C or relative to the lower band at 34°C with standard deviation of at least three experiments. Bands of Uba1B at 34°C in each cell are selected as a control to compare the amount of Uba1 because it appeared to be permanent. P values were calculated by Student t -test. Significant differences from values at 34°C (B, gray bars; C, solid gray bars) are shown by asterisks. Incubation at 39°C resulted in a significant decrease in Uba1A in tsTM3 cells, although the proportion of Uba1A to Uba1B in mutant cells at 34°C was smaller than that of wild-type CHO-K1.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Western Blot, Mutagenesis, Incubation, Standard Deviation

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Isoform of Uba1 tagged with GFP and its localization. Cell lines expressing GFP-Uba1 or Uba1-GFP constructs were isolated from the ts mutant cell line, tsTM3. Uba1 tagged with GFP complements deficiencies in tsTM3 cells and allows them to grow normally at 39°C. Cells were counterstained with Hoechst 33342. The fluorescent Uba1 in the GFP-Uba1 derivative is mainly nuclear, whereas the other derivative, Uba1-GFP, contains higher concentrations in both nucleus and cytoplasm. Bar, 10 µm. (B) Western blot analysis of Uba1 tagged with GFP. Cells expressing GFP-Uba1 or Uba1-GFP, as well as parental (tsTM3) and grandparental (CHO-K1) cells were lysed and the proteins resolved on acrylamide gels. Six samples of each distinct GFP clone were analyzed: lanes 3–8, GFP-Uba1; lanes 9–14, Uba1-GFP. Different forms of Uba1 were detected by immunoblotting with antibodies directed against Uba1 and GFP. Only relevant parts of the blot are shown. α-tubulin was included as a loading control. Positions of endogenous and hybrid Uba1 are shown. Band intensities relative to that of the endogenous cytoplasmic form of Uba1 in each of the clones are presented below the blot of Uba1. A derivative of tsTM3 cells, tm3UG16, appears to express very little Uba1-GFP, suggesting the possibility of spontaneous reversion. It is also possible that the rescue depends on a cleaved form of hybrid that resembles the endogenous form but which can no longer be detected via fluorescence. Slightly different migration of bands is observed in tm3UG11. (C) Images of living tsTM3 cells expressing Uba1 tagged with GFP. Cells were counterstained with Hoechst 33342. Upper and middle rows represent living interphase cells expressing the GFP-Uba1 construct. The fluorescent Uba1 in a derivative, tm3GU1, is predominantly in the nucleus. Living mitotic cells (bottom). Bar, 10 µm.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Expressing, Construct, Isolation, Mutagenesis, Western Blot, Clone Assay, Fluorescence, Migration

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Diagram of the structures of the full-length and truncated forms of Uba1. Boxes represent the domains of Uba1 in . (B) Photographs of colonies of tsTM3 cells after transfection of 2 µg of plasmid DNAs encoding GFP hybrids of Uba1 or Uba1D1-40 and vectors. After 14 days of incubation at 39°C or 34°C with 400 µg/ml G418, the colonies on the dishes were stained with methylene blue. Cells survived at 39°C after transfection of Uba1 derivatives; however, relatively few cells and no cells grew after transfection of Uba1D1-40 derivatives and vectors, respectively. There was no difference in the colony formation between plasmid DNAs encoding GFP hybrids of Uba1 and Uba1D1-40 after the selection for resistance to G418. (C) Quantitative analysis of colony formation at 39°C. Colonies such as those shown in panel (B) were counted. The colony formation efficiencies of 39°C were normalized to those of 34°C with G418 and are expressed with a standard deviation of at least three experiments. P values were calculated by Student t -test. Significant differences from the efficiency of GFP-Uba1 are shown by asterisks.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Transfection, Plasmid Preparation, Incubation, Staining, Selection, Standard Deviation

Journal: PLoS ONE

Article Title: Characterization of Ubiquitin-Activating Enzyme Uba1 in the Nucleus by Its Mammalian Temperature-Sensitive Mutant

doi: 10.1371/journal.pone.0096666

Figure Lengend Snippet: (A) Western blot analysis of Fucci in CHO-K1 or tsTM3 cells. Wild-type CHO-K1 or ts mutant tsTM3 cells expressing Fucci-G 1 Orange or Fucci-S/G 2 /M Green were grown at 34°C or incubated at 39°C for the times shown in the figure and lysed. The proteins resolved on acrylamide gels and were detected by immunoblotting with antibodies directed against fluorescent tags (mKO2 or mAG1) and Uba1. Only relevant parts of the blot are shown. α-tubulin is included as a loading control. Temperature-dependent increases in the amount of Fucci with the reduction of Uba1 were found in tsTM3 cells. (B) Quantitative analysis of bands in blots of tsTM3 cells expressing Fucci. Band intensities of Fucci and Uba1, like those shown in panel (A), were measured and are expressed relative to that of each at 34°C or relative to the lower band at 34°C with standard deviation of at least three experiments. P values were calculated by Student t -test. Significant differences from values at 34°C are shown by asterisks. Incubation at 39°C resulted in a significant increase of Fucci (mKO2-hCdt1 or mAG1-hGem) with decrease in Uba1A in tsTM3 cells.

Article Snippet: The Bgl II- Sal I restriction fragment containing the coding sequence of hamster Uba1 cDNA was cloned into the vectors pEGFP-C1 and pEGFP-N3 (Clontech Laboratories, Palo Alto, CA) to yield the plasmids GFP-Uba1 and Uba1-GFP, respectively.

Techniques: Western Blot, Mutagenesis, Expressing, Incubation, Standard Deviation