Journal: Nature Communications

Article Title: Dynamic arrest and aging of biomolecular condensates are modulated by low-complexity domains, RNA and biochemical activity

doi: 10.1038/s41467-022-30521-2

Figure Lengend Snippet: a The addition of 0.05 mg/ml structured RNA (600 nucleotides) to a solution of 11 µM full-length Dhh1 and 5 mM ATP/MgCl 2 lead to the formation of droplets with a high fraction (47 ± 4%) of glass-/gel-like droplets, in contrast with droplets formed in presence of polyU (0%). b Fluorescence recovery after photobleaching (FRAP) measurements showed a decrease in the mean recovery of three different droplets over a time course of 100 min. Simultaneously, the morphology of the droplets changed from an initially spherical to an irregular shape. Error bars represent the standard deviation of FRAP signals of three different droplets. c Condensates formed in presence of structural RNA cannot be dissolved by dilution. d Different droplets imaged by using confocal microscopy at the same time point exhibited different values of mCherry-tagged Dhh1 and Fluorescein-12-labeled RNA intensities. Intensity values represent the whole droplet mean intensity of individual droplets. e Effect of droplet turnover on material properties of biomolecular condensates. Schematic illustration of the ATP-hydrolysis-regeneration system. f Fractions of the different droplet subpopulations characterized by DDM over 100 min of incubation: high-diffusive liquid (green), low-diffusive liquid (blue), and dynamically arrested (gray). Error bars represent the standard error of the mean of at least 15 different droplets per condition. g Mean mobile fraction extracted from FRAP measurements at time 0 (dark red) and after 100 min incubation (light red). In presence of polyU, the mobile fraction was about 91 ± 3% and remains almost constant over time. A similar behavior was observed for the Dhh1 DQAD variant. When polyU was replaced with structured RNA, the mobile fraction decreased to 5 ± 5% over time, and this decrease could be partially rescued when coupled to an active system. Error bars represent standard deviation of mobile fractions of three different droplets. Source data for panels b , d , f , g are provided in the file.

Article Snippet: For labeling, 0.9 mM of Fluorescein-12-labeled UTP (Jena Bioscience, Germany) was added.

Techniques: Fluorescence, Standard Deviation, Confocal Microscopy, Labeling, Incubation, Variant Assay

Journal: Genome Research

Article Title: Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms

doi: 10.1101/gr.257857.119

Figure Lengend Snippet: Nanopore sequencing–based isoform dynamics (nano-ID) combines metabolic RNA labeling with long-read nanopore sequencing of native RNA molecules. ( A ) Experimental schematic of 5E U-labeled RNA isoforms subjected to direct RNA long-read nanopore sequencing. Metabolic labeling of human K562 cells with the nucleoside analog 5-ethynyluridine ( 5E U) in vivo. Newly synthesized RNA isoforms will incorporate 5E U instead of standard uridine (U) residues. Newly synthesized RNA isoforms (labeled) can then be distinguished from pre-existing RNA isoforms (unlabeled) in silico after sequencing the native full-length molecules on an array of nanopores . 5E U-containing RNA isoforms are computationally traceable and can thus be classified. Identification and quantification of single molecules subsequently enable assessment of exon usage, poly(A)-tail length, and RNA isoform stability. ( B ) Experimental schematic to derive synthetic RNAs for nucleoside analog benchmark. RNAs were in vitro transcribed: Either the standard bases A, U, C, and G were used as a control, or one of the natural bases was exchanged for a nucleoside analog (shown for 5E U). ( C ) Barplot showing the probability of a single-nucleoside analog to cause a mismatch in the alignment (compared with natural U or G, Methods) of all tested nucleoside analogs ( 5E U). ( 5Br U) 5-bromouridine; ( 5I U) 5-iodouridine; ( 4S U) 4-thiouridine; ( 6S G) 6-thioguanine. ( D ) Box plots showing the electric current readout (averaged per read) of the nanopore in pico-Amperes (pA; y -axis) associated with different nucleoside analogs 5E U, 5Br U, 5I U, 4S U, and 6S G (center position in 5-mer) in comparison to A, C, G, and U . Black horizontal lines indicate median raw electric current readout associated with G and U nucleosides. ( E , top ) Base miscalls (colored vertical bars) of the standard base-calling algorithm for synthetic RNAs containing 5E U instead of U (‘- 5E U-,’ 7756 molecules) and synthetic control RNAs (‘-U-,’ 17,149 molecules) in comparison to the original sequence (reference) of an exemplary region on synthetic RNA “Spike-in 8” (Methods) ( Supplemental Table S3 ). ( Middle ) Synthetic RNA sequences with (- 5E U-) and without 5E U (-U-). ( Bottom ) Alignment of the raw signal readout of the nanopore in pico-Amperes (pA) to the reference sequence (nt). Synthetic control RNAs (-U-, 17,149 molecules) are shown in black. 5E U-containing synthetic RNAs are shown in red (- 5E U-, 7756 molecules). Traces represent the average signal of all molecules. 5E U-containing synthetic RNAs show a clear deviation from the expected signal level in blue. Blue boxes indicate mean and standard deviation of 5-mers in the nanopore (provided by ONT).

