Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A ) Domain composition of tRNA-LC subunits. RecA, RecA-like domain; CH, calponin homology-like domain; CC, coiled coil; LR, leucine-rich region; NLS, nuclear localization signal. ( B ) Deletion analysis of tRNA-LC subunits. StrepTactin (upper left), Amylose (upper right), and Ni-NTA (bottom left) affinity pull-down assays using lysates of Sf9 cells expressing full-length tRNA-LC subunits (affinity tags as indicated in the figure). The input (bottom right) and bound fractions were analyzed by SDS-PAGE and visualized by in-gel GFP (middle gel) or mCherry (bottom gel) fluorescence followed by Coomassie Blue staining (upper gel). tRNA-LC = RTCB:DDX1:FAM98B:CGI-99:ASW, omitted subunits in the deletion constructs are indicated (Δ). ( C ) Size-exclusion chromatography interaction analysis of the core tRNA-LC (RTCB:DDX1:FAM98B:CGI-99) and Archease. SDS-PAGE analysis of the elution peak components is shown in the inset. The estimated molecular weights of the peaks based on their elution volumes are indicated.

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: Expressing, SDS Page, Fluorescence, Staining, Construct, Size-exclusion Chromatography

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A ) SDS-PAGE analysis of the purified human tRNA-LC containing RTCB, DDX1, FAM98B, CGI-99, and ASHWIN. ( B ) In vitro RNA circularization ligation assay. Recombinant full-length tRNA-LC (RTCB:DDX1:FAM98B:CGI-99:ASHWIN), core tRNA-LC (RTCB:DDX1:FAM98B:CGI-99), minimal tRNA-LC (RTCB:DDX1(696-740):FAM98B(200-239):CGI-99(102-244)), and RTCB were incubated with radiolabeled 21-nt single-stranded RNA in the presence of Archease. RNA circularization was analyzed by denaturing PAGE and visualized by autoradiography. ( C ) In vitro RNA circularization ligation assay as in ( B ) with or without ATP.

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: SDS Page, Purification, In Vitro, Ligation, Recombinant, Incubation, Autoradiography

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A ) Intra-molecular cross-links identified in the five-subunit tRNA-LC sample are shown as dashed lines. ( B ) Inter-molecular cross-links identified in the four-subunit core tRNA-LC containing RTCB, DDX1, FAM98B, and CGI-99 subunits. Domain composition is indicated as in . The dashed lines indicate cross-links observed at least once in two replicates.

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques:

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A ) Inter-subunit cross-links between RTCB, DDX1, CGI-99, and FAM98B subunits within the five-subunit full tRNA-LC are shown as dashed lines. ( B ) Inter-subunit cross-links between ASW and the remaining subunits of the full tRNA-LC. Domain schematic as in . The dashed lines indicate cross-links observed at least once in two replicates. Figure 2—source data 1. Cross-links identified in the cross-linking and mass spectrometric (XL-MS) analysis of the full tRNA-LC. Figure 2—source data 2. Cross-links identified in the cross-linking and mass spectrometric (XL-MS) analysis of the core tRNA-LC.

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques:

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A ) Crystal structure of the N-terminal region of human CGI-99 (residues 2–101). The N- and C-termini, as well as the N-terminal extension of CGI-99 (residues 2–18), are indicated. ( B ) DALI pairwise alignment of CGI-99(2-101) (yellow) with human NDC80 (blue, PDB ID: 2ve7). ( C ) Pull-down assays using pre-immobilized GST-CGI-99(2-101) with lysates from HEK293T cells transiently overexpressing HA-(StrepII) 2 -GFP-FAM98B constructs. The input (I) and bound (B) fractions were analyzed by SDS-PAGE and visualized by in-gel GFP fluorescence (upper panel) followed by Coomassie Blue staining (lower panel). The ‘no bait’ control contained HEK293T lysate incubated with GSH resin in the absence of GST-CGI-99, ‘no prey ctrl’ contained GSH resin incubated with GST-CGI-99(2-101).

