Journal: The EMBO Journal

Article Title: Determining the effects of pseudouridine incorporation on human tRNAs

doi: 10.1038/s44318-025-00443-y

Figure Lengend Snippet: ( A ) Cloverleaf representations of a human tRNA Gln UUG with all uridines (U) replaced by Ψ (blue) or m 1 Ψ (violet). Melting temperatures (°C) of tRNA Gln UUG with various modifications (Tm is indicated). Biological replicates, N = 3. ( B ) 2D representation of the structure of tRNA with site-specific Ψ (circles) added by individual PUS. PUS are shown in cartoon representation: PUS1, PUS3, and PUS4 are obtained from Alphafold2 prediction while PUS7 (PDB 5KKP) and PUS10 (PDB 2V9K) are crystal structures. ( C ) Detection of PUS-dependent Ψ formation on tRNA Gln UUG . The reverse‐transcribed cDNA products were resolved in a 15% urea gel and the CMC‐Ψ mediated short cDNAs product are indicated by double-arrows (Ψ 13 orange, Ψ 28 blue, Ψ 39 green, Ψ 55 red) while the Cy5-primer is indicated by a triangle. A short exposure of the same gel is shown in the dashed box. The Ψ 13 and Ψ 28 -corresponding cDNAs are detected using a different set of site-specific primers (shown in a dash lined box on the right). ( D ) Melting temperature changes (ΔTm, °C) of tRNA Gln UUG , tRNA Glu UUC , tRNA Asp GUC by Ψ modifications at specific positions in comparison to corresponding tRNA treated with a mixture of inactive PUS. Gray dots on the plot indicate unmodified tRNA and colored dots indicate introduced Ψ sites. Biological replicates, N = 3. ( E ) Melting temperature changes (ΔTm, °C) of tRNA Gly CCC by Ψ modifications at specific positions in comparison to corresponding tRNA treated with a mixture of inactive PUS. Gray dots on the plot indicate unmodified tRNA and colored dots indicate introduced Ψ sites. Biological replicates, N = 3. ( F ) Melting temperature changes (ΔTm, °C) of tRNA Gly CCC modified only by PUS4, only by PUS7, by PUS4 and PUS7 and after mixing the individual PUS4 and PUS7 samples at a 1:1 ratio (left). Melting temperature changes (ΔTm, °C) of tRNA Gly CCC after mixing samples modified individually by PUS4 or PUS4/7 at different ratios (middle). Melting temperature changes (ΔTm, °C) of tRNA Gly CCC after mixing samples modified individually by PUS7 or PUS4/7 at different ratios (middle). Biological replicates, N = 3. ( G ) Melting temperature changes (ΔTm, °C) of tRNA Gly CCC , after incubation with PUS4 (left) PUS7 (middle) and PUS4/7 (right) for the indicated time in comparison to corresponding tRNA not incubated with any PUS. Detection of PUS-dependent Ψ formation on tRNA Gly CCC (below). Biological replicates, N = 3. ( H ) Melting temperature changes (ΔTm, °C) of unmodified (left) and PUS3, PUS4, and PUS7 modified (right) tRNA Gly CCC , after repeated heating and cooling cycles. Biological replicates, N = 3. The distribution of the data is represented in box plots. The central line inside the box indicates the median and the square the mean. The lower and upper edges correspond to the first (Q1) and third (Q3) quartiles, respectively, defining the interquartile range (IQR). The whiskers extend to the minimal and maximal values within the 1.5 IQR. Points beyond this range are considered outliers and are shown as transparent points. All statistical analyses were performed using one-way ANOVA ( α = 0.05) with a Bonferroni multiple comparisons test. Statistically significant differences are indicated (ns: no significance, **** p ≤ 0.0001). Inactive PUS variants are indicated by *. .

Article Snippet: 5-Propargylamino-CTP-Cy5 , Jena Bioscience , NU-831-CY5.

Techniques: Reverse Transcription, Comparison, Modification, Incubation

Journal: The EMBO Journal

Article Title: Determining the effects of pseudouridine incorporation on human tRNAs

doi: 10.1038/s44318-025-00443-y

Figure Lengend Snippet: ( A ) Cartoon presentation of domains of each PUS (upper-left). The length is labeled, and domain is highlighted where the catalytic residue is indicated by a triangle. The truncated regions of PUS1 and PUS7 are shown as dashed lines. An SDS-PAGE gel of recombinant Hs PUS enzymes (lower-left). Catalytic mutants marked with stars. SEC profiles of purified human PUS enzymes (upper-right) and the collected fraction for each purification were resolved in SDS-PAGE gels (lower-right). ( B ) MST analyses of PUS, including wild type and inactive form (DA), binding to tRNA Gln UUG . The calculated Kds of wild-type PUS and inactive forms (DA), binding to tRNA Gln UUG . ( C , D ) Detection of PUS-dependent Ψ formation on tRNA Gln UUG , tRNA Gly CCC , tRNA Glu UUC and tRNA Asp GUC . The reverse‐transcribed cDNA products were resolved in a 15% or 18% urea gel and the CMC‐Ψ mediated short cDNAs are indicated by double-arrows (Ψ 13 orange, Ψ 27/28 blue, Ψ 39 green, Ψ 54 yellow Ψ 55 red). Each tRNA primer (labeled with Cy5) is indicated by a triangle. In the case of tRNA Asp GUC , the signal for primer is obtained from a short exposure shown in the dash lined box while the Ψ 13 -dependent cDNA is obtained using a site-specific primer (shown in a dash lined box on the side). ( E ) Cryo-EM reconstructions of unmodified and Ψ 13 - and Ψ 39 -modified tRNA Gln UUG . The modified sites are highlighted by color code in the 2D cartoon and the model (Ψ 13 orange and Ψ 39 green).

