poly a mrna  (TaKaRa)

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) Representative fluorescent micrographs of gonads (pachytene region) from live worms co-expressing PGL-3::mCherry and GFP::WAGO-4 (upper row) or RFP::ZNFX-1 and GFP::WAGO-4 (lower row) in WT and 3×FG mutant backgrounds at 20 °C. For each condition, a single confocal slice is shown for the indicated proteins and merged channels. Scale bar: 5 µm. ( B ) Quantification of GFP::WAGO-4 mean intensity signal inside granules vs. the cytoplasm. Ratios were calculated from images of live worms expressing GFP::WAGO-4 together with PGL-3::mCherry (P granules, purple hues) or RFP::ZNFX-1 (Z granules, blue hues) in WT and 3×FG backgrounds. Average ratios with SD are plotted, and numbers above bars indicate average values. Analysis was performed on the pachytene region, covering approximately 35 nuclei for P granules (35.3 ± 2.8) and 30 nuclei for Z granules (29.7 ± 2.7) from three live worms grown at 20 °C. To ensure precise granule volume definition and accurate intensity ratio measurement, the granule vs. cytoplasm signal ratios were calculated for each of 54 confocal planes (0.1 µm). Statistical analysis was performed using a two-tailed Mann–Whitney test, P values indicated in the graph. Representative fluorescence micrographs are shown in ( A ). ( C ) Schematic representation of the in vitro phase separation experiment. Two types of condensates were formed to approximate minimal P granules. Each condensate contained recombinant purified PGL-1, PGL-3, GLH-1, and GLH-4 proteins from an insect cell expression system, along with commercial poly(A)+ mRNA. The first type of condensate contained WT GLH-1 and GLH-4, while the second contained GLH-1 and GLH-4 with F → A mutations in FG dipeptides (identical to those introduced in live worms, shown in Fig. ). To each condensate type, the same amount of worm-purified mScarlet::WAGO-4 was added. For negative controls, the same amount of WAGO-4 storage buffer was added to the condensates. ( D ) Graph representing the ratio of mScarlet::WAGO-4 signal intensity between the condensed and dilute phases for WT and FG-mutant condensates. Results from a single experiment are shown; five to six ROIs per image were defined inside and outside condensates, with six images analyzed for each condition ( n = 32–36). Bars represent average values with SD and numbers above bars indicate average values. Statistical analysis was performed using a two-tailed Mann–Whitney test, P values indicated in the graph. A second independent experiment is shown in Fig. . mScarlet::WAGO-4 storage buffer was used as a negative control, and ratios were calculated in the same manner. ( E ) Schematic representation of WAGO-4 constructs expressed in worms. Fusions or point mutations were introduced at the native wago-4 locus using CRISPR-Cas. Introduced fusions or WAGO-4 predicted domains, identified based on sequence alignment with human Ago2, are depicted in different colors, as indicated. ( F ) Representative fluorescence micrographs of gonads (pachytene region) from live worms expressing different fluorescently labeled WAGO-4 proteins: GFP::WAGO-4, MTS::GFP::WAGO-4, PH::GFP::WAGO-4, and GFP::WAGO-4(Y611E). For each expressed protein, a single confocal slice is shown for worms grown at 20 °C. Scale bar: 5 µm. ( G ) Graph representing the number of progeny for worms of the indicated genotypes grown at 26 °C for one generation. Each dot represents the number of progeny from a single worm ( n = 17–19). Horizontal lines represent mean values, and error bars indicate SD. Statistical analysis was performed using the Kruskal–Wallis test, and the corresponding P values are reported in Dataset . Groups sharing at least one letter above the plot are statistically indistinguishable ( P < 0.05). ( H ) Quantification of unfertilized oocytes in the uteri of worms grown at 26 °C, categorized into three groups: (1) worms containing exclusively embryos, (2) worms containing at least one embryo and at least one unfertilized oocyte, and (3) worms containing exclusively unfertilized oocytes. Data represent the average proportion of worms in each category from four independent experiments ( n = 52.8 ± 6.1 worms per experiment). Dots represent values from individual experiments and error bars indicate SD. ( I ) Mortal germline assay at 25 °C comparing the 3×FG mutant, MTS::GFP::WAGO-4-expressing strain, and wago-4(ko) , with N2 as a control. For each strain, five replicates with five P 0 worms were used. .

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Expressing, Mutagenesis, Two Tailed Test, MANN-WHITNEY, Fluorescence, In Vitro, Recombinant, Purification, Negative Control, Construct, CRISPR, Sequencing, Labeling, Control

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) Table showing the number of up- and downregulated genes in 3×FG, wago-4(Y611E) , and wago-4(ko) worms grown at 20 °C or 26 °C ( p < 0.05). ( B ) PCA plot based on the top 500 most variable genes. Three biological replicates were analyzed for each genotype and temperature condition, with each replicate individually plotted. The legend on the right indicates the symbol for each condition. ( C ) Table listing all histone genes downregulated in 3×FG vs. WT based on mRNA sequencing at 20 °C. Each column represents a histone gene, and rows indicate the histone type it encodes. Genes highlighted in bold also showed increase in WAGO-4-associated 22G-RNAs in 3×FG compared to WT (Fig. ). ( D ) Volcano plot showing fold enrichment of proteins from total worm lysates of worms grown at 26 °C (3×FG vs. WT), determined by mass spectrometry ( n = 3 biological replicates). Statistical significance was calculated using limma (Ritchie et al, ). Detected histone proteins or clusters and known germ granule proteins are highlighted.

