Nuclear bodies: concentrating at an aqueous site, with momentum

ABSTRACT

The second FASEB conference on Nuclear Bodies: Hubs of Genome Activity was held June 2–7, 2024, at Niagara Falls, NY. The central theme was how these protein and RNA–protein complexes that are situated in the nucleus outside the genome support and regulate gene expression. Topics included their relevance to disease, especially cancer, their molecular dynamics, and their phase separation transition properties. The meeting included numerous trainees as speakers and session chairs, hosted a memorable Keynote talk on diversity, and a vibrant career development session.

In the late 19th and early 20th centuries, various nuclear bodies were observed by light microscopy in stained preparations and later by electron microscopy, revealing them to be situated apart from the chromatin. In the modern era, these bodies have been linked to on-process and even regulatory roles in gene expression. An inaugural meeting in this field was held in 2022, supported by the Federation of American Societies for Experimental Biology (FASEB) [1]. This second meeting, also supported by FASEB, bore witness to an expanding scale of this field and continued the meeting’s theme of having all attendees be speakers, whether lab heads or trainees. This report summarizes the main invited talks as well as other details of the meeting. Readers wishing more in-depth coverage of the current Nuclear Body field are referred to a recent reviews collection [2].

RNAs are key components

RNA plays a crucial role in various biological processes, including the maintenance and modulation of nuclear structures and functions. Noriko Saitoh (Tokyo, Japan) presented studies on a cluster of long noncoding RNAs (lncRNAs) named ELEANORS. These lncRNAs have expression levels that correlate with the risk of late-onset secondary disease in patients with estrogen receptor (ER)-positive breast cancer by forming nuclear clouds that help maintain 3D genome structure. Among the ELEANORS, ELEANOR2 is a natural antisense lncRNA derived from an ultraconserved sequence within an intron of the CCDC170 gene, which is located near the estrogen receptor 1 gene. ELEANOR2 binds to and regulates the activity of an adjacent super-enhancer close to CCDC170, playing a role in the progression of ER-positive breast cancer.

MALAT1 is a well-studied and abundant nuclear lncRNA specifically localized to splicing speckles. Prasanth Kannanganattu (Urbana-Champaign, US) presented studies on how MALAT1 promotes hypoxia-responsive cancer progression. Hypoxia, a state of low oxygen tension, is commonly observed in solid malignant tumors. His group found that the genome-wide distribution of hypoxia-responsive gene hubs is at nuclear speckles, and that MALAT1 regulates the expression and RNA processing of many hypoxia-responsive genes. Mechanistically, MALAT1 regulates the splicing of these genes by acting as a scaffold, facilitating the association and interaction of RNA-binding proteins (RBPs), including SRSF1, with hypoxia-responsive genes.

Francesca Losi (Reggio Emilia, Italy) presented studies on a novel set of lncRNAs, known as FRG2A, which are overexpressed in the muscles of individuals with Facioscapulohumeral Muscular Dystrophy (FSHD). FSHD is associated with a reduction in the copy number of the D4Z4 macrosatellite at the chromosome 4q telomere. Interestingly, FRG2A predominantly localizes to the Dense Fibrillar Compartment (DFC) of nucleoli and is involved in the organization of nuclear chromatin architecture. FRG2A is essential for the compaction of nucleolar-associated domain (NAD) regions by anchoring centromere satellites to the nucleoli, which subsequently impacts ribosome function and protein synthesis in muscle cells.

Ling-Ling Chen (Shanghai, China) presented studies of identifying a novel pathophysiological role of nuclear stress bodies (nSBs) in restraining acute inflammatory responses. nSBs are transient, membrane-less organelles that transcribe the constitutive heterochromatin Satellite III (SatIII) DNAs, primarily from chromosome 9q12, and are present only in primates under severe stress. Upon sensing stress, the rapid expansion and activation of SatIII heterochromatin loci within a few hours significantly shortens the spatial distance between adjacent genes, such as NFIL3 to SatIII. This allows NFIL3 loci to access transcription factors newly recruited by SatIII RNAs at nSBs, resulting in enhanced transcription of NFIL3. NFIL3 is a key transcription suppressor that prevents the excessive expression of critical inflammatory cytokines. Importantly, NFIL3 expression positively correlates with SatIII RNAs in PBMCs from septic patients, and high expression of SatIII RNAs appears to benefit septic patient survival.

