EMBO/soda-nel huggingface dataset:
A Salmonella protein causes macrophage cell death by inducing autophagy. (2003)
Figure 8.

The S. typhimurium-induced macrophage death does not require caspase activity. BMDPM were infected with wild-type S. typhimurium or the TTSS-defective invG mutant in the presence or absence of 50 μM of the pan-caspase inhibitor Z-VAD, and cell death was measured as indicated in the legend to Fig. 1.
Selective Autophagy of RIPosomes Maintains Innate Immune Homeostasis during Bacterial Infection (2022)
Figure 2

Figure 2. RIPK2 oligomers provide a platform for NODs oligomerization

(A) Left panel, the domain organization map of NOD1, NOD2, and RIPK2. Right panel, representative confocal images of immunofluorescence assays performed in HEK293T cells transfected with GFP-NOD1, GFP-NOD2, and GFP-RIPK2 plasmids for 9 h. Scale bar 5 µm.

(B) Left panel, representative high-content microscopy images of HEK293T cells transfected with GFP-NOD1, GFP-NOD2, and GFP -RIPK2 plasmids for 9 h. Right panel, the graph depicts the average number of RIPosomes per GFP-positive cell. About 15000 cells were plated per well and RIPosomes were screened in 35 fields per well. Mean ± SD, n=3 (biological replicates), ****p < 0.00005, ordinary one-way ANOVA (Tukey's multiple comparisons test).

(C) Western blot analysis of soluble and insoluble fractions of HEK293T cells transfected with the CARD domain-containing region of NOD1, NOD2, and RIPK2.

(D) Representative confocal images of HEK293T cells transfected with GFP-NOD1CARD, GFP-NOD2CARD, and GFP- RIPK2CARD. Scale bar 5 µm.

(E) The soluble and insoluble fractions of S. flexneri infected THP-1 (MOI 1:25, 8 h) cell lysates were subjected to Western blot analysis with indicated antibodies.

(F) Representative confocal images of immunofluorescence assays conducted with THP-1 cells infected with RFP expressing S. flexneri (pseudo-colored, blue) (MOI 1:25, 8h). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications.

(G) Representative confocal images of immunofluorescence assays performed with HEK293T cells transfected with GFP-RIPK2 and Flag-NOD2 (upper panel) or Flag-NOD1 (lower panel). Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 or 10 µm as indicated in figures. Inset zoom panels are digital magnifications.

(H-I) HEK293T cells were transfected with (H) full length and (I) CARD domains of NOD1, NOD2, and RIPK2 followed by Western blot analysis with soluble and insoluble fractions using indicated antibodies.

(J-K) Western blot analysis of soluble and insoluble fractions of RIPK2 +/+ and RIPK2-/- HT-29 cells infected with S. flexneri (MOI 1:25, 8 h) with indicated antibodies.

(L-M) Representative immunofluorescence images of doxycycline-inducible GFP-RIPK2 expressing HeLa cells. (L) Upper panel, uninfected. Lower panel, S. flexneri -infected MOI 1:25, 4h). Immunostaining was performed with the p65 antibody (red) and DNA stained with DAPI (Blue). (M) The graph indicates % of cells that are RIPosomes positive or negative with nuclear/cytoplasmic p65 (5 fields (each group), Mean ± SD, n=3). ****p < 0.00005, Student's unpaired t-test.

.
Selective Autophagy of RIPosomes Maintains Innate Immune Homeostasis during Bacterial Infection (2022)
Figure 4

Figure 4. Autophagy protein IRGM interacts and colocalizes with NODs, RIPK2, and RIPosomes.

(A-B) The THP-1 cell lysates were subjected to immunoprecipitation analysis (A) untreated and treated with iE-DAP (40 μg/ml, 6 h), (B) untreated and treated with MDP (40 μg/ml, 6 h). IP was performed with isotype control IgG or IRGM antibody and Western blotting was performed with indicated antibodies. IgG (H); IgG heavy chain.

(C-D) Co-IP analysis of the interactions between (C) GFP-RIPK2 and Flag-IRGM or (D) Flag-NOD1 and GFP-IRGM in HEK293T cell lysates.

(E) GST pull-down assay using purified GST and GST-IRGM and in-vitro translated 35S radiolabeled myc-NOD2, myc-NOD1, and myc-RIPK2.

(F) Representative confocal images of HEK293T cells expressing GFP-RIPK2 and mCherry or mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm.

