Preface The 4D Nucleome Network aims to develop and apply approaches to map the structure and dynamics of the human and mouse genomes in space and time with the goal of gaining deeper mechanistic understanding of how the nucleus is organized and functions. The project will develop and benchmark experimental and computational approaches for measuring genome conformation and nuclear organization, and investigate how these contribute to gene regulation and other genome functions. Validated experimental approaches will be combined with biophysical modeling to generate quantitative models of spatial genome organization in different biological states, both in cell populations and in single cells.
Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by f luorescence correlation spectroscopy (FCS) and f luorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 ؋ 10 ؊7 cm 2 ͞s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (<1 ؋ 10 ؊7 cm 2 ͞s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 ؋ 10 ؊7 cm 2 ͞s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.An understanding of the physical environment inside the cell nucleus is central to a coherent view of gene expression. It is important to know how the viscosity and molecular diffusion rates in the nucleus of a living cell compare with experimental conditions in vitro, where interactions between nucleic acids and proteins are studied at high dilution in aqueous solution. For example, it is not clear whether ribonucleoprotein (RNP) complexes can diffuse freely about the nucleus, impeded only by locally high concentrations of macromolecules or, alternatively, are bound or compartmentalized in such a way as to constrain their motion. Some observations suggest that premRNA transcripts are tethered to elements of the transcriptional, splicing, and͞or polyadenylation machinery (1-3), and it has been proposed that processed mRNAs make their way out of the nucleus by molecular diffusion (4, 5). However, mediated processes have not been ruled out, and the functional relationships between RNA export and nuclear structure remain unclear (6).Recently, Politz et al. (7) characterized nucleic acid uptake and hybridization in living cells by in situ reverse transcription and found that fluorescently labeled oligo(dT) can be taken up by living cells and form hybrid...
Poly(A) RNA can move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion.
The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and͞or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves. T he nucleolus long has been known as a dense subnuclear structure at which ribosomal genes are clustered and ribosomal transcription, rRNA processing, and ribosomal subunit assembly occurs (1). More recently, it has become clear that the RNA components of other ribonucleoproteins also may localize to the nucleolus (2, 3), including the signal recognition particle (SRP) (4). Protein components of both RNase P and RNase MRP also recently have been shown to be localized in the nucleolus (5). The hypothesis has been advanced that the nucleolus not only has evolved to carry out ribosome synthesis but, in fact, is the site of assembly of and͞or interaction between multiple ribonucleoprotein machines, many of which are involved in protein synthesis (6). However, no information regarding the potential nucleolar localization of the other components of the SRP has been available.The SRP is a ribonucleoprotein that arrests translation of secretory or membrane proteins and docks the nascent polypeptide-ribosome complex at receptors on the endoplasmic reticulum (ER), whereupon translation is resumed to direct the protein into the membrane assembly or secretory pathways (7-9). In mammalian cells, the SRP contains six proteins (7) and an Ϸ300-nt RNA (10) that previously had been known as 7S RNA or 7SL RNA (11-13). In an early study it was found that ...
The repressive compartment of the nucleus is comprised primarily of telomeric and centromeric regions, the silent portion of ribosomal RNA genes, the majority of transposable element repeats, and facultatively repressed genes specific to different cell types. This compartment localizes into three main regions: the peripheral heterochromatin, perinucleolar heterochromatin, and pericentromeric heterochromatin. Both chromatin remodeling proteins and transcription of noncoding RNAs are involved in maintenance of repression in these compartments. Global reorganization of the repressive compartment occurs at each cell division, during early development, and during terminal differentiation. Differential action of chromatin remodeling complexes and boundary element looping activities are involved in mediating these organizational changes. We discuss the evidence that heterochromatin formation and compartmentalization may drive nuclear organization.
Chromatin, which consists of DNA and associated proteins, contains genetic information and is a mechanical component of the nucleus. Heterochromatic histone methylation controls nucleus and chromosome stiffness, but the contribution of heterochromatin protein HP1α (CBX5) is unknown. We used a novel HP1α auxin-inducible degron human cell line to rapidly degrade HP1α. Degradation did not alter transcription, local chromatin compaction, or histone methylation, but did decrease chromatin stiffness. Single-nucleus micromanipulation reveals that HP1α is essential to chromatin-based mechanics and maintains nuclear morphology, separate from histone methylation. Further experiments with dimerization-deficient HP1αI165E indicate that chromatin crosslinking via HP1α dimerization is critical, while polymer simulations demonstrate the importance of chromatin-chromatin crosslinkers in mechanics. In mitotic chromosomes, HP1α similarly bolsters stiffness while aiding in mitotic alignment and faithful segregation. HP1α is therefore a critical chromatin-crosslinking protein that provides mechanical strength to chromosomes and the nucleus throughout the cell cycle and supports cellular functions.
The classical view of the nucleolus as solely committed to ribosome biosynthesis has been modified by recent studies pointing to additional roles for this nuclear domain. These newly recognized features include the nucleolar presence of several nonribosomal RNAs transcribed by RNA polymerase III, as well as nucleolar roles in gene silencing, cell cycle progression, and cellular senescence. The signal recognition particle (SRP) 1 RNA, and several protein components of the SRP also recently have been detected in the nucleolus. Thus, the large and small ribosomal subunits, the 5S rRNA-ribonucleoprotein complex, and now the SRP, are known to be assembled in or pass through the nucleolus. These findings, together with the recent observations that some transfer RNA precursor molecules and the pretransfer RNA processing enzyme, RNase P, are also found in the nucleolus, raise the possibility that these translational components are congressed in the nucleolus in order to probatively interact with one another, perhaps as a test of proper conformational fit. We hypothesize that such interactions may be an important checkpoint during nucleolar assembly of the translational machinery at steps ranging from the regulation of nascent transcript processing to a possible transient preassembly of the entire translational apparatus.
The perinucleolar compartment (PNC) is a nuclear substructure present in transformed cells. The PNC is defined by high concentrations of certain RNA binding proteins and a subset of small RNAs transcribed by RNA polymerase III (pol III), including the signal recognition particle RNA and an Alu RNA as reported here. To determine if the PNC is dependent on pol III transcription, HeLa cells were microinjected with the selective pol III inhibitor, Tagetin. This resulted in disassembly of the PNC, whereas inhibition of pol I by cycloheximide or pol II by ␣-amanitin did not significantly affect the PNC. However, overexpression of one of the PNC-associated RNAs from a pol II promoter followed by injection of Tagetin blocked the Tagetin-induced PNC disassembly, demonstrating that it is the RNA rather than pol III activity that is important for the PNC integrity. To elucidate the role of the PNC-associated protein PTB, its synthesis was inhibited by siRNA. This resulted in a reduction of the number of PNC-containing cells and the PNC size. Together, these findings suggest, as a working model, that PNCs may be involved in the metabolism of specific pol III transcripts in the transformed state and that PTB is one of the key elements mediating this process.
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