Extended Data Fig. 1: Sub-nucleolar localization of pre-rRNA intermediates by designed smFISH probes.
From: Pre-rRNA spatial distribution and functional organization of the nucleolus
a. Schematic of spatially organized nucleolar subdomains and corresponding steps of rRNA biogenesis. Transcription initiates in the FC, followed by modifications in the DFC, and processing with pre-ribosomal particle assembly in the PDFC and GC. The steps of pre-rRNA processing and RNP assembly are simplified. b. Diagram summarizing pre-rRNA processing steps and intermediates. The smFISH probes used in this study are indicated in magenta, highlighting their target regions and the pre-rRNA intermediates expected to detect. c. NB confirming 5′ ETS-1 and 3′ ETS probes mainly recognize the full-length 47S pre-rRNA. See also Fig. 1a. d. The ITS1-targeted probe sets recognize the early-stage pre-rRNAs, including mostly 47S and some 41S pre-rRNAs. Representative images in (g) and Fig. 1c. e. The 5′ ETS-targeted probe sets recognize the early-stage pre-rRNAs and 18S rRNA precursors, including 47S and 30S pre-rRNAs. Representative images in Fig. 1d. f. The ITS2-targeted probe sets recognize 5.8S/28S rRNA precursors. The probe ITS2 in Fig. 1e mainly recognizes the 32S and 12S pre-rRNAs. The probe spanning site 3′ in (h) mainly recognizes the 32S pre-rRNA. g. The Site 2 probe signals localize to the DFC–PDFC region. Left, probe schematic. Middle, representative single-slice and averaged SIM images of Site 2-detected 47S pre-rRNA (magenta) and FBL (green) in HeLa cells. Right, line intensity plot of Site 2 and FBL signals. n = 50 DFC units from 17 cells. h. The Site 3′-detected LSU pre-rRNAs localize mainly to the PDFC–GC region. Left, probe schematic. Middle, representative single-slice and averaged SIM images of the Site 3′-detected 32S pre-rRNA (magenta) and FBL (green) in HeLa cells. Right, line intensity plot of Site 3′ and FBL signals. n = 50 DFC units in 17 cells. i. U13 snoRNA that binds the 3′ end of 18S pre-rRNA is mainly localized to the DFC. Left, schematic depicting the targeted position of U13 snoRNA on the pre-rRNA. Middle, representative single-slice and averaged SIM images of U13 snoRNA (magenta) and FBL (green) in HeLa cells. Right, line intensity plot of U13 snoRNA and FBL signals. n = 53 DFC units from 10 cells. j. E2 snoRNA that binds the 3′ end of 18S pre-rRNA is largely localized to the DFC. Left, schematic depicting the targeted position of E2 snoRNA on the pre-rRNA. Middle, representative single-slice and averaged SIM images of E2 snoRNA (magenta) and FBL (green) in HeLa cells. Right, line intensity plot of E2 snoRNA and FBL signals. n = 54 DFC units from 13 cells. c,d,e,f, For gel source data, see Supplementary Fig. 1.
