Journal: Science Advances

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and shape their functional biology

doi: 10.1126/sciadv.adu6354

Figure Lengend Snippet: ( A ) Schematic depicting functional relationships between UBQLN2, RTL8, and PEG10. Both UBQLN2 and PEG10 interact with RTL8, forming a hypothetical trimolecular complex (I) that may affect UBQLN2-dependent proteasomal degradation of PEG10 (II) and SG localization of both proteins (III). ( B ) Volcano plot of proteins significantly decreased (blue) or increased (red) in RTL8 KO HEK293 cells compared to control cells (combination of HEK293 WT and NTC guide RNA–treated cells). n = 3 biological replicates from one WT, two NTC, and two RTL8 KO clonal lines. Cutoff was set at P < 0.05 and an absolute fold change of 2. Also, see table S1. ( C ) Phylogenetic tree of human RTL proteins, with branch lengths proportional to evolutionary distances. Colored ovals highlight RTL proteins studied here. ( D ) Domain organization of PEG10, RTL8, and LDOC1 showing shared retrovirus-like features. PEG10 gag-pol is generated by ribosomal frameshifting at the frameshift site. ( E ) Immunoblot of lysates from WT, NTC, and RTL8 KO HEK293 cells. Relative PEG10 gag-pol levels, normalized to levels in WT cells, are shown below. Also, see fig. S1 (A to D). ( F ) Immunoblot of lysates from various human cell lines treated with siRNA against RTL8 or UBQLN2 or control (Ctrl) siRNA. Relative PEG10 gag-pol levels, normalized to levels in control siRNA-treated cells, are shown below. Also, see fig. S1 (E to G). ( G ) Immunoblot of lysates from NTC or RTL8 KO HeLa cells expressing HA-tagged RTL8C, HA-RTL8C ΔN, Myc-tagged LDOC1, FLAG-tagged UBQLN2, or empty vector (Vec). Relative PEG10 gag-pol levels, normalized to levels in RTL8 KO cells transfected with an empty vector, are shown below. Also, see figs. S1H and S2. In [(E) to (G)], data represent the means ± SD ( n = 3), analyzed with one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. GAPDH, loading control. Third-party graphical elements in (A) are incorporated from Bioicons under a CC-0 license.

Article Snippet: HEK293 T-rex control and UBQLN1, UBQLN2, and UBQLN4 knockout (TKO) cell lines were maintained in complete DMEM supplemented with blasticidin S (10 μg/liter; Thermo Fisher Scientific), and hygromycin (100 μg/ml; Thermo Fisher Scientific).

Techniques: Functional Assay, Control, Generated, Western Blot, Expressing, Plasmid Preparation, Transfection, Comparison

Journal: Science Advances

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and shape their functional biology

doi: 10.1126/sciadv.adu6354

Figure Lengend Snippet: ( A ) Lysates from NTC or RTL8 KO HEK293 cells were immunoprecipitated with control or α-UBQLN2 antibody in the absence or presence of the cross-linker DTBP. The amount of PEG10 or RTL8 coimmunoprecipitated, relative to immunoprecipitated UBQLN2 and normalized to NTC HEK293 without DTBP addition, is shown below. ( B ) PLA assessing interaction between endogenous UBQLN2 and PEG10 in the indicated HeLa cell lines. Nuclei and cell membranes are labeled with DAPI and WGA, respectively. Quantification of PLA foci/cell is shown below. The number of individual cells quantified per line from two independent replicates is indicated. Scale bars, 10 μm. Lysates from ( C ) NTC or RTL8 KO HeLa cells expressing full-length (FL) HA-RTL8C, HA-RTL8C ΔN, or empty vector or ( D ) NTC or PEG10 KO HeLa cells expressing FL V5-PEG10 gag-pol, V5-PEG10 gag-pol ΔPPR, or empty vector were immunoprecipitated with α-UBQLN2 Ig. The amount of coimmunoprecipitated PEG10 or RTL8, relative to immunoprecipitated UBQLN2 and normalized to NTC HeLa, is shown below. Also, see fig. S3. In [(A) to (D)], data were analyzed using one-way ANOVA with Tukey’s multiple comparison test. In [(A), (C), and (D)], data represent the means ± SD ( n = 3). GAPDH, loading control.

