Abstracts Membraneless organelles (MLOs) often exhibit internal architecture, yet whether the local transcriptome differentially partitions across MLO subdomains remains largely uncharacterized. Here we combine super-resolution imaging with in situ reverse transcription-based sequencing to profile transcriptomes within MLO subdomains. Using the human tripartite nucleolus as a model system, we identify distinct RNA populations in the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC). Pre-rRNA processing intermediates demonstrate a layered progression across nucleolar subdomains, reflecting the temporal order of the processing steps. Processing steps involved in large-small subunit separation show increased retention in the DFC in highly differentiated cells. Mature small nucleolar RNAs (snoRNAs) are preferentially enriched in the DFC and spatially segregated from their precursor transcripts. Many non-snoRNA-related transcripts, often derived from nucleolus-proximal genes, show modest enrichment in the GC. These results illustrate functional RNA organization across nucleolar subdomains and provide a framework for nanoscale transcriptome mapping of biomolecular condensates.
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