Extended Data Fig. 3: Characterization of the nascent pre-rRNA associated nucleolar proteins.

a. Pearson’s correlation of protein LFQ intensity from three MS repeats at each chase time point (0, 30, 60 min) after batch effect correction. The protein pull-down experiment was performed as described in Extended Data Fig. 2a in HeLa cells. b. Dynamics of protein binding intensities across nucleolar subdomains. LOESS regression curves show averaged z-scores of log2[LFQ intensity] of proteins localized in FC, DFC, PDFC, and GC regions, respectively. The normalized log2[LFQ intensity] was derived from three independent MS replicates. c. Heatmap of SSU- and LSU- precursor-bound protein dynamics (0-, 30-, 60 min chase) in HeLa cells. SSU-precursor-bound proteins with peak enrichment time at early time points (0- and 30 min) and LSU-precursor-bound proteins with peak enrichment time at late time point (60 min) are marked by black boxes and highlighted in Fig. 1j. Values represent mean z-scores of log2-transformed LFQ intensities, first normalized within each replicate across time points and then averaged across three MS replicates. d. Published time-resolved RNA interactome data using pulse-chase metabolic labelling with 4-thio-uridine also revealed a similar outflow pattern of nucleolar protein localization, consistent with the findings in this study: FC, DFC, and PDFC proteins bound nascent rRNAs sequentially within the first 40 min followed by GC proteins presented thereafter. e. Top, schematic of the nucleolar sub-domains with representative marker proteins: FC (RPA194), DFC (FBL), PDFC (DDX21) and GC (B23). Bottom, Western blot (WB) analysis of proteins precipitated with 5-EU-labelled nascent pre-rRNAs in Extended Data Fig. 2a in HeLa cells. FBL is mostly enriched at the early chase time points (0 min and 30 min), DDX21 is mostly enriched at 30 min, and B23 is mostly enriched at 60 min mirroring the spatiotemporal distribution of nascent pre-rRNAs in Fig. 1h. For gel source data, see Supplementary Fig. 1. f. The enrichment dynamics of representative proteins (RPA194, FBL, DDX21, and B23) in distinct nucleolar sub-domains (FC, DFC, PFDC, and GC) derived from MS data in HeLa cells. Values represent mean z-scores of log2-transformed LFQ intensities, first normalized within each replicate across time points and then averaged across three MS replicates. Results align with WB validation in (e). g. A proposed model summarizing the spatial distribution patterns of distinct pre-rRNA segments and their association with SSU and LSU pre-ribosomes.