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Sequencing, Mass Spectrometry

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) MA plot representing differential gene expression (3×FG vs.WT) based on mRNA sequencing from worms grown at 20 °C. Red dots indicate upregulated genes, blue dots indicate downregulated genes, and gray dots represent genes without significant changes in expression. The number of regulated genes is indicated at the bottom ( P < 0.05). ( B ) Enrichment analysis of genes classified as oogenic, spermatogenic, or germline-constitutive (Ortiz et al, ) compared to genes up- or downregulated in 3×FG vs. WT based on mRNA sequencing. Log₂ enrichment is indicated by color according to the scale shown, and significant enrichment or depletion (p < 0.05, Fisher’s exact test) is highlighted in bold and underlined. ( C ) Venn diagram showing the overlap of upregulated genes ( P < 0.05) from mRNA sequencing for three genotypes: 3×FG, wago-4(Y611E) , and wago-4(ko) . Numbers in brackets indicate the size of each gene group. ( D ) Venn diagram showing the overlap of downregulated genes ( P < 0.05) from mRNA sequencing for three genotypes: 3×FG, wago-4(Y611E) , and wago-4(ko) . Numbers in brackets indicate the size of each gene group. ( E ) Table presenting the percentage of up- or downregulated genes in 3×FG, wago-4(Y611E) , and wago-4(ko) that are identified as WAGO-4 targets according to Seroussi et al . Percentages are highlighted with yellow and green hues. The total number of up- and downregulated genes is indicated for each condition.

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Gene Expression, Sequencing, Expressing

Journal: The EMBO Journal

Article Title: Germ granule localization of nematode Argonaute WAGO-4 ensures fidelity in small RNA loading

doi: 10.1038/s44318-025-00606-x

Figure Lengend Snippet: ( A ) Bar plots showing the 5’ nucleotide composition and length distribution of sRNAs in each Argonaute IP. Pie charts depict the proportion of sRNAs corresponding to each genetic element (biotype). Data represent the average of three biological replicates. IPs were performed using 3xFLAG::GFP::WAGO-4 in young adult worms. ( B ) Volcano plot showing Log 2 fold enrichment of 22G-RNAs targeting WAGO-4 targets in 3×FG vs. WT worms grown at 20 °C. Each dot represents the average Log 2 fold change ( n = 3 biological replicates). Differential expression and statistical analysis were performed using DESeq2 (Love et al, ). Targets with an average Log 2 fold change of <−1 or >1 and P < 0.01 were considered to have a significant decrease or increase in WAGO-4-associated 22G-RNAs, respectively (indicated by red dots). ( C ) Enrichment of WAGO-4 sRNA targets in four groups: WT-specific targets, 3×FG-specific targets, targets with a decrease in WAGO-4-associated sRNAs in 3×FG, and targets with an increase in WAGO-4-associated sRNAs in 3×FG. These groups were compared to genes up- or downregulated in 3×FG/WT based on mRNA sequencing. All data represent worms grown at 20 °C. Log₂ enrichment is indicated by color according to the scale shown, and significant enrichment or depletion ( P < 0.05, Fisher’s exact test) is highlighted in bold and underlined. ( D ) Venn diagram showing the overlap of WAGO-4 targets in WT worms (this study) with CSR-1 targets (Seroussi et al, ). Both datasets were derived from sRNA sequencing after Ago IP from adult worms grown at 20 °C. Numbers in brackets indicate the size of each group. ( E ) Metagene profiles for sRNAs complementary to protein-coding gene WAGO-4 targets (all identified targets in WT and 3×FG worms grown at 20 °C). Size-normalized targets were partitioned into 100 bins, and the mean coverage (RPM) for each bin was plotted. Solid lines represent IP values, and dashed lines represent input values. The number of identified targets for each genotype is indicated in brackets. ( F ) Metagene profiles for sRNAs complementary to protein-coding gene WAGO-4 and CSR-1 targets. CSR-1 targets were identified from CSR-1 IP in WT worms (Seroussi et al, ). Size-normalized targets were partitioned into 100 bins, and mean coverage (RPM) for each bin was plotted. Solid lines represent IP values, and dashed lines represent input values. The total number of genes analyzed was determined as WAGO-4 and CSR-1 shared targets based on published data (Seroussi et al, ), and the number of identified targets for each genotype is indicated in brackets.

Article Snippet: poly-A+ mRNA , Takara , 636101.

Techniques: Quantitative Proteomics, Sequencing, Derivative Assay

Journal: iScience

Article Title: Induced hepatic stem cells are suitable for human hepatocyte production

doi: 10.1016/j.isci.2022.105052

Figure Lengend Snippet:

Article Snippet: Human Fetal Liver Poly A + RNA , Clontech Laboratories, Inc. , Cat# 636108.

Techniques: Affinity Purification, Recombinant, Activity Assay, Cell Culture, Transfection, Plasmid Preparation, Lysis, Real-time Polymerase Chain Reaction, Staining, Software, Imaging, Fluorescence, Knock-Out