Iain A. Sawyer

Cajal bodies are linked to genome conformation

The mechanisms underlying nuclear body (NB) formation and their contribution to genome function are unknown. Here we examined the non-random positioning of Cajal bodies (CBs), major NBs involved in spliceosomal snRNP assembly and their role in genome organization. CBs are predominantly located at the periphery of chromosome territories at a multi-chromosome interface. Genome-wide chromosome conformation capture analysis (4C-seq) using CB-interacting loci revealed that CB-associated regions are enriched with highly expressed histone genes and U small nuclear or nucleolar RNA (sn/snoRNA) loci that form intra- and inter-chromosomal clusters. In particular, we observed a number of CB-dependent gene-positioning events on chromosome 1. RNAi-mediated disassembly of CBs disrupts the CB-targeting gene clusters and suppresses the expression of U sn/snoRNA and histone genes. This loss of spliceosomal snRNP production results in increased splicing noise, even in CB-distal regions. Therefore, we conclude that CBs contribute to genome organization with global effects on gene expression and RNA splicing fidelity.

Nuclear bodies: Built to boost

The classic archetypal function of nuclear bodies is to accelerate specific reactions within their crowded space. In this issue, Tatomer et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201504043) provide the first direct evidence that the histone locus body acts to concentrate key factors required for the proper processing of histone pre-mRNAs.

Cajal body function in genome organization and transcriptome diversity

Nuclear bodies contribute to non-random organization of the human genome and nuclear function. Using a major prototypical nuclear body, the Cajal body, as an example, we suggest that these structures assemble at specific gene loci located across the genome as a result of high transcriptional activity. Subsequently, target genes are physically clustered in close proximity in Cajal body-containing cells. However, Cajal bodies are observed in only a limited number of human cell types, including neuronal and cancer cells. Ultimately, Cajal body depletion perturbs splicing kinetics by reducing target small nuclear RNA (snRNA) transcription and limiting the levels of spliceosomal snRNPs, including their modification and turnover following each round of RNA splicing. As such, Cajal bodies are capable of shaping the chromatin interaction landscape and the transcriptome by influencing spliceosome kinetics. Future studies should concentrate on characterizing the direct influence of Cajal bodies upon snRNA gene transcriptional dynamics. Also see the video abstract here.

Chromatin loops and causality loops: the influence of RNA upon spatial nuclear architecture

An intrinsic and essential trait exhibited by cells is the properly coordinated and integrated regulation of an astoundingly large number of simultaneous molecular decisions and reactions to maintain biochemical homeostasis. This is especially true inside the cell nucleus, where the recognition of DNA and RNA by a vast range of nucleic acid-interacting proteins organizes gene expression patterns. However, this dynamic system is not regulated by simple “on” or “off” signals. Instead, transcription factor and RNA polymerase recruitment to DNA are influenced by the local chromatin and epigenetic environment, a gene’s relative position within the nucleus and the action of noncoding RNAs. In addition, major phase-separated structural features of the nucleus, such as nucleoli and paraspeckles, assemble in direct response to specific transcriptional activities and, in turn, influence global genomic function. Currently, the interpretation of these data is trapped in a causality dilemma reminiscent of the “chicken and the egg” paradox as it is unclear whether changes in nuclear architecture promote RNA function or vice versa. Here, we review recent advances that suggest a complex and interdependent interaction network between gene expression, chromatin topology, and noncoding RNA function. We also discuss the functional links between these essential nuclear processes from the nanoscale (gene looping) to the macroscale (sub-nuclear gene positioning and nuclear body function) and briefly highlight some of the challenges that researchers may encounter when studying these phenomena.

Specific genomic cues regulate Cajal body assembly.

ABSTRACT The assembly of specialized sub-nuclear microenvironments known as nuclear bodies (NBs) is important for promoting efficient nuclear function. In particular, the Cajal body (CB), a prominent NB that facilitates spliceosomal snRNP biogenesis, assembles in response to genomic cues. Here, we detail the factors that regulate CB assembly and structural maintenance. These include the importance of transcription at nucleating gene loci, the grouping of these genes on human chromosomes 1, 6 and 17, as well as cell cycle and biochemical regulation of CB protein function. We also speculate on the correlation between CB formation and RNA splicing levels in neurons and cancer. The timing and location of these specific molecular events is critical to CB assembly and its contribution to genome function. However, further work is required to explore the emerging biophysical characteristics of CB assembly and the impact upon subsequent genome reorganization.

Spectral imaging to visualize higher-order genomic organization.