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: Construct, SDS Page, Fluorescence, Staining, Incubation

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A, B ) DALI structural alignment of the N-terminal domain of CGI-99 (yellow) with ( A ) Chlamydomonas reinhardtii IFT54 (green, PDB ID: 5fmt) and ( B ) Escherichia coli Lon protease (pink, PDB ID: 3ljc). N- and C-termini are indicated. ( C, D ) Pull-down assays of immobilized ( C ) GST-CGI-99(1-244) or ( D ) GST-CGI-99(101-244) with lysates of HEK293T cells transiently overexpressing HA-(StrepII) 2 -GFP-FAM98B constructs. The input (I) and bound (B) fractions were analyzed by SDS-PAGE and visualized by in-gel GFP fluorescence (upper panel) followed by Coomassie Blue staining (lower panel). The ‘no bait’ control contained HEK293T lysate incubated with GSH resin in the absence of GST-CGI-99, ‘no prey ctrl’ contained GSH resin incubated with one of the GST-CGI-99 constructs, and ‘CGI-99(1-244) control’ is the bound fraction of GSH-immobilized GST-CGI-99(1-244) incubated with HEK293T cell lysate expressing HA-(StrepII) 2 -GFP-FAM98B(2-301).

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: Construct, SDS Page, Fluorescence, Staining, Incubation, Expressing

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A ) Size-exclusion chromatogram of the complex before (blue) and after (orange) limited proteolysis. Elution fractions from SEC were resolved by SDS-PAGE and stained with Coomassie Blue (right and lower panels). Unidentified bands are indicated with an asterisk. ( B ) Masses identified by electrospray ionization mass spectrometry analysis of the RTCB:DDX1(436-740):FAM98B:CGI-99 (upper panel), peak A (middle panel) and peak B (bottom panel) from limited proteolysis of RTCB:DDX1(436-740):FAM98B:CGI-99.

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: SDS Page, Staining, Mass Spectrometry

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: ( A–C ) Truncation analysis of the core tRNA-LC (RTCB:DDX1:FAM98B:CGI-99). Pull-down assays on lysates of Sf9 cells expressing constructs of the core tRNA-LC with truncated DDX1 ( A ), CGI-99 ( B ), or FAM98B ( C ). The input and bound fractions were analyzed by SDS-PAGE and visualized by in-gel GFP (middle panel) or mCherry (bottom panel) fluorescence followed by Coomassie Blue staining (upper panel). ( D ) Top: size-exclusion chromatography purification of the minimal tRNA-LC, RTCB:DDX1(696-740):FAM98B(200-239):CGI-99(102-244). The estimated molecular weight of the peak according to its elution volume is indicated. Bottom: SDS-PAGE analysis of the final sample. The collected fractions are indicated by gray dashed lines. Figure 4—source data 1. Mass spectrometry analysis of protein fragments resulting from limited proteolysis of RTCB:DDX1(436-740):FAM98B:CGI-99.

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: Expressing, Construct, SDS Page, Fluorescence, Staining, Size-exclusion Chromatography, Purification, Molecular Weight, Mass Spectrometry

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: (A) Recombinant core tRNA-LC (RTCB:DDX1:FAM98B:CGI-99) and RTCB were incubated with radiolabeled single-stranded RNA substrate in the presence of Archease and divalent metal ions. Substrate RNA circularization was analyzed by denaturing PAGE and visualized by autoradiography. (B) Fraction of substrate conversion in (A) as measured by densitometric quantification of band intensities.

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: Recombinant, Incubation, Autoradiography

Journal: eLife

Article Title: Molecular architecture of the human tRNA ligase complex

doi: 10.7554/eLife.71656

Figure Lengend Snippet: Affinity purifications from Sf9 cells expressing RTCB(64-505):DDX1:FAM98B:CGI-99. The input and bound fractions were analyzed by SDS-PAGE and visualized by in-gel GFP (middle panel) or mCherry (bottom panel) fluorescence followed by Coomassie Blue staining (upper panel).

Article Snippet: Similarly, DNA encoding CGI-99 (UniProt Q9Y224) was cloned into site 1 of the UC Berkeley MacroLab 5A vector (gift from Scott Gradia, Addgene plasmid #30121), while DNA sequence encoding FAM98B (UniProt Q52LJ0) N-terminally fused to StrepII tag, GFP, and TEV protease cleavage site (amplified from the UC Berkeley MacroLab 438-Rgfp vector, Addgene plasmid #55221) was cloned into site 2 of the same vector.

Techniques: Expressing, SDS Page, Fluorescence, Staining