Journal: Nature Communications

Article Title: Loss of TRIM29 mitigates viral myocarditis by attenuating PERK-driven ER stress response in male mice

doi: 10.1038/s41467-024-44745-x

Figure Lengend Snippet: a Immunoblot (IB) analysis of endogenous proteins TRIM29 and PERK precipitated with anti-PERK or immunoglobulin G (IgG) from whole-cell lysates of mouse primary neonatal cardiomyocytes from Trim29 fl/fl and WTMyHC-Cre mice with or without (Mock) CVB3 and EMCV infection for 6 h at an MOI of 5. Schematic diagram showing full-length PERK (Full, b ) or TRIM29 (Full, c ) and serial truncations of PERK ( b ) or TRIM29 ( c ) with deletion (Δ) of various domains (left margin); numbers at ends indicate amino acid positions (top). Luminal, ER luminal domain; TM, transmembrane domain; Kinase, protein kinase domain; BBOX, the B-box zinc-finger domain; CC, the coil-coil domain; OmpH, the outer membrane protein H domain. Immunoblot analysis of purified HA-tagged full-length PERK and serial truncations of PERK with deletion of various domains alone ( d ) or purified HA-tagged full-length TRIM29 and serial truncations of TRIM29 with deletion of various domains alone ( e ) with anti-HA antibody (top blot) or after incubation with Myc-tagged TRIM29 ( d ) or Myc-tagged PERK ( e ) and immunoprecipitation with anti-Myc antibody (second blot), and immunoblotting analysis of purified Myc-tagged TRIM29 ( d ) or Myc-tagged PERK ( e ) with anti-Myc antibody (third blot) or after incubation with Myc-tagged TRIM29 ( d ) or Myc-tagged PERK ( e ) and immunoprecipitation with anti-Myc antibody (bottom blot). f Immunoblot analysis of endogenous phosphorylated PERK (pPERK), HA-tagged PERK and Myc-tagged TRIM29 from whole-cell lysates of HEK293T cells co-transfected with HA-PERK and vector or Myc-TRIM29 for 24 h. g Immunoblot analysis of endogenous proteins TRIM29 and phosphorylated PERK (pPERK) precipitated with anti-pPERK or immunoglobulin G (IgG) from whole-cell lysates of mouse primary neonatal cardiomyocytes from Trim29 fl/fl mice followed by CVB3 infection at an MOI of 5 and treatment with the PERK inhibitor GSK2656157 or DMSO for 6 h. The position of protein markers (shown in kDa) is indicated on the right. Data are representative of three independent experiments. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used for immunoblot analysis: anti-TRIM29 (1:1000; A301-210A; Bethyl), anti-TRIM29 (1:1000; sc-33151; Santa Cruz), anti-ANP (1:1000; sc-515701; Santa Cruz), anti-FITC (1:2000; 71–1900; Thermo Fisher Scientific), antibody to phosphorylated PERK (1:1000; MA5-15033; Thermo Fisher Scientific), anti-PERK (1:1000; 3192 S; Cell Signaling Technology), anti-IRE1α (1:1000; 3194 S; Cell Signaling Technology), anti-ATF6 (1:1000; 65880 S; Cell Signaling Technology), anti-CHOP (1:1000; 2895 S; Cell Signaling Technology), anti-cleaved caspase-3 (1:1000; 9664 S; Cell Signaling Technology), anti-BCL2 (1:1000; 3498 S; Cell Signaling Technology), anti-BAX (1:1000; 2772 S; Cell Signaling Technology), anti-STING (1:1000; 13647 S; Cell Signaling Technology), anti-IRF3 (1:1000; 4302 S; Cell Signaling Technology), anti-TBK1 (1:1000; 3504 S; Cell Signaling Technology), anti-SUMO1 (1:1000; 4930 S; Cell Signaling Technology), anti-SUMO2/3 (1:1000; 4971 S; Cell Signaling Technology), antibody to phosphorylated IRF3 at Ser396 (1:1000; 4947 S; Cell Signaling Technology), antibody to phosphorylated TBK1 at Ser172 (1:1000; 5483 S; Cell Signaling Technology), antibody to phosphorylated IRE1α (1:1000; ab48187, Abcam), anti-His (1:5000; 652504; BioLegend), anti-Flag (1:5000; A8592; Sigma), anti-GAPDH (1:20,000; G9295; Sigma), anti-HA (1:5000; H6533; Sigma) and anti-Myc (1:5000; 16–213; Sigma).

