g-probe  (Iris Medical Inc)

Journal: Nature Communications

Article Title: JULGI coordinates vascular development and leaf patterning through non-cell-autonomous regulation of miR165/166

doi: 10.1038/s41467-025-67339-7

Figure Lengend Snippet: a REMSA of the G-rich element within pri-MIR165a and its mutant variants with GST-JUL1 and GST-JUL1(RA) at increasing protein concentrations. The G-rich element contains three G blocks. Blue letters indicate G to A substitutions. b Quantification of probe-JUL1 binding shown in ( a ). The Y -axis shows the percentage of JUL1 bound probe relative to the total probe, and the X -axis indicates JUL1 concentration. Band intensities were measured using ImageJ. c REMSA of full-length pri-MIR165a with GST-JUL1 in the presence of increasing amounts of various cold competitors. d Quantification of JUL1 binding shown in ( c ). The relative ratio of protein-probe complex to free probe is presented. Band intensities were determined using ImageJ. e Co-localization of pri-MIR165a with JUL1 proteins in Arabidopsis protoplasts. (Left) Schematic diagram of the MS2 coat protein/24×MS2 hairpin system fused to pri-MIR165a for in vivo visualization. (Right) MS2-GFP signals represent the localization of MS2 coat protein which profile MS2 hairpin-fused pri-MIR165a, and mRFP signals show the cellular distributions of JUL1 or JUL1(RA). Scale bars, 10 µm. f Intensity profiles of GFP (MS2) and RFP (JUL1) fluorescence in the cytosolic compartment indicated by arrows in e . g Association of JUL1 with pri-MIR165a in planta . JUL1 was immunoprecipitated with an anti-HA antibody from 4-week-old Col-0 and jul1 proJUL1:JUL1-HA leaves. JUL1 proteins and pri-MIR165a transcripts were determined by immunoblot and qRT-PCR, respectively. Enrichment values of pri-MIR165a were normalized to % input. The bar graph represents the mean ± SEM with individual data points ( n = 6, biological replicates). Different letters indicate statistically significant differences ( P < 0.05), as determined by one-way ANOVA with a post hoc Tukey’s HSD test. All experiments were independently repeated three times with consistent results. The data shown are from a representative experimental set.

Article Snippet: For RNA EMSA with G-rich element, single-stranded RNA (ssRNA) oligonucleotides of the G-rich region of primary MIR165a (pri-MIR165a_G-rich), various G to A mutants (mut ALL , mut #1 , mut #2 , and mut #3 ) and anti sensed counter part of G-rich element (anti-probe) were synthesized (Bioneer).

Techniques: Mutagenesis, Binding Assay, Concentration Assay, In Vivo, Fluorescence, Immunoprecipitation, Western Blot, Quantitative RT-PCR

Journal: Nature Communications

Article Title: JULGI coordinates vascular development and leaf patterning through non-cell-autonomous regulation of miR165/166

doi: 10.1038/s41467-025-67339-7

Figure Lengend Snippet: a Bead-binding assay for interaction between the G-rich element and its antisense counterpart (anti-probe) in the presence of GST-JUL1 or GST-JUL1(RA). The schematic diagram illustrates the setup: G-rich element-coated streptavidin beads were incubated with or without the anti-probe and GST-JUL1. The G-rich element is Cy5-labeled (red fluorescence), while the anti-probe is Cy3-labeled (green fluorescence). Hybridization of the anti-probe and the G-rich element, mediated by Watson-Crick base pairing, is depicted by a yellow fluorescence signal. b Förster resonance energy transfer (FRET) analysis using Cy5-labeled G-rich element and Cy3-labeled anti-probe in the presence of GST-JUL1 or GST-JUL1(RA) ( n = 3). The fluorescence intensities of Cy3 and Cy5 were measured at 560 nm and 660 nm, respectively, with an excitation at 520 nm. c Altered association of DCL1 with pri-MIR165a by JUL1 ( n = 6). In protoplasts, pri-MIR165a was co-expressed with the dsRNA-binding domain of DCL1 ( dsRBD-HA ), either alone or together with JUL1-FLAG . dsRBD-HA was immunoprecipitated with an anti-HA antibody, and the associated levels of pri-MIR165a were determined by qRT-PCR. d In vitro pri-MI165a processing assay performed with isolated nuclei from YFP-DCL1 expressing protoplasts. Nuclear protein extracts were incubated with pri-MIR165a in the presence of increasing amounts of GST-JUL1. Red, blue, and green sequences indicate G blocks, mature miR165, and mutated nucleotides, respectively. Red, blue, and green arrowheads denote the primary, precursor, and mature forms of pri-MIR165a, respectively. The bar graphs in b and c represent the mean ± SEM with individual data points. n indicates biological replicates. Different letters indicate statistically significant differences ( P < 0.05), as determined by two-way ANOVA with a post hoc Tukey’s HSD test ( b ) and one-way ANOVA with a post hoc Tukey’s HSD test ( c ). All experiments were independently repeated three times with consistent results. The data shown are from a representative experimental set.

Article Snippet: For RNA EMSA with G-rich element, single-stranded RNA (ssRNA) oligonucleotides of the G-rich region of primary MIR165a (pri-MIR165a_G-rich), various G to A mutants (mut ALL , mut #1 , mut #2 , and mut #3 ) and anti sensed counter part of G-rich element (anti-probe) were synthesized (Bioneer).

Techniques: Binding Assay, Incubation, Labeling, Fluorescence, Hybridization, Förster Resonance Energy Transfer, Immunoprecipitation, Quantitative RT-PCR, In Vitro, Isolation, Expressing