No class of nuclear bodies has had more confluence with RNA, both as to content and commerce, than nuclear ‘speckles’, initially named interchromatin granule clusters based on their ultrastructure and presence between chromosome territories. Opening his talk, Andrew Belmont (Urbana-Champaign, US) reminded the audience of the initial contrasting ideas about these bodies, viz. that they represent sites at which mRNA splicing factors are stored versus that they are the sites of splicing, these two notions not being mutually exclusive. He also alluded to more recent work by his group and others showing that subsets of active genes lie at the edges of speckles. He presented further interrogations of this using the TSA-seq method his lab has developed, which has a resolution of about 50 nm based on the chemistry involved. It was found that the subsets of active genes in closest proximity to speckles tend to be GC-rich, conserved, and residing in dense arrays, i.e., are small genes, and that they also are enriched in super-enhancers. Related studies revealed a phenomenon in which gene expression levels are amplified upon contact with a speckle, with the speckle enlarging. He presented additional results showing that promoter type is a greater factor in determining speckle proximity than a gene’s expression level. Additional tracking data indicated a degree of tethering between pairs of vicinal speckles and that the SON protein may be involved. Directed mass transfer between speckles was implicated and he further speculated on ‘dark matter’ that may serve to keep speckles restricted to chromatin-deficient regions.

In a second speckle-based talk, Mitchell Guttman (Pasadena, US) presented data showing that genes near speckles have higher concentrations of gene-vicinal splicing factors as well as higher co-transcriptional splicing levels than genes which are located further from speckles. He also reported that experimental recruitment of a specific pre-mRNA to speckles is sufficient to boost its level of splicing. His and Andrew Belmont’s talks show how new methods, deployed in both of their labs, can lead to a more coherent understanding of a longstanding issue.

Other presentations provided new insights into pre-rRNA processing and microRNA functions in the nucleolus. Sofia Quinodoz (Princeton, US) presented studies on dissecting pre-rRNA processing in space and time within the nucleolus. Using metabolic labeling coupled with RNA-sequencing and imaging analyses, she measured the location and timing of various processing steps of pre-rRNA cleavage and modifications within the nucleolus. Notably, perturbation of U3 snoRNA-mediated 18S rRNA cleavage blocks 18S rRNA processing and results in the ‘inside-out’ nucleoli. In contrast, 28S rRNA processing is not affected by U3 knockdown and leads to an accumulation of 28S rRNA inside the ‘inverted’ nucleoli. These findings suggest that the spatiotemporal processing of pre-rRNA can impact the multiphase organization of the nucleolus.

In addition to investigating critical steps of pre-rRNA processing in nucleoli, Susan Baserga (New Haven, US) presented studies identifying 15 microRNAs involved in dysregulated pre-rRNA processing. They found that RPS28 is a direct target of the MIR-28 siblings, hsa-miR-28-5p, and hsa-miR-708-5p, which can cause a severe pre-18S rRNA processing defect. Intriguingly, this RPS28-MIR-28 microRNA interaction appears to be conserved solely among humans and great apes, indicating a potentially underappreciated connection between microRNAs and the modulation of nucleolar pre-rRNA processing.

Vikram Paralkar (Philadelphia, US) addressed the question of how rRNA transcription rates are set in different hematopoietic cell types. His team optimized an assay called FISH-FLOW that allows simultaneous detection of nascent and mature rRNAs. Key findings were that different cell types have precisely controlled rates of rRNA transcription, that this rate is important for maintenance of cell identity, and that cell-type-specific transcription factors (TFs) bind rDNA repeats and control Pol I activity. In a myeloid progenitor cell line, a 30% reduction in rRNA transcription was enough to impair cell growth and induce differentiation. These observations suggest that cell-type-specific TFs control rRNA transcription in different cell types, and that this regulation is required for maintaining normal cell identity.

Nuclear bodies and cancer

In an opening Keynote talk, David Spector (Cold Spring Harbor, US) presented an in-depth summary of his 40 years of work on the long non-coding RNA (lncRNA) MALAT1, which is critical for metastasis. The Spector lab previously identified a novel mechanism of 3'-end processing of this RNA. More recent studies have revealed that increased levels of the MALAT1 lncRNA impact breast cancer progression and metastasis. Knockout or antisense oligonucleotide knockdown of MALAT1 results in the differentiation of mammary tumors and a significant reduction in metastasis. The Spector laboratory is partnering with several biotechnology companies to begin clinical trials for MALAT1 anti-sense oligonucleotides.