Techniques: Western Blot, Infection, Membrane, Purification, Incubation, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Commonality and diversity in tRNA substrate recognition in t 6 A biogenesis by eukaryotic KEOPSs

doi: 10.1093/nar/gkac056

Figure Lengend Snippet: t 6 A is introduced to mature tRNAs in the cytoplasm. Time course curves of the t 6 A modification of four transcripts, tRNA Arg (UCU) (blue filled squares), pre-tRNA Arg (UCU) (green squares), hctRNA Ile (UAU) (peach triangles), pre-hctRNA Ile (UAU) (purplish red triangles), and controls (no tRNA addition, black circles), by YRDC and hKEOPS ( A ) or by Sua5 and Sc KEOPS ( B ). The data represent the average of three independent replicates and the standard deviations. Subcellular localization of endogenous OSGEP and TP53RK ( C ) or overexpressed OSGEP-FLAG ( D ) and TP53RK-FLAG ( E ) analyzed by nucleocytoplasmic separation assays. The red arrow represents OSGEP in (C). Cytoplasmic (Cyto) and nuclear (Nuc) fractions were separated from HEK293T cells. GAPDH and Lamin A/C were used as markers of the Cyto and Nuc fractions, respectively. ( F ) Immunofluorescence determination of endogenous OSGEP and TP53RK in HEK293T cells. ( G ) Fluorescence determination of the localization of the five relevant subunits (OSGEP-EGFP, TPRKB-EGFP, GON7-EGFP, TP53RK-EGFP and LAGE3-EGFP). Thu nucleus was stained with DAPI in (F) and (G).

Article Snippet: Anti-GAPDH (60004-1-Ig) and anti-OSGEP (15033-1-AP) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Modification, Immunofluorescence, Fluorescence, Staining

Journal: Nucleic Acids Research

Article Title: Commonality and diversity in tRNA substrate recognition in t 6 A biogenesis by eukaryotic KEOPSs

doi: 10.1093/nar/gkac056

Figure Lengend Snippet: t 6 A is a critical positive determinant in aminoacylation of tRNA Ile (AAU) and tRNA Ile (GAU) but not tRNA Ile (UAU) isoacceptors by human cytoplasmic IleRS. ( A ) Western blot results showing OSGEP protein levels in HEK293T cells infected with OSGEP-specific (shRNA1, shRNA2) or control (Scramble) shRNAs. The red arrow represents OSGEP. ( B ) UPLC-MS/MS quantification of t 6 A modification levels in tRNA Lys (UUU) purified from WT and two KD (shRNA1 and shRNA2) cells. The amounts of t 6 A and N (= A+C+G+U) were calculated based on the area under the t 6 A or N peaks in the UPLC-MS/MS chromatogram. The data were two independent replicates and the corresponding standard deviations. The P values were determined using two-tailed Student's t test for paired samples. * P < 0.05; ** P < 0.01. ( C ) Determination of tRNA charging levels of three tRNA Ile isoacceptors in wild-type (Scramble) and OSGEP knockdown HEK293T cell lines by acid northern blots. The relative aminoacylation levels were calculated based on two independent replicates and their corresponding standard deviations. DA, de-aminoacylated tRNA. The P values were determined using two-tailed Student's t test for paired samples. * P < 0.05; ** P < 0.01. ( D ) UPLC-MS/MS analysis of the digested products of hypo- or modified tRNA Ile (UAU). Aminoacylation of hctRNA Ile (AAU) (red filled squares) and t 6 A-hctRNA Ile (AAU) (orange filled triangles) ( E ); or hctRNA Ile (GAU) (red filled squares) and t 6 A-hctRNA Ile (GAU) (orange filled triangles) ( F ) or hctRNA Ile (UAU) (red filled squares) and t 6 A-hctRNA Ile (UAU) (orange filled triangles) ( G ) by hIleRS. Controls (black filled circles) represent assays in which no tRNAs were added. The data represent the averages of three independent experiments and the corresponding standard deviations.

Article Snippet: Anti-GAPDH (60004-1-Ig) and anti-OSGEP (15033-1-AP) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Western Blot, Infection, Control, Tandem Mass Spectroscopy, Modification, Purification, Two Tailed Test, Knockdown, Northern Blot

Journal: Nucleic Acids Research

Article Title: Commonality and diversity in tRNA substrate recognition in t 6 A biogenesis by eukaryotic KEOPSs

doi: 10.1093/nar/gkac056

Figure Lengend Snippet: Effects of t 6 A deficiency on codon-anticodon pairing and +1 frameshifting. ( A ) Schematic showing the dual-luciferase system, in which 6× ANN codons were inserted downstream of the F-luc gene ATG start codon in a pmirGLO plasmid, and a separate R-luc gene was used as a control. ( B ) Effects of t 6 A deficiency on ANN-codon (in black in the x-axis) decoding efficiency by various tRNAs (in red in the x-axis) in WT (Scramble) and two OSGEP KD cell lines. 6× TCT codons decoded by non-t 6 A-modified tRNA Ser (AGA) were included as controls. The data represent the average of two independent replicates and the standard deviations. The P values were determined using two-tailed Student's t test for paired samples. * P < 0.05; ** P < 0.01 and *** P < 0.001. ( C ) Schematic showing the +1 frameshifting assay at the ATT codon decoded by tRNA Ile (AAU). An “A” nucleotide (indicated by red) was inserted upstream of the ATT codon at position 7 of the Ile codon of F-luc (AATT-F-Luc). The data represent the average of three independent replicates and the standard deviations. The P values were determined using two-tailed Student's t test for paired samples. * P < 0.05; ** P < 0.01 and *** P < 0.001.

Article Snippet: Anti-GAPDH (60004-1-Ig) and anti-OSGEP (15033-1-AP) antibodies were purchased from Proteintech (Wuhan, China).

Techniques: Luciferase, Plasmid Preparation, Control, Modification, Two Tailed Test