Advanced surface passivation for high-sensitivity studies of biomolecular condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, broadly applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multipoint attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time fluorescence recovery after photobleaching imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Advanced Surface Passivation for High-Sensitivity Studies of Biomolecular Condensates.

Biomolecular condensates are cellular compartments that concentrate biomolecules without an encapsulating membrane. In recent years, significant advances have been made in the understanding of condensates through biochemical reconstitution and microscopic detection of these structures. Quantitative visualization and biochemical assays of biomolecular condensates rely on surface passivation to minimize background and artifacts due to condensate adhesion. However, the challenge of undesired interactions between condensates and glass surfaces, which can alter material properties and impair observational accuracy, remains a critical hurdle. Here, we introduce an efficient, generically applicable, and simple passivation method employing self-assembly of the surfactant Pluronic F127 (PF127). The method greatly reduces nonspecific binding across a range of condensates systems for both phase-separated droplets and biomolecules in dilute phase. Additionally, by integrating PF127 passivation with the Biotin-NeutrAvidin system, we achieve controlled multi-point attachment of condensates to surfaces. This not only preserves condensate properties but also facilitates long-time FRAP imaging and high-precision single-molecule analyses. Using this method, we have explored the dynamics of polySIM molecules within polySUMO/polySIM condensates at the single-molecule level. Our observations suggest a potential heterogeneity in the distribution of available polySIM-binding sites within the condensates.

Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.

An optimized fixation method containing glyoxal and paraformaldehyde for imaging nuclear bodies.

The mammalian cell nucleus contains different types of membrane-less nuclear bodies (NBs) consisting of proteins and RNAs. Microscopic imaging has been widely applied to study the organization and structure of NBs. However, current fixation methods are not optimized for such imaging: When a fixation method is chosen to maximize the quality of the RNA fluorescence in situ hybridization (FISH), it often limits the labeling efficiency of proteins or affects the ultrastructure of NBs. Here, we report that addition of glyoxal (GO) into the classical paraformaldehyde (PFA) fixation step not only improves FISH signals for RNAs in NBs via augmented permeability of the fixed nucleus and enhanced accessibility of probes, but also largely preserves protein fluorescent signals during fixation and immunostaining. We also show that GO/PFA fixation enables the covisualization of different types of nuclear bodies with minimal impact on their ultrastructures under super-resolution microscopy.

Dynamic Imaging of RNA in Living Cells by CRISPR-Cas13 Systems.

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.

Nascent Pre-rRNA Sorting via Phase Separation Drives the Assembly of Dense Fibrillar Components in the Human Nucleolus.

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.

Cellular functions of long noncoding RNAs.

A diverse catalog of long noncoding RNAs (lncRNAs), which lack protein-coding potential, are transcribed from the mammalian genome. They are emerging as important regulators in gene expression networks by controlling nuclear architecture and transcription in the nucleus and by modulating mRNA stability, translation and post-translational modifications in the cytoplasm. In this Review, we highlight recent progress in cellular functions of lncRNAs at the molecular level in mammalian cells.

Linking RNA Processing and Function.

RNA processing is critical for eukaryotic mRNA maturation and function. It appears there is no exception for other types of RNAs. Long noncoding RNAs (lncRNAs) represent a subclass of noncoding RNAs, have sizes of >200 nucleotides (nt), and participate in various aspects of gene regulation. Although many lncRNAs are capped, polyadenylated, and spliced just like mRNAs, others are derived from primary transcripts of RNA polymerase II and stabilized by forming circular structures or by ending with small nucleolar RNA-protein complexes. Here we summarize the recent progress in linking the processing and function of these unconventionally processed lncRNAs; we also discuss how directional RNA movement is achieved using the radial flux movement of nascent precursor ribosomal RNA (pre-rRNA) in the human nucleolus as an example.

Genome-wide screening of NEAT1 regulators reveals cross-regulation between paraspeckles and mitochondria.

