Fig 1.
Workflow for labeling nascent RNAs in the C. elegans intestine.
(A) Worms are grown on NGM plates to the desired life-stage (L4, adult, or aged animal). (B) Using a dissecting scope, animals are dissected in Dissection Media (DM). A hypodermic needle is used to make a single cut between the pharynx and the mouth, causing the intestine to extrude out of the worm body. (C) An example of an extruded intestine. Intestinal lumen is marked (*). (D) Dissected intestines are transferred to a tube. (E) DM is removed, and nascent RNA is labeled using RNA Labeling Media, followed by (F) fixation and click chemistry. (G) Intestines are mounted on a slide for imaging. (H) Click chemistry covalently combines the azide (fluorophore) and alkyne (EU).
Fig 2.
EU-click can measure changes in nascent RNA transcription in the C. elegans intestine.
Images and quantitative measurements of nascent transcription (5 min pulse) with and without transcription inhibition. (A) Example nucleoli from int1 cells after treatment with DMSO (top) or ActD (bottom). Nucleolar vacuole is marked (*). Columns from left to right: GARR-1::GFP (FZ marker), EU conjugated with AF594 (EU-AF594), composite image of GARR-1::GFP (green) and EU-AF594 (orange), and intensity profiles of GFP and EU-AF594 signal along the line shown on the composite image. EU-AF594 pixel values in the grayscale and composite images and the line plots are normalized using the same range for DMSO and ActD. (B) Transcription rate (EU incorporated/5 min) in the nucleolus decreases upon transcription inhibition by ActD. Box plot comparing the sum of pixel values in the nucleolar mask for DMSO- and ActD-treated int1 cells. Points represent measurements from different worms; color indicates batch number. Significance was tested by two-way ANOVA using batch number and treatment as independent variables (p < 1 × 10-8). (C) Example nucleoli from int1 cells from L4440 (control) and tif-1a KD. Similar to A. (D) Transcription rate in nucleolus decreases upon tif-1a KD. Measurements and test of significance are similar to B (p < 1 × 10-14).
Fig 3.
Nascent RNA transcription overlaps with the FZ compartment of nucleoli.
(A) Example nucleoli of int1 cells from worm lines expressing different GFP labeled protein markers localized to Fibrillar Zone (FZ) or Granular Zone (GZ) compartments. Top to bottom: RPOA-2::GFP (FZ), GARR-1::GFP (FZ), DAO-5::GFP (FZ), and NUCL-1::GFP (GZ). Columns from left to right: nucleolar protein::GFP (::GFP), EU-AF594, composite image of ::GFP (green) and EU-AF594 (orange), and intensity profiles of GFP and AF594 signal along the line shown on the composite image. GFP and EU-AF594 pixel values in the line plots are normalized (min/max) along each line. (B) Box plots comparing the Spearman correlation of pixel values between GFP (nucleolar protein) and EU-AF594 (nascent RNA). Points represent measurements from different worms. Significance was tested using two-way ANOVA and Tukey-Kramer test. P-values for each pairwise comparison: not significant (n. s.) for DAO-5 vs. GARR-1, p < 0.001 (***) for NUCL-1 vs. RPOA-2, DAO-5, and GARR-1, and p < 0.01 (**) for RPOA-2 vs. DAO-5 and GARR-1. (C) Nascent rRNA transcription localizes to the FZ compartment of the nucleolus in the C. elegans intestine. GZ: Granular Zone; FZ: Fibrillar Zone; NV: Nucleolar Vacuole.