Maralice Conacci-Sorrell (Dallas, US) reported on her group’s study of cancer-specific nucleolar regulators, via an unbiased screen for nucleolus-centered growth-promoting pathways in myc-transformed cells. One hit was the zinc finger protein (692 ZNF692), knockdown of which disrupts nucleolar integrity, reduced ribosome synthesis and translational efficiency. She also reported that this protein and its interactors are elevated in kidney tumors including ones in which the patients have a germline mutation in the TCA cycle enzyme fumarate hydratase. In a parallel talk, Maria del Carmen Lafita-Navarro from this group described additional findings on ZNF692, namely that it interacts with nucleophosmin 1 in ribosome assembly, forming a hub in the nucleolar granular component that facilitates the late steps of ribosome assembly in rapidly growing cells

Marikki Laiho (Baltimore, US) presented an unbiased drug sensitivity screen with Pol I inhibitors in a large cancer cell-line panel. Her results point to a Pol I link to splicing via the ribosomal large subunit protein RPL22L1 in many cancer lines and, in particular, a disruption of this link in microsatellite instable cancer cells. This revealed ribotoxic axis has implications for therapeutic innovation. Miguel Rivera (Boston, US) reported new functions for EWS cancer fusion proteins, applying a new method called DisP-seq (disordered protein precipitation followed by DNA sequencing). This maps the genomic locations of bound proteins with intrinsically disordered regions (IDRs), many of which turn out to be transcription factors. Application of this method to Ewing sarcoma cells revealed how chromatin clusters of IDR-containing proteins control the binding of disordered TFs and set up long-range oncogene sequestration, a new perspective on the agency of chromatin-bound IDR-containing proteins and oncogenesis.

Two talks on PML bodies significantly expanded our understanding of these intriguing nuclear structures. Hugues de Thé (Paris, France) presented a Keynote lecture describing decades of work that have defined how PML protein function is controlled by SUMOylation to regulate the phase separation dynamics of these bodies. He also presented his group’s work that has defined the mechanisms by which arsenic functions to cure rare leukemias through the regulation of PML, which constitutes a major clinical advance for these hematological malignancies. Huaiying Zhang (Pittsburgh, US) described how SUMO mediates phase separation between DNA repair factors, thus enriching SUMO on telomeres and leading to the formation of ALT telomere bodies (ABPs), including the recruitment of PML to these bodies or their fusion with PML bodies. This work reveals a bifurcating role of SUMO in enabling the ALT pathway through promotion of DNA repair, protein-phase separation and ABP formation.

Form and function of nuclear bodies

Nuclear bodies are thought to coalesce via a common set of principles and mechanisms, but they carry out a variety of different functions within the cell. Three paradigmatic nuclear substructures participate in the biogenesis of certain classes of ribonucleoproteins (RNPs): the Cajal Body (CB), the Histone Locus Body (HLB), and the Paraspeckle (PS). Eight talks at the meeting involved discussion of these three bodies and the biological processes carried out by factors that concentrate therein.

Greg Matera (Chapel Hill, US) led off this session with an overview of spliceosomal snRNP assembly, a process that is chaperoned by the Survival Motor Neuron (SMN) protein complex. Proteomic analyses carried out by his lab have identified new functions for the SMN complex in innate immune signaling, with connections to the larger proteostasis network of heat shock (folding) chaperones. Misfolded or misassembled SMN complexes are thought to trigger a proteotoxic stress response that has important consequences for the study of motor neuron diseases like ALS and SMA, posing the question: Who chaperones the chaperones?

Following on from SMN, Karla Neugebauer (New Haven, US) spoke about her laboratory’s recent work on coilin, an SMN binding partner, and the primary scaffolding factor for CBs. Applying a suite of ‘omics techniques to coilin, these investigators have found that all expressed small nucleolar RNAs traffic through CBs, adding a major class of ncRNAs to the list of CB constituents. Additional studies identified coilin-proximal proteins that serve to regulate CB number, composition, and structure.

Nathalie Escande-Beillard (Istanbul, Turkiye) presented exciting findings about another denizen of the CB, a small nuclear RNP transport factor called Snurportin1 (SPN1). The PI has identified a cohort of patients that have point mutations in the gene encoding SPN1 that present with a novel form of muscular dystrophy. Cell biology studies show that loss-of-function mutations in SPN1 cause CBs to disassemble and coilin to re-localize to the nucleolus. Structural modeling and biochemical pull-downs showed that SPN1 uses its C-terminal domain to multimerize, and that truncation of this domain is particularly problematic in muscle cells.