Article Snippet: HEK293 T-rex control and UBQLN1, UBQLN2, and UBQLN4 knockout (TKO) cell lines were maintained in complete DMEM supplemented with blasticidin S (10 μg/liter; Thermo Fisher Scientific), and hygromycin (100 μg/ml; Thermo Fisher Scientific).

Techniques: Immunoprecipitation, Control, Labeling, Expressing, Plasmid Preparation, Comparison

Journal: Science Advances

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and shape their functional biology

doi: 10.1126/sciadv.adu6354

Figure Lengend Snippet: ( A ) Schematic depicting possible SG localization of the UBQLN2-RTL8-PEG10 complex given the known SG localization of UBQLN2 and PEG10. ( B ) PLA assessing the interaction between endogenous UBQLN2 and PEG10 in HeLa cells induced to form SGs by sodium arsenite or heat shock. Nuclei and cell membranes are labeled with DAPI and WGA, respectively, and arrowheads in insets mark SGs stained with G3BP1. Quantification of PLA foci per square micrometers within SGs and in the whole cell is shown below. The number of individual cells quantified per condition from two independent replicates is indicated. One-way ANOVA with Tukey’s multiple comparison test was performed. Scale bars, 10 μm and 5 μm for insets. ( C ) B-isox–mediated precipitation of RNA granule proteins from three human cell lines. WCL, whole-cell lysate before B-isox addition; S, supernatant; P, pellet. G3BP1 and TIAR are positive controls, and GAPDH is a negative control. Ponceau S was used to normalize loading. ( D ) Three-color dSTORM of HeLa cells treated with sodium arsenite. Numbers identify the zoomed regions shown below. Arrowheads mark regions of overlap between PEG10 and G3BP1. The Jaccard index between G3BP1 clusters and UBQLN2 or PEG10 is shown at the top right. Dotted lines connect data points from an individual cell. An unpaired t test was performed. n = 15 cells across three independent replicates. Scale bars, 10 μm and 0.5 μm for zoomed regions.

Article Snippet: HEK293 T-rex control and UBQLN1, UBQLN2, and UBQLN4 knockout (TKO) cell lines were maintained in complete DMEM supplemented with blasticidin S (10 μg/liter; Thermo Fisher Scientific), and hygromycin (100 μg/ml; Thermo Fisher Scientific).

Techniques: Labeling, Staining, Comparison, Negative Control

Journal: Science Advances

Article Title: Endogenous retrovirus-like proteins recruit UBQLN2 to stress granules and shape their functional biology

doi: 10.1126/sciadv.adu6354

Figure Lengend Snippet: ( A ) After treatment with sodium arsenite, the indicated HeLa cell lines were immunostained for endogenous UBQLN2, PEG10, and the SG marker G3BP1. Arrowheads in insets mark SGs. UBQLN2 or PEG10 intensity within SGs was quantified and is shown to the right. Also, see figs. S4 and S5A. ( B ) NTC and RTL8 KO HeLa cells expressing HA-RTL8C, HA-RTL8C ΔN, or control protein (HA-mFAP10) treated with sodium arsenite. UBQLN2 intensity within SGs is shown at the bottom left. The Mander’s overlap coefficient (MOC) between HA and G3BP1 is shown at the bottom right. Also, see fig. S5B. ( C ) U-2 OS G3BP1-GFP cells expressing V5-PEG10 gag, gag-pol, gag-pol ΔPPR, or control protein (V5-Nanoluciferase) treated with sodium arsenite. The immunoblot of lysates from HeLa and U-2 OS G3BP1-GFP cells is shown at the bottom left. UBQLN2 intensity within SGs is shown at the bottom right. Also, see fig. S5C. In all panels, nuclei are labeled with DAPI. Scale bars, 10 μm and 5 μm for insets. In [(B) and (C)], filled arrowheads in insets indicate SGs with UBQLN2, and unfilled arrows indicate SG without UBQLN2. In all panels, one-way ANOVA with Tukey’s multiple comparison test was performed. The number of individual cells quantified per line/plasmid from three independent replicates is indicated.

Article Snippet: HEK293 T-rex control and UBQLN1, UBQLN2, and UBQLN4 knockout (TKO) cell lines were maintained in complete DMEM supplemented with blasticidin S (10 μg/liter; Thermo Fisher Scientific), and hygromycin (100 μg/ml; Thermo Fisher Scientific).

Techniques: Marker, Expressing, Control, Western Blot, Labeling, Comparison, Plasmid Preparation