(G-H) Representative confocal images of THP-1 cells infected with RFP expressing S. flexneri, (MOI 1:25, 8 h) and immunostained with, (G) IRGM antibody (H) IRGM and RIPK2 antibody. Line profile: co-localization analysis using line intensity profiles. Scale bar, 5 µm. Zoom panels are digital magnifications. In image H, for better contrast, RFP expressing Shigella is pseudocolored to blue.

(I) Representative confocal images of THP-1 cells infected with RFP expressing S. flexneri, (MOI 1:25, 20 min) and immunostained with IRGM and EEA1 antibodies. DNA stained with DAPI. Scale bar, 5 µm. Zoom panels are digital magnifications.

(J) Representative confocal images of HEK293T cells expressing GFP-RIPK2, Flag-NOD1, and mCherry-IRGM. Line profile: co-localization analysis using line intensity profiles. Scale bar, 3 µm. Zoom panels are digital magnifications.

(K) Representative confocal images of proximity ligation assay (PLA) in HEK293T transfected with GFP or GFP-RIPK2 and Flag-IRGM plasmid. Scale bar 3 or 5 µm as indicated.

(L) The domain organization map of RIPK2 and deletion construct cloned as GFP-tagged proteins.

(M) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of RIPK2 and IRGM to map the domain/s of RIPK2 interacting with IRGM. Asterisk indicate the main band of overexpressed protein.

(N) The domain organization map of NOD1 and deletion construct cloned as GFP-tagged proteins

(O) A co-IP analysis is performed with HEK293T cell lysates expressing various domains of NOD1 and IRGM to map the domain/s of NOD1 interacting with IRGM. Asterisk indicate the main band of overexpressed protein
Selective Autophagy of RIPosomes Maintains Innate Immune Homeostasis during Bacterial Infection (2022)
Figure 7

Figure 7. RIPK2 inhibition ameliorates shigellosis- and DSS-induced gut inflammation in Irgm1 knockout mice

(A-B) Upper panels, schematic representation of shigellosis- and DSS-induced colitis models used. In lower panels, the graph depicts percentage loss in body weight in Irgm1+/+ and GSK583 untreated or treated Irgm-/- mice when (A) infected with S. flexneri (n=6 mice) or (B) administrated with DSS (n=3 mice). Mean ± SD, *p < 0.05, **p < 0.005, Student's unpaired t-test.

(C) Left panel, representative pictures of colons of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 infected with S. flexneri. Right panel, the graph depicts the average colon lengths of the mice groups. Mean ± SD, n=6, **p < 0.005, ***p < 0.0005, Student's unpaired t-test.

(D) Representative microscopic images of H&E staining of colon tissues of Irgm1+/+ and Irgm-/- mice untreated or treated with GSK583 administrated with DSS. The graph depicts the combined histological scores. Mean ± SD, n=3 (DSS), **p < 0.005, Student's unpaired t-test. Scale bar, 200 µm

(E) The soluble and insoluble fractionations of lysates from colon tissues of Shigella-infected or DSS-treated Irgm1+/+ and Irgm-/- mice treated with GSK583 as indicated, were subjected to immunoblot analysis with indicated antibodies.

(F) The qRT-PCR analysis with total RNA isolated from the colon tissues of S. flexneri infected Irgm1+/+ or Irgm1-/- or GSK583 treated Irgm1-/- mice. Mean ± SD, n=6-8, *p < 0.05, **p < 0.005, ***p < 0.0005, ordinary one-way ANOVA (Tukey's multiple comparisons test).

(G) Representative confocal images of IL-1β immunostained colon tissues of DSS-treated Irgm1+/+ or Irgm1-/- or GSK583 treated Irgm1-/- mice. Scale bar, 10 µm

(H-I) Western blot analysis with the colon tissue lysates of DSS-treated or S. flexneri infected mice groups as indicated.

Figure Expanded View Legends
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex (2020)
Figure 1

Figure 1. Rdh54 binds tightly to the PSC.

Schematic of ssDNA curtain assay.

Widefield images of RPA-mCherry-ssDNA molecules (magenta) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).

Widefield images of Rad51-ssDNA molecules (unlabeled) in the presence of GFP-Rdh54 (green; 1, 3, 10 and 30 nM).

Kymograph illustrating the dissociation of GFP-Rdh54 from Rad51-ssDNA; arrowhead highlights the time point at which free GFP-Rdh54 was flushed from the sample chamber.

Graph depicting the dissociation rate of GFP-Rdh54 from Rad51-ssDNA. Error bars represent s.d. of individual ssDNA molecules (N=25).

Kymograph showing the association of GFP-Rdh54 with a Rad51-ssDNA molecule; arrowhead highlights the injection time point for GFP-Rdh54.

