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Keeping the nucleolus a liquid condensate The nucleolus is a multilayered, membraneless nuclear condensate in which DNA polymerase I (Pol I)–mediated ribosomal DNA (rDNA) transcription and pre-rRNA processing occur in fibrillar center and dense fibrillar component (FC/DFC) units. How its biophysical properties are regulated has remained elusive. Wu et al . found that the RNA helicase DDX21 forms a shell coating each FC/DFC unit in the nucleolus (see the Perspective by Yamazaki and Hirose). The authors found that a long noncoding RNA called SLERT facilitates the transition from the open to the closed configuration of the helicase using a chaperone-like mechanism. DDX21 in the closed conformation forms loose clusters that confer the FC/DFC unit sufficient liquidity and space required for Pol I processivity. In addition, DDX21 within the loose clusters cannot approach and wrap rDNA, thus licensing rDNA for transcription. —DJ

Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR–RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d–BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR–RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels. This paper describes a CRISPR–Cas13 system to effectively target circRNAs and screen their functions in vitro and in vivo, which enables the study of relevant circRNA phenotypes in human cell proliferation and in mouse embryogenesis.

Fibrillar centers (FCs) and dense fibrillar components (DFCs) are essential morphologically distinct sub-regions of mammalian cell nucleoli for rDNA transcription and pre-rRNA processing. Here, we report that a human nucleolus consists of several dozen FC/DFC units, each containing 2-3 transcriptionally active rDNAs at the FC/DFC border. Pre-rRNA processing factors, such as fibrillarin (FBL), form 18-24 clusters that further assemble into the DFC surrounding the FC. Mechanistically, the 5' end of nascent 47S pre-rRNA binds co-transcriptionally to the RNA-binding domain of FBL. FBL diffuses to the DFC, where local self-association via its glycine- and arginine-rich (GAR) domain forms phase-separated clusters to immobilize FBL-interacting pre-rRNA, thus promoting directional traffic of nascent pre-rRNA while facilitating pre-rRNA processing and DFC formation. These results unveil FC/DFC ultrastructures in nucleoli and suggest a conceptual framework for considering nascent RNA sorting using multivalent interactions of their binding proteins.

Visualizing the location and dynamics of RNAs in live cells is key to understanding their function. Here, we identify two endonuclease-deficient, single-component programmable RNA-guided and RNA-targeting Cas13 RNases (dCas13s) that allow robust real-time imaging and tracking of RNAs in live cells, even when using single 20- to 27-nt-long guide RNAs. Compared to the aptamer-based MS2-MCP strategy, an optimized dCas13 system is user friendly, does not require genetic manipulation, and achieves comparable RNA-labeling efficiency. We demonstrate that the dCas13 system is capable of labeling NEAT1, SatIII, MUC4, and GCN4 RNAs and allows the study of paraspeckle-associated NEAT1 dynamics. Applying orthogonal dCas13 proteins or combining dCas13 and MS2-MCP allows dual-color imaging of RNAs in single cells. Further combination of dCas13 and dCas9 systems allows simultaneous visualization of genomic DNA and RNA transcripts in living cells.