The long noncoding RNA NEAT1 (nuclear enriched abundant transcript 1) nucleates the formation of paraspeckles, which constitute a type of nuclear body with multiple roles in gene expression. Here we identify NEAT1 regulators using an endogenous NEAT1 promoter-driven enhanced green fluorescent protein reporter in human cells coupled with genome-wide RNAi screens. The screens unexpectedly yield gene candidates involved in mitochondrial functions as essential regulators of NEAT1 expression and paraspeckle formation. Depletion of mitochondrial proteins and treatment of mitochondrial stressors both lead to aberrant NEAT1 expression via ATF2 as well as altered morphology and numbers of paraspeckles. These changes result in enhanced retention of mRNAs of nuclear-encoded mitochondrial proteins (mito-mRNAs) in paraspeckles. Correspondingly, NEAT1 depletion has profound effects on mitochondrial dynamics and function by altering the sequestration of mito-mRNAs in paraspeckles. Overall, our data provide a rich resource for understanding NEAT1 and paraspeckle regulation, and reveal a cross-regulation between paraspeckles and mitochondria.

Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection.

Circular RNAs (circRNAs) generated via back-splicing are enhanced by flanking complementary sequences. Expression levels of circRNAs vary under different conditions, suggesting participation of protein factors in their biogenesis. Using genome-wide siRNA screening that targets all human unique genes and an efficient circRNA expression reporter, we identify double-stranded RNA-binding domain containing immune factors NF90/NF110 as key regulators in circRNA biogenesis. NF90/NF110 promote circRNA production in the nucleus by associating with intronic RNA pairs juxtaposing the circRNA-forming exon(s); they also interact with mature circRNAs in the cytoplasm. Upon viral infection, circRNA expression is decreased, in part owing to the nuclear export of NF90/NF110 to the cytoplasm. Meanwhile, NF90/NF110 released from circRNP complexes bind to viral mRNAs as part of their functions in antiviral immune response. Our results therefore implicate a coordinated regulation of circRNA biogenesis and function by NF90/NF110 in viral infection.

SLERT Regulates DDX21 Rings Associated with Pol I Transcription.

Dysregulated rRNA synthesis by RNA polymerase I (Pol I) is associated with uncontrolled cell proliferation. Here, we report a box H/ACA small nucleolar RNA (snoRNA)-ended long noncoding RNA (lncRNA) that enhances pre-rRNA transcription (SLERT). SLERT requires box H/ACA snoRNAs at both ends for its biogenesis and translocation to the nucleolus. Deletion of SLERT impairs pre-rRNA transcription and rRNA production, leading to decreased tumorigenesis. Mechanistically, SLERT interacts with DEAD-box RNA helicase DDX21 via a 143-nt non-snoRNA sequence. Super-resolution images reveal that DDX21 forms ring-shaped structures surrounding multiple Pol I complexes and suppresses pre-rRNA transcription. Binding by SLERT allosterically alters individual DDX21 molecules, loosens the DDX21 ring, and evicts DDX21 suppression on Pol I transcription. Together, our results reveal an important control of ribosome biogenesis by SLERT lncRNA and its regulatory role in DDX21 ring-shaped arrangements acting on Pol I complexes.

Unusual Processing Generates SPA LncRNAs that Sequester Multiple RNA Binding Proteins.

We identify a type of polycistronic transcript-derived long noncoding RNAs (lncRNAs) that are 5' small nucleolar RNA (snoRNA) capped and 3' polyadenylated (SPAs). SPA processing is associated with nascent mRNA 3' processing and kinetic competition between XRN2 trimming and Pol II elongation. Following cleavage/polyadenylation of its upstream gene, the downstream uncapped pre-SPA is trimmed by XRN2 until this exonuclease reaches the co-transcriptionally assembled snoRNP. This snoRNP complex prevents further degradation, generates a snoRNA 5' end, and allows continuous Pol II elongation. The imprinted 15q11-q13 encodes two SPAs that are deleted in Prader-Willi syndrome (PWS) patients. These lncRNAs form a nuclear accumulation that is enriched in RNA binding proteins (RBPs) including TDP43, RBFOX2, and hnRNP M. Generation of a human PWS cellular model by depleting these lncRNAs results in altered patterns of RBPs binding and alternative splicing. Together, these results expand the diversity of lncRNAs and provide additional insights into PWS pathogenesis.

Shedding light on paraspeckle structure by super-resolution microscopy.

The nuclear body paraspeckle is built on the lncRNA Neat1 and plays important roles in gene regulation. In this issue, West et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601071) use super-resolution structured illumination microscopy to show that paraspeckles are organized in a core-shell spheroidal structure composed of Neat1 and seven proteins.