Association rates of GFP-Rdh54 on individual Rad51 PSCs (N=44). Red lines represent the mean and s.d.
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex (2020)
Figure 2

Figure 2. Rad54 and Rdh54 can co-occupy the PSC.

Images of Rad51-ssDNA molecules bound by both mCherry-Rdh54 (30 nM; magenta) and GFP-Rad54 (green; 30 nM).

Quantification of bound GFP-Rad54 (10 nM) signal intensity for Rad51-ssDNA molecules with and without bound mCherry-Rdh54 (10 nM). Error bars represent s.d. of individual ssDNA molecules (N = 21).

Images showing increasing concentrations of GFP-Rad54 (green) only or mCherry-Rdh54 (magenta) only bound separately to Rad51-ssDNA molecules.

Images of GFP-Rad54 (green) and mCherry-Rdh54 (magenta) co-bound to the same Rad51-ssDNA molecules.

Quantification of GFP-Rad54 binding in the absence and presence of mCherry-Rdh54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).

Quantification of mCherry-Rdh54 binding in the absence or presence of GFP-Rad54. The data was fit by non-linear regression and error bars represent s.d. of individual molecules (N = 43).
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex (2020)
Figure 3

Figure 3. Rad54 and Rdh54 have distinct binding sites on the PSC.

Schematic illustration of full-length and chimeric Rad54 and Rdh54 protein constructs.

Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules.

Images of GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 (1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules in the presence of 30 nM Hed1.

Quantification of the integrated GFP signal intensity for GFP-tagged Rad54 (N = 75), Rdh54NRad54 (N = 46), Rdh54 (N = 50) and Rad54NRdh54 (N = 35; 1, 3, 10 and 30 nM each) bound to Rad51-ssDNA molecules. The data was fit by non-linear regression and error bars represent s.d. for individual DNA molecules. Note GFP-Rdh54 and GFP-Rad54 data are reproduced from Figures 1 and 2, respectively, for comparison.

Comparison of fold binding inhibition for GFP-tagged Rad54, Rdh54NRad54, Rdh54 and Rad54NRdh54 in reactions conducted in the presence of 30 nM Hed1. The dot represents the mean and the error bars represent the range for two independent experiments.
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex (2020)
Figure 4

Figure 4. Rdh54 does not promote PSC association with dsDNA during the homology search.

Schematic of the dsDNA curtain assay used for homology search assays.

Schematic of the Atto565-labeled tailed duplex substrate and PSC assembly procedure for homology search assays.

Widefield images of Atto565-labeled PSCs (magenta) bound to dsDNA curtains stained with YOYO1 (green). The PSCs were prepared with the indicated combinations of Rad51, Rdh54 and GFP-Rad54.

Number of Atto565-labeled PSC binding events per dsDNA molecule for PCS prepared with Rad54 and Rdh54 only (minus Rad51; N = 372), Rad51 plus Rad54 only (N= 117), Rad51 plus Rdh54 only (N = 258), Rad51 plus Rdh54 and Rad54 (N = 303). The error bars represent the s.d. for three independent experiments.
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex (2020)
Figure 5

Figure 5. Cooperative action of Rad54 and Rdh54 during the homology search.

Kymographs of Rad51-tailed duplex PSCs (magenta) containing the indicated combinations of Rad54 and Rdh54 proteins.

Translocation velocities for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). The error bars represent s.d.

Translocation distances for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 86), Rad54 plus Rdh54 (lane 2; N = 144), Rad54-K341R plus Rdh54 (lane 3; N = 47), Rad54 plus Rdh54-K318R (lane 4; N = 112), or Rad54-K341R plus Rdh54-K318R (lane 5; N = 64). Error bars were generated by bootstrapping.

First passage recognition efficiency for tailed duplex PSCs prepared with Rad54 only (lane 1; N = 145), Rad54 plus Rdh54 (lane 2; N = 115), Rad54-K341R plus Rdh54 (lane 3; N = 45), Rad54 plus Rdh54-K318R (lane 4; N = 39), or Rad54-K341R plus Rdh54-K318R (lane 5; no translocation observed). Error bars (s.d.) were generated from three independent experiments.

PSC binding distributions following 10 min reactions for PSCs prepared with Rad54 plus Rdh54 (N= 181); Rad54 plus Rdh54-K318R (N= 115); Rad54-K341R plus Rdh54 (N = 90); or Rad54-K341R plus Rdh54­-K318R (N = 54), as indicated. Error bars were generated by bootstrapping. The asterisks denote the targeted region of homology on the donor dsDNA molecules.
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex (2020)
Figure 6

Figure 6. Correct spatial positioning is required for Rad54 function in vitro.

