rRNA intermediates dictate nucleolar architecture in C. elegans

The nucleolus is a multilayered, membraneless organelle essential for rRNA transcription, processing and ribosome assembly; however, it remains largely unclear whether and how these molecular events coordinate the organization of the multilayered nucleolar structure. We previously reported that the accumulation of 27SA2 rRNA upon the loss of a particular subset of RPL proteins elicits nucleolar reshaping, resulting in the formation of nucleolar rings and vacuoles in C. elegans. Here, we systematically investigated the effects of pre-18S rRNA processing and rRNA transcription on nucleolar structure and reported that defects in these molecular events result in reorganization of the nucleolar architecture. In particular, the depletion of a special subset of small ribosomal subunit proteins (RPSs), namely, class I RPSs, induces a concentric three-layered nucleolar architecture; the inhibition of rRNA transcription induces another three-layered architecture, a sandwich-like nucleoli. Via fluorescence imaging, we found that nucleolar proteins undergo demixing and spatial redistribution into distinct subnucleolar regions during nucleolar structure reorganization. Tracking the subcellular localization of rDNA via a lacI::tagRFP/lacO system revealed that rDNA also redistributed upon nucleolar reorganization. We further identified the Ce.22S rRNA intermediate, which accumulates upon the loss of class I RPSs and regulates the reorganization of the nucleolar structure. Moreover, the conserved nucleolar protein NUCL-1/nucleolin is essential for nucleolar architecture reorganization and regulates the development of nematodes during impaired rRNA production. Together, these findings reveal that the nucleolar structure is orchestrated by the status of rRNA biogenesis and that the accumulation of particular rRNA intermediates can direct the organization of specific nucleolar architectures and the spatial mixing/demixing behavior of the nucleolar proteome.