Article Snippet: For IVT of labeled synthetic RNAs, 100% of UTP (resp. GTP) was substituted with either 5-ethynyl-UTP ( 5E U; Jena Bioscience), 5-bromo-UTP ( 5Br U; Sigma-Aldrich), 5-iodo-UTP ( 5I U; TriLink BioTechnologies), 4-thio-UTP ( 4S U; Jena Bioscience), or 6-thio-GTP ( 6S G; Sigma-Aldrich).

Techniques: Nanopore Sequencing, Labeling, In Vivo, Synthesized, In Silico, Sequencing, In Vitro, Control, Comparison, Standard Deviation

Journal: Genome Research

Article Title: Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms

doi: 10.1101/gr.257857.119

Figure Lengend Snippet: Direct RNA long-read nanopore sequencing of 5E U-labeled RNA isoforms in human K562 cells. ( A ) Multilayered data collection scheme. Parameter collection of samples was realized on three different layers: raw signal (electric current), base-call trace values, and alignment-derived mismatch properties (Methods). ( B ) In this study, data were collected in human K562 cells: control (three replicates) and 5E U 24 h (three replicates), as well as 5E U 60 min (six replicates) ( Supplemental Tables S1, S2 ). The neural network was trained on the 5E U 24 h versus control samples and used to classify reads of the 5E U 60 min samples into 5E U labeled and unlabeled. ( C ) ROC analysis of fivefold cross-validated neural network training with an accuracy of 0.87 and a false-discovery rate (FDR) of 0.1. Plot shows ROC curves (1 – specificity versus sensitivity) for all reads of the test set (black; alignment length ≥0 nt, AUC = 0.94) (Methods; Supplemental Table S5 ), for reads with an alignment length >500 nt (gray; alignment length ≥500 nt, AUC = 0.95), and for reads with an alignment length >1000 nt (dashed gray; alignment length ≥1000 nt, AUC = 0.96). ( D ) Genome browser view of classified direct RNA long-read nanopore sequencing reads of the human GAPDH gene locus on Chromosome 12 (∼8 kbp; Chr12: 6532405–6540375) visualized with the Integrative Genomics Viewer (IGV; version 2.4.10; human hg38) . From top to bottom , raw nanopore sequencing reads (unlabeled reads are shown in gray, 5E U-labeled reads are shown in red, and poly(A)-tail is shown in green; shown are typical aligned raw reads below the accumulated coverage of all measured reads), and corrected and collapsed isoforms (dark gray) determined with the FLAIR algorithm based on raw reads and RefSeq GRCh38 annotation (blue).

Article Snippet: For IVT of labeled synthetic RNAs, 100% of UTP (resp. GTP) was substituted with either 5-ethynyl-UTP ( 5E U; Jena Bioscience), 5-bromo-UTP ( 5Br U; Sigma-Aldrich), 5-iodo-UTP ( 5I U; TriLink BioTechnologies), 4-thio-UTP ( 4S U; Jena Bioscience), or 6-thio-GTP ( 6S G; Sigma-Aldrich).

Techniques: Nanopore Sequencing, Labeling, Derivative Assay, Control

Journal: Genome Research

Article Title: Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms

doi: 10.1101/gr.257857.119

Figure Lengend Snippet: Nano-ID monitors RNA isoform dynamics during heat shock (HS). ( A ) Experimental set-up of the HS treatment (60 min at 42°C) in human K562 cells. 5E U labeling was performed for 60 min. ( B ) Box plot showing half-lives (min) of significantly up-regulated RNA isoforms in HS ( 5E U 60 min HS) against control samples ( 5E U 60 min). ( C ) As in B for significantly down-regulated RNA isoforms. ( D ) Bar plot showing correlation (Spearman's rank correlation coefficient ρ) of RNA half-lives and poly(A)-tail lengths before and after HS (1310 loci). The third bar shows the correlation of RNA half-life fold change upon HS and poly(A)-tail length fold change upon HS. ( E ) Half-life fold changes upon HS ( y -axis) depicted for individual RNA isoforms alongside the half-life fold change derived from combined RNA (gene level; all RNAs encoded by the entire gene loci regardless of isoform assignment). Shown are 478 high-confident loci ( x -axis). All estimates are supported across biological replicates ( n ≥ 5) and conditions (HS; control). Half-life estimates for combined RNA (gene level) are depicted as a black line (sorted in decreasing order). Blue dots represent individual RNA isoform half-life estimates (1988 isoforms in total). All RNA isoform half-life estimates as well as one respective combined RNA half-life estimate (in black) sharing a common x -axis coordinate belong to the same gene loci. Vertical blue and black lines represent standard deviations of individual estimates. For individual RNA isoform half-life estimates, standard deviations are only shown if not overlapping with the standard deviation of the respective combined half-life estimates (black).

Article Snippet: For IVT of labeled synthetic RNAs, 100% of UTP (resp. GTP) was substituted with either 5-ethynyl-UTP ( 5E U; Jena Bioscience), 5-bromo-UTP ( 5Br U; Sigma-Aldrich), 5-iodo-UTP ( 5I U; TriLink BioTechnologies), 4-thio-UTP ( 4S U; Jena Bioscience), or 6-thio-GTP ( 6S G; Sigma-Aldrich).

Techniques: Labeling, Control, Derivative Assay, Standard Deviation