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In contrast to the well-studied condensation and folding of chromosomes during mitosis, their dynamics during interphase are less understood. We deployed a CRISPR-based DNA imaging system to track the dynamics of genomic loci situated kilobases to megabases apart on a single chromosome. Two distinct modes of dynamics were resolved: local movements as well as ones that might reflect translational movements of the entire domain within the nucleoplasmic space. The magnitude of both of these modes of movements increased from early to late G1, whereas the translational movements were reduced in early S phase. The local fluctuations decreased slightly in early S and more markedly in mid-late S. These newly observed movements and their cell cycle dependence suggest the existence of a hitherto unrecognized compaction-relaxation dynamic of the interphase chromosome fiber, operating concurrently with changes in the extent of overall movements of loci in the 4D genome., (© 2019 Ma et al.)

The bacterial CRISPR-Cas9 system has been repurposed for genome engineering, transcription modulation, and chromosome imaging in eukaryotic cells. However, the nuclear dynamics of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) guide RNAs and target interrogation are not well defined in living cells. Here, we deployed a dual-color CRISPR system to directly measure the stability of both Cas9 and guide RNA. We found that Cas9 is essential for guide RNA stability and that the nuclear Cas9-guide RNA complex levels limit the targeting efficiency. Fluorescence recovery after photobleaching measurements revealed that single mismatches in the guide RNA seed sequence reduce the target residence time from >3 h to as low as <2 min in a nucleotide identity- and position-dependent manner. We further show that the duration of target residence correlates with cleavage activity. These results reveal that CRISPR discriminates between genuine versus mismatched targets for genome editing via radical alterations in residence time., (© 2016 Ma et al.)

The life of the nucleolus has proven to be more colorful and multifaceted than had been envisioned a decade ago. A large number of proteins found in this subnuclear compartment have no identifiable tie either to the ribosome biosynthetic pathway or to the other newly established activities occurring within the nucleolus. The questions of how and why these proteins end up in this subnuclear compartment remain unanswered and are the focus of intense current interest. This review discusses our thoughts on the discovery of nonribosomal proteins in the nucleolus.

The idea that actin may have an important function in the nucleus has undergone a rapid transition from one greeted with skepticism to a now rapidly advancing research field. Actin has now been implicated in transcription by all three RNA polymerases, but the structural form it adopts in these processes remains unclear. Recently, a claim was made that monomeric nuclear actin plays a role in signal transduction, while a just-published study of RNA polymerase I transcription has implicated polymeric actin, consorting with an isoform of its classical partner myosin. Both studies are critically discussed here, and although there are several issues to be resolved, it now seems reasonable to start thinking about functions for both monomeric and assembled actin in the nucleus.

The Cajal body is an intriguing nuclear structure present in a great variety of plant, animal, and some fungal cells. Recent work on the ribonucleoprotein enzyme telomerase has indicated an unanticipated degree of intranuclear dynamics of both its RNA and protein subunits. In this issue, Jady et al. place the Cajal body on the intranuclear traffic route of telomerase RNA