D-loop stimulation by Rad54 or Rdh54NRad54 in reactions with Rad51 (300 nM), tailed duplex substrate (15 nM) and a pUC19 plasmid dsDNA (0.3 nM).

Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.

D-loop stimulation by Rdh54 and Rad54NRdh54.

Quantification of D-loop formation for Rad54 and Rdh54NRad54. Error bars represent s.d. of three independent experiments.

ATPase stimulation for GFP-tagged Rad54, Rdh54NRad54, Rdh54 or Rad54NRdh54 in the presence of Rad51-ssDNA. Error bars represent s.d. of three independent experiments.

Quantification of Rad51 Atto565-tailed duplex PSCs bound per dsDNA molecule in dsDNA curtain homology search assays with Rad54 (N = 117), Rdh54NRad54 (N = 150) or Rad54NRdh54 (N=175). The error bars represent the s.d. for three independent experiments.

Fraction translocating Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 117), Rdh54NRad54 (N = 150), Rad54NRdh54 (N = 0) and Rdh54 (N = 0).

Translocation velocities of Rad51 Atto565-tailed duplex PSCs in reactions with Rad54 (N = 86) and Rdh54NRad54 (N = 18). The error bars represent the s.d. of the data.
Rad54 and Rdh54 occupy spatially and functionally distinct sites within the Rad51-ssDNA presynaptic complex (2020)
Figure 7

Figure 7. The correct binding sites of Rad54 is required for DNA repair.

Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rad54∆ yeast strains.

Yeast spot growth assays to monitor the ability of Rad54, Rdh54, Rad54NRdh54, and Rdh54NRad54 to complement MMS sensitivity in rdh54∆ yeast strains.

Models depicting the relationship between binding sites and functional status for Rad54 and Rdh54 bound to a mitotic Rad51-ssDNA presynaptic complex.

Speculative model for the distribution of Rad54 and Rdh54 within meiotic presynaptic complexes. Note, the Rad54 and Rdh54 binding sites are shown at the "top" and "bottom" of the Rad51-ssDNA filaments for convenience only to illustrate that they occupy different sites within the PSC; a more detailed understanding of their binding sites will require high resolution structural data. Additional details of the model are presented in the text.

Expanded View Figures
TUT-DIS3L2 is a mammalian surveillance pathway for aberrant structured noncoding RNAs (2016)
Figure 1

Figure 1. DIS3L2 CLIP identifies broad repertoire of uridylated RNAs

A. SDS-PAGE analysis of the D391N DIS3L2-RNA complexes. RNAs were radioactively labeled at the 5' end and DIS3L2-bound RNAs were detected by autoradiography. The negative control was performed with HEK293T-Rex cells (Control)

B. DIS3L2 CLIP reads possess oligo(U) 3' termini. Histograms of reads with 3'-terminal stretches of identical nucleotides. Only oligo(U) tails longer than 4 nucleotides are observed.

C. The untemplated uridylation is mostly found in the proximity of mature 3' RNA ends. The heat map shows the coverage of various RNA types by uridylated reads. RNA class and number of uridylated genes/transcripts for each category are indicated on the left. The length of each mature RNA was scaled to 100%.

D. List of DIS3L2-bound uridylated miRNAs with the number of uridylated reads identified in all three replicate CLIPs. Members of the let-7 family are shown in bold.

E. Identification of hybrid reads consisting of the 5' and 3' arm of pre-let-7 miRNA. Profile of read coverage along the pre-let-7 genomic locus is indicated above.

F. Secondary structure of pre-hsa-let-7i, with both 5p and 3p mature products highlighted in bold. The intramolecular ligation position is indicated in dash line between nt 28 and 61. It corresponds to cleavage positions of RNaseT1, which leaves 3'-terminal guanosine residues.

G. D391N DIS3L2 overexpression does not cause abundance changes of CLIPed uridylated RNAs. Scatter plot of transcript abundances estimated from RNA-seq from the cell line overexpressing D391N DIS3L2 and control HEK293T-Rex cells. CLIPed transcripts are marked in white.
TUT-DIS3L2 is a mammalian surveillance pathway for aberrant structured noncoding RNAs (2016)
Figure 2

Figure 2. TUT-DIS3L2 is a surveillance pathway for structured ncRNAs produced by all three RNA Polymerases

A. Types of uridylated ncRNAs identified by DIS3L2 CLIP-seq.

B. U+ read coverage in downstream regions of mature U12 snRNA, Vault1 and 7SL ncRNAs. The full dark box is representing mature ncRNA, the thin line indicates the region downstream of the mature 3' end (extension).