Mirek Dundr (Chicago, US) addressed the question of where CB formation is nonrandomly initiated in the nucleus. He reported data showing that nascent pre-snRNA transcripts recruit critical CB components. Additional studies revealed that CBs have a non-uniform mesh-like structure with multiple, partially miscible phases involved in snRNP and snoRNP biogenesis residing at different sites within the CB interior. Another distinct phase of CB assembly/homeostasis involves transcription of snRNA genes located at the CB periphery.

Much like its larger sibling the nucleolus, the HLB differs from other NB subtypes in that it is organized around a particular group of genes. James Kemp and Mark Geisler presented two talks from Robert Duronio’s laboratory (Chapel Hill, US) focused on histone genes and HLBs. Kemp used an impressive array of high-resolution microscopy-based techniques together with genetics to show that cell-cycle dependent histone gene expression within the HLB is largely regulated by promoter-proximal pausing of RNA Pol II. Outside of the S-phase, Pol II fails to elongate into the 3’-end of histone genes, as measured by RNA in situ hybridization. Geisler investigated the role of a putative repressor protein, called Mute. This protein is thought to help turn down histone expression at the end of S-phase by interfering with phosphorylation of the HLB scaffolding factor Mxc/NPAT. The Mute protein was also shown to be important for regulating the self-renewing asymmetrical division neuroblasts and other stem cells.

Another important HLB component is the transcription factor called CLAMP. This GA-rich binding protein plays numerous roles in the cell, some of which are involved in histone gene transcription, whereas others are not. Mukulika Ray and Joseph Aguilera presented two talks from Erica Larschan’s laboratory (Providence, US) describing completely different aspects of CLAMP function. Aguilera presented work showing how CLAMP binds to different GA-rich regions of the genome (including the Histone locus) and that it may serve as a dosage compensation factor that mediates 3D folding of chromatin domains on the Drosophila X chromosome. CLAMP binds to specific sites called MREs (MSL recognition elements) and competes with another GA-rich binding protein called GAF. Ray spoke about a novel role for CLAMP as an RNA binding protein that regulates sex-specific alternative splicing in the early embryo. Using global cross-linking and immunoprecipitation approaches, Ray showed that the N-terminal region of CLAMP contains a prion-like domain (PrLD) that binds RNA that is essential for organismal development. This novel RNA binding domain, which is located within a large intrinsically disordered region of CLAMP, is thought to modulate the dynamics of hnRNP-like proteins in the formation of splicing condensates.

Paul Kaufman (Worcester, US) described the formation of the nuclear bodies known as paraspeckles (PSs) in macrophages when their pro-inflammatory differentiation is activated by bacterial lipopolysaccharide, but not during their anti-inflammatory differentiation elicited by interleukin-4. The pro-inflammatory assembly involves known PS RNA-binding proteins and the long non-coding RNA NEAT1–2. In the second part of his talk he discussed the biological activities and biochemical properties of the chromatin-binding domain of Ki-67, which coats mitotic chromosomes and becomes nucleolar during interphase. This domain is predicted to be completely disordered. It binds naked DNA with a sigmoidal binding curve in fluorescence polarization experiments, but its interactions with nucleosomes are complex.

In and near the nucleolus

Brian McStay (Galway, Ireland) presented new finding on the Nucleolar Organizer Regions (NORs), the repeated arrays of rDNA whose expression creates this venerable nuclear body. But as he has long emphasized, a NOR does not necessarily mean a nucleolus, which is related to rDNA silencing. He described a system in which single human NOR-bearing chromosomes harbored in mouse cell hybrids. The former are silent due to an inter-species incompatibility of a rDNA transcription factor, SL1, but by introducing the gene for human SL1 into these test cells, the human NORs are activated and can then be moved into human cells for analysis. By introducing sequence tags into the rDNA of these transferred NOR’s his group was able to track nucleolar assembly and synthesis of these ‘customized’ ribosomes. In addition, he presented evidence that, in contrast to the prevailing view that factors collating around NOR’s lead these nascent nucleoli to fuse into larger, normal nucleoli, the NOR-bearing p-arms of the acrocentric chromosomes possess a property of self-association, independent of the rDNA itself or any influence by existing nucleoli.

Sui Huang (Chicago, US) described studies showing that the transcription and processing of pre-rRNA synthesis are closely coordinated. AID degron-mediated degradation of the Pol I subunit PAF53 induced a prompt morphological segregation of nucleoli. However, the removal of the pre-rRNA processing factor UTP4 blocked the PAF53-induced nucleolar segregation. Furthermore, the loss of PAF53 led to a shutdown of the SSU processome via proteasome degradation of a subset of its constituents. These results point to a proteolytic component of the control pathway that inhibits pre-rRNA processing upon the cessation of its transcription.