ABSTRACT A concern in the field of genomics is the proper interpretation of large, high-throughput sequencing datasets. The use of DNA FISH followed by high-content microscopy is a valuable tool for validation and contextualization of frequently occurring gene pairing events at the single-cell level identified by deep sequencing. However, these techniques possess certain limitations. Firstly, they do not permit the study of colocalization of many gene loci simultaneously. Secondly, the direct assessment of the relative position of many clustered gene loci within their respective chromosome territories is impossible. Thus, methods are required to advance the study of higher-order nuclear and cellular organization. Here, we describe a multiplexed DNA FISH technique combined with indirect immunofluorescence to study the relative position of 6 distinct genomic or cellular structures. This can be achieved in a single hybridization step using spectral imaging during image acquisition and linear unmixing. Here, we detail the use of this method to quantify gene pairing between highly expressed spliceosomal genes and compare these data to randomly positioned in silico simulated gene clusters. This is a potentially universally applicable approach for the validation of 3C-based technologies, deep imaging of spatial organization within the nucleus and global cellular organization.

Integrator subunit 4 is a ‘Symplekin-like’ scaffold that associates with INTS9/11 to form the Integrator cleavage module

Abstract Integrator (INT) is a transcriptional regulatory complex associated with RNA polymerase II that is required for the 3′-end processing of both UsnRNAs and enhancer RNAs. Integrator subunits 9 (INTS9) and INTS11 constitute the catalytic core of INT and are paralogues of the cleavage and polyadenylation specificity factors CPSF100 and CPSF73. While CPSF73/100 are known to associate with a third protein called Symplekin, there is no paralog of Symplekin within INT raising the question of how INTS9/11 associate with the other INT subunits. Here, we have identified that INTS4 is a specific and conserved interaction partner of INTS9/11 that does not interact with either subunit individually. Although INTS4 has no significant homology with Symplekin, it possesses N-terminal HEAT repeats similar to Symplekin but also contains a β-sheet rich C-terminal region, both of which are important to bind INTS9/11. We assess three functions of INT including UsnRNA 3′-end processing, maintenance of Cajal body structural integrity, and formation of histone locus bodies to conclude that INTS4/9/11 are the most critical of the INT subunits for UsnRNA biogenesis. Altogether, these results indicate that INTS4/9/11 compose a heterotrimeric complex that likely represents the Integrator ‘cleavage module’ responsible for its endonucleolytic activity.

Pre-mRNA Splicing: Function and Dysfunction

Most precursor messenger RNAs (pre-mRNAs) have noncoding introns that intervene exons. Introns must be removed and exons ligated together to form a mature mRNA. This process, called pre-mRNA splicing, allows an additional layer of genetic complexity as alternative splicing of exons into the final mRNA can control transcript abundance and protein isoform production. The essential role of splicing in normal cellular function is exemplified by human diseases that are caused by mutations in proteins involved in splicing. Here, we outline the process of splicing and alternative splicing regulation, describe some of the human diseases caused by splicing factor mutation and discuss the relationship between function and phenotype.

Membraneless nuclear organelles and the search for phases within phases

Cells are segregated into two distinct compartment groups to optimize cellular function. The first is characterized by lipid membranes that encapsulate specific regions and regulate macromolecular flux. The second, known collectively as membraneless organelles (MLOs), lacks defining lipid membranes and exhibits self‐organizing properties. MLOs are enriched with specific RNAs and proteins that catalyze essential cellular processes. A prominent sub‐class of MLOs are known as nuclear bodies, which includes nucleoli, paraspeckles, and other droplets. These microenvironments contain specific RNAs, exhibit archetypal liquid–liquid phase separation characteristics, and harbor intrinsically disordered, multivalent hub proteins. We present an analysis of nuclear body protein disorder that suggests MLO proteomes are significantly more disordered than structured cellular features. We also outline common MLO ultrastructural features, exemplified by the three sub‐compartments present inside the nucleolus. A core‐shell configuration, or phase within a phase, is displayed by several nuclear bodies and may be functionally important. Finally, we summarize evidence indicating extensive RNA and protein sharing between distinct nuclear bodies, suggesting functional cooperation and similar nucleation principles. Considering the substantial accumulation of specific coding and noncoding RNA classes inside MLOs, evidence that RNA buffers specific phase transition events, and the absence of a clear correlation between total intrinsic protein disorder and MLO accumulation, we conclude that RNA biogenesis may play a key role in MLO formation, internal organization, and function.

10 – Nuclear Bodies

The cell nucleus is nonrandomly organized and lacks defining internal membranes. As such, prominent domains composed of DNA, RNA, and protein complexes within the nucleus are easily detectable by microscopy and are crucial for maintaining spatial and temporal control of cellular homeostasis. Of these domains, nuclear bodies (NBs) are a diverse range of protein and RNA-based structures that regulate gene transcription and silencing, RNA processing and retention, protein modifications, and other key cellular processes. Well-known NBs include the nucleolus, promyelocytic NBs, Cajal bodies, nuclear speckles, and histone locus bodies. These regions are phase separated from the surrounding nucleoplasm and within their confined spaces they concentrate essential but limited factors that accelerate specific catalytic or assembly reactions. Here, we describe how these nuclear structures are assembled and structurally maintained, highlight key NBs and their clinical relevance. We also provide a brief overview of recent developments regarding biophysical studies that are beginning to uncover the dynamics of NB nucleation and function.