C. Quantification of DIS3L2 D391N RIP analysis of U12 and U5D snRNAs showing number of snRNA clones with mature (Mat), extended (ext) or trimmed (Trim) 3' end. +U is number of clones with untemplated 3' oligo(U) ends. For more details see Figure S2. (D) An example of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) for RNU5D snRNA.

E. U+ reads mapped to the rDNA locus by using the artificial chromosome U13369.1.
TUT-DIS3L2 is a mammalian surveillance pathway for aberrant structured noncoding RNAs (2016)
Figure 3

Figure 3. TUT-DIS3L2 pathway targets precursor and aberrant forms of tRNAs

A. Schematic representation of the pre-tRNA secondary structure.

B. Uridylated tRNA reads overlap mainly with 3' trailers, T-loop and anticodon loop regions. Heat map showing the coverage of uridylated reads on pre-tRNAs. The pre-tRNA sub-regions (as indicated in (A)) are in small letters on the x-axis.

C. Northern blot analysis of RNAs immunoprecipitated with Flag-tagged WT (DIS3L2) and D391N (MUT) DIS3L2, AGO2 and DIS3, respectively. The blots were probed with radioactively labeled probes for 5' and 3' ends of mature tRNAVal, respectively.

D. Sequencing of MUT DIS3L2-bound tRNAVal and tRNALeu confirmed the uridylation status of the tRNA fragments identified by CLIP-seq. Genomic sequence for each tRNA is indicated on top with the 3' trailer region separated by a vertical line. Non-templated uridine residues are in red.

E. Identification of hybrid reads (produced by intra-strand ligation of RNaseT1 products during CLIP protocol) mapping to tRNAPhe(GAA) and tRNAAsn(GTT) genes. To the right: the schematic representation of selected hybrid reads with corresponding color-code.
TUT-DIS3L2 is a mammalian surveillance pathway for aberrant structured noncoding RNAs (2016)
Figure 4

Figure 4. DIS3L2 targets short uridylated transcripts originating from mRNA transcription start sites.

A. DIS3L2-bound uridylated mRNA reads map to the 5’ and 3’ UTRs.

B. Most 3' UTR U+ reads match the stem-loop structure of histone mRNAs. Heat map representation of the uridylation position.

C. The uridylated aberrant RNAs are stabilized in cells with nonfunctional DIS3L2. Small RNAs were modified with 3' end linker, and linker-specific primer was used for cDNA synthesis. Uridylated RNAs were PCR amplified in a semi-quantitative way with the same amount of input RNA used for RT reaction. We used a combination of the gene- and linker-specific primers, -RT ctrl is RT-PCR control, where no reverse transcriptase was added; PCR control is a reaction with no cDNA added. The graph on the right summarizes the percentage of uridylated and nonuridylated 5' fragments of OAT mRNA in HEK293T-Rex cells (Ctrl) and cells overexpressing mutant DIS3L2 (D391N).

D. DIS3L2-bound uridylated RNAs are degraded by DIS3L2 in vivo. RT-PCR amplification of 5'mRfs of OAT and RPL12 genes and 3' extended forms of U12 snRNA (RNU12 ext) from HEK293T cell lines with modified expression of DIS3L2 (as marked on top) was performed as described in C. Regions of mature 5S rRNA and 7SL RNA were used as loading controls. DIS3L2 KO is a DIS3L2 knock-out cell line. DIS3L2 rescue is DIS3L2 KO cells with stably integrated mutant (D391N) and wild type (WT) DIS3L2 expressing constructs, respectively. Bands marked with * are unspecific primer-dimer PCR products.

E. Metagene analysis of the position of DIS3L2 D391N clipped U+ reads peak maximum around mRNA transcription start sites (TSS) and of the ChIP-seq data for RNAP II.

F. U+5'mRfs and aberrant forms of U12 snRNA are highly enriched in the cytoplasm. RT-PCR detection of 5'mRfs of the OAT and RPL12 and aberrant form of U12 snRNA was performed as described in C with RNA isolated from nuclear and cytoplasmic fractions, respectively. The RT-PCR amplification of the body of 5S rRNA was used as a positive control for RNA isolation and RT reaction in both fractions. The identity and uridylation status of cytoplasmic OAT U+5'mRfs was confirmed by sequencing (shown on the bottom). In red are untemplated nucleotides, in green region corresponding to the first intron of OAT pre-mRNA. The efficiency of the subcellular fractionation was monitored by western blot (right panel, WB). DDX5 is nucleoplasmic, ACO2 is mitochondrial, and DIS3L2 and TUBα are cytoplasmic markers.