The meeting’s EMBO Keynote lecture was given by Raffaella Santoro (Zurich, Switzerland). She presented a method, termed NoLMseq, in which laser-capture microdissection and DNA sequencing allow mapping of Nucleolus Associated Domains (NADs) in single cells. Applied to embryonic stem cells, this revealed considerable cell-to-cell heterogeneity of NADs, falling into populations with distinct chromatin states. Further results showed that NADs typically contact nucleoli in a monoallelic manner and that contact frequency sets NAD gene expression and their chromatin states. Additional applications of NoLMseq revealed how NADs reorganize during stress.

Condensation in, on, and around the genome

Biomolecular condensation is intricately linked to studies of nuclear bodies. The session on condensates was connected by the central theme of how protein- and RNA-rich condensates interact with and are nucleated and regulated by the genome. Two speakers focused on aberrant nuclear condensation that occurs with cancer-associated mutations.

Dong Li (Beijing, China) described the use of super-resolution live imaging to investigate mislocalization of cancer-associated translocation products, which often form clusters or condensates in inappropriate locations. FUS-CREB3L2 forms ER membrane-associated clusters in an orientation-dependent manner, when the FUS end of the translocation is presented to the cytoplasmic side, but not ER lumen side of the transmembrane protein. Endogenous CREB3 is tethered on the outside of the ER, where the tail gets cleaved and goes to the nucleus to act as a transcription factor. The fusion protein can follow this process as well and create nuclear puncta of cleaved FUS-CREB3L2 at DNA loci.

Shasha Chong (Pasadena, US) presented her work on condensation of chromatin-binding factors, in particular the cancer-associated chromosomal translocation product EWS-FLI1. Using single-molecule counting and tracking in live cells, she finds that EWS-FLI1 proteins use their low-complexity domains to cluster into groups of approximately 24 proteins, though only 8 DNA binding sites are present linearly. The fusion protein shows more hub formation propensity than either parent protein, which may contribute to the aberrant transcriptional profile associated with Ewing Sarcoma.

Two speakers shared their focus on the ‘sequence grammar’ that underlies the specificity of interactions between protein intrinsically disordered regions (IDRs) or low complexity domains (LCDs) that can create condensates.

Matthew King (St Louis, US) described this language for nucleolar proteins, finding that fibrillar center (FC) proteins exhibit lysine-rich blocks, while dense fibrillar component (DFC) proteins instead have tracts rich in negatively charged glutamic and aspartic acids. This high concentration of charges within a condensate can result in a very different pH within the condensate than outside of it, which is mollified by mutating the D/E tracts of DFC proteins. Interestingly, pH gradients formed by condensates can drive enzymatic reactions like dephosphorylation without enzymes.

On the theme of specificity of IDR and LCD interactions, Nicolas Fawzi (Providence, US) reported the lack of specificity of a chemical commonly thought to disrupt condensates: 1,6-hexanediol. The cautionary tale warned researchers that this agent does solely act only condensates. Instead, he employs NMR spectroscopy to study the structure of low complexity domains, focusing especially on the ALS-associated protein TDP-43. The C-terminal domain of TDP-43 mediates liquid-like or aggregative behaviors, but it was unknown whether it acts as a dimer or multimer. A 10-residue helix was found in the CTD of TDP- 43 to act as a tetramer, with methionine, leucine, and tryptophan residues in this helix stabilizing its oligomerization. This newly identified putative helix is near the ALS-associated A321V mutation, and altering the helix affects phase separation propensity and nuclear localization of TDP-43.

Two other speakers highlighted the connection between condensates and physical forces in the nucleus. Marie-Hélène Verlhac (Paris, France) showed how force transference from cytoplasmic cytoskeletal elements in mouse oocytes results in reorganization of Cajal bodies, nuclear speckles and nucleoli within the nucleus. These start out as multiple and widely distributed nuclear bodies, and then through the extreme nuclear deformations imparted by cytoskeletal elements, they encounter one another and fuse. The altered distribution of functional nuclear bodies influences their function, shown by higher efficiency of splicing in the mature oocyte with one large speckle than in the early oocyte with many distributed speckles.