Article Snippet: 5-Propargylamino-CTP-Cy5 , Jena Bioscience , NU-831-CY5.

Techniques: Labeling, Residue, SDS Page, Recombinant, Purification, Binding Assay, Reverse Transcription, Cryo-EM Sample Prep, Modification

Journal: The EMBO Journal

Article Title: Determining the effects of pseudouridine incorporation on human tRNAs

doi: 10.1038/s44318-025-00443-y

Figure Lengend Snippet: ( A ) Melting temperature changes (ΔTm, °C) of tRNA His GUG , tRNA Lys UUU , tRNA Ser UGA , tRNA Arg UCU by Ψ modifications at specific positions in comparison to corresponding tRNA treated with a mixture of inactive PUS. Gray dots on the plot indicate unmodified tRNA and colored dots indicate introduced Ψ sites. Biological replicates, N = 3. ( B ) Local resolution estimations for tRNA Gln UUG . The relative resolution scale is shown in the inset. Ψ sites are highlighted by circles (Ψ 13 orange, Ψ 39 green, Ψ 55 red). The arrows indicate the local conformational changes. All cryo-EM maps are contoured to RMSD = 8. ( C ) tRNA representation with Ψ sites and the positions of Cy5 probe are highlighted (green). Global melting temperatures (Tm, °C) measured with RiboGreen for unlabeled and U 48 or 5ʹ-Cy5 labeled tRNA Gln UUG . Melting temperature changes (ΔTm, °C) of tRNA Gln UUG labeled with Cy5 at 5ʹ (left) or U 48 (right) upon Ψ modifications. The temperature (Tm) scale is shown in the inset. Biological replicates, N = 3. Inactive PUS are indicated by *. The distribution of the data is represented in box plots. The central line inside the box indicates the median and the square the mean. The lower and upper edges correspond to the first (Q1) and third (Q3) quartiles, respectively, defining the interquartile range (IQR). The whiskers extend to the minimal and maximal values within the 1.5 IQR. Points beyond this range are considered outliers and are shown as transparent points. All statistical analysis was performed using one-way ANOVA (α = 0.05) with a Bonferroni multiple comparisons test. Statistically significant differences are indicated (ns: no significance, **** p ≤ 0.0001. For tRNA Ser UGA * p = 0.01517, for tRNA Arg UCU * p = 0.04746). .

Article Snippet: 5-Propargylamino-CTP-Cy5 , Jena Bioscience , NU-831-CY5.

Techniques: Comparison, Cryo-EM Sample Prep, Labeling

Journal: bioRxiv

Article Title: Distinct Mechanisms of Recognition of Phosphorylated RNAPII C- Terminal Domain by BRCT Repeats of the BRCA1–BARD1 Complex: Insights from Structural and Functional Analyses

doi: 10.1101/2025.01.22.634233

Figure Lengend Snippet: The BRCA1-BARD1 complex forms liquid-like condensates in vitro , which accommodate phosphorylated CTD domain of RNAPII and RNA. A) Schematic representation of BRCA1 and BARD1 domains. Positions of investigated binding mutants are indicated. B) Liquid-liquid phase separation (LLPS) assays with purified, Alexa488-labelled BRCA1- BARD1. BRCA1-BARD1 (at 1.25 µM, 2.5 µM, and 5 µM) was mixed with the crowding agent (10% dextran). Bar chart (top) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with BRCA1-BARD1. Statistical significance was determined by unpaired t-test. A nested scatterplot (middle) represents quantification (n = 3) of an area of individual droplets from three independent experiments with BRCA1-BARD1, with median area determined per dataset. Statistical significance was determined by nested t- test. Representative images from three experiments (bottom) are depicted as an overlay of differential interference contrast (DIC) and GFP. Where indicated, hexane-1,6-diol (hex; at 10%) was added to inhibit hydrophobic interactions or ATP (at 5mM) to inhibit electrostatic interactions. Scale bars, 10 µm. C) LLPS assays with purified, Alexa488-labelled BRCA1-BARD1, pS5pS7 mCherry-hCTD and Cy5-RNA. BRCA1-BARD1 (at 5 µM) was mixed with phosphorylated CTD (2.5 µM) or Cy5-ITS1 RNA (at 15 nM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC), Alexa488, and Cy5. Scale bars, 10 µm. D) Bar chart (top) representing quantification (n = 3) of the number of droplets per frame from the LLPS experiments with the BRCA1-BARD1 complex shown in (C). Statistical significance was determined by unpaired t-test. A nested scatterplot (bottom) represents quantification (n = 3) of an area of individual droplets from three independent experiments with the BRCA1- BARD1 complex shown in (C), with median area determined per dataset. Statistical significance was determined by nested t-test. E) LLPS assays with purified, BRCA1-BARD1, pS5pS7 GFP-(CTD) 26 and Cy5-RNA. BRCA1-BARD1 (at 5 µM) was mixed with phosphorylated CTD (2.5 µM) and Cy5-ITS1 RNA (at 15 nM) in the presence of a crowding agent (10% dextran). Representative images from three experiments are depicted as an overlay of differential interference contrast (DIC), GFP, and Cy5. Scale bars, 10 µm.

Article Snippet: For the preparation of the Cy5-labelled ITS1 RNA, 8 µg of the template DNA was mixed with ATP, UTP, and GTP (at 2mM), CTP (at 1mM), Cy5-labelled CTP (5- Propargylamino-CTP-Cy5, Jena Bioscience, at 0.05 mM), and T7 RNA polymerase (at 1 µM) in a buffer containing 0.1 M Tris-HCl, pH 8.1; 1% Triton X-100; 16 mM MgCl 2 ; 10 mM spermidine; and 50 mM DTT.

Techniques: In Vitro, Binding Assay, Purification