Amy Strom (Princeton, US) introduced a technology that utilizes light-controlled engineered condensates to push and pull on genomic loci. She emphasized that liquid-like condensates can utilize surface tension to apply force on the genome, without the use of ATP-driven motors. This force, in her system applied by synthetic condensates, is sufficient to rearrange loci over microns of nuclear space and create targeted pairwise genomic interactions. She suggested that the endogenous condensates may be sites of force generation in genome organization, acting as anchors or ‘motors’ to orchestrate long-range or even inter-chromosomal interactions.

New methods for studying nuclear substructures

Novel technologies drive research fields forward and the work presented at this conference was no exception, as three interesting new methods were presented. Kathleen Collins (Berkeley, US) described a two-component RNA system to deliver large insert transgenes via target-primed reverse transcription (RT) at specific sites in the genome. This method offers considerable promise for many different gene therapy applications as it evades surveillance by the innate immune system. Hadi Najafi (Worcester, US) described how he has incorporated Collins’ RT element system into his work aiming to capture and identify ternary complexes of microRNAs, mRNAs, and Argonaute 2 protein in the nucleolus

David Shechner (Seattle, US) described a novel approach that allows for detailed characterization of microenvironments surrounding nearly any RNA of interest, coding and non-coding. This new proximity-labeling approach is called O-MAP (Oligonucleotide-mediated Proximity-Interactome Mapping), and uses many off-the-shelf components that can be combined in innovative ways. One application involved testing the idea that nascent pre-mRNAs might serve as scaffolding elements to generate novel nuclear subdomains that can bring together (or sequester away) RNA binding proteins, and this was clearly illustrated using the Titin (TTN) gene locus as a model.

Yet another novel method was presented by Mei Zhang of Syncell Inc. (Taipei, Taiwan). This is a turnkey system named ‘Microscoop’ for proximity labeling that utilizes a photoactivatable biotin compound to enable high-content in situ photolabeling. The novel element of the system is to microscopically train the photoactivation beam to trigger photochemical conversion of the compound specifically within a delimited site within the nucleus.

Diversity and careers

The organizers were committed not only to engaging trainees as speakers and session chairs (all of the latter were) but also to having a session on Diversity. This was achieved by our good fortune to have a Keynote talk by George Langford (Syracuse, US), an acclaimed leader in advocating for minority biological scientists. He began his lecture with an engaging portrayal of Black culture in America and then proceeded to present his highly successful ‘Pair-Up’ program in which minority scientists are hosted for week-long visits by centers of excellence in the imaging field. Dr Langford emphasized, among several key points, that this program differs from other efforts in that it is not ‘remedial’ but rather is ‘catalytic’. His talk was extremely well received and then was followed by a robust open forum on diversity and career development, including their overlap.

Of the 37 invited main speakers, 17 were female and 20 were male (self-assigned). Fourteen of the main speakers were trainees (post-docs or graduate students) as were all of the session chairs. The meeting included 11 short talks, most of them by trainees, and parallel poster presentations. The countries represented were China, France, Ireland, Italy, Japan, Switzerland, Taiwan, Turkey, and the United States. The organizers did not ‘target’ any genders, ethnicities, or countries in creating the list of invitees. We simply invited those whom we believed were doing the most interesting and relevant work. That this resulted in the diversity it did is reason for optimism.

Conclusion

The organizers had not known until later in the planning stages that we would be at Niagara Falls as opposed to other venues that FASEB commissions for these conferences but were delighted to have this opportunity. The conference center was only a 20-min walk to the falls, and we all visited it – most of us more than once. As one of the ‘most aqueous’ places in the world (it flows at up to 1.7 cubic meters per min), we were reminded that water may be 54.5 M inside the cell, that the equilibrium affinity constants between/among certain proteins and with RNAs are what they are (i.e., are constants), that the ever-present hydrophobic effect rules to the same degree it always has, but that newly recognized physical chemistry informs our expanding understanding of nuclear bodies. This FASEB meeting being at Niagara Falls reminded us that this water’s mass x velocity is a fitting metaphor for the present momentum of the Nuclear Body field.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

Three of the authors have relationships with sponsors of the conference. L-LC is a scientific cofounder of Ribo-X Therapeutics, and AGM and TP are employees of the University of North Carolina and the University of Massachusetts Chan Medical School, respectively. EL and AS have no disclosures to make.

Author contributions

L.L.C., E.L., A.G.M., A.S., and T.P. all contributed to the writing and editing of the manuscript. All authors have read and approved the final version of this manuscript.

Data-availability statement

Data sharing is not applicable to this article as no new data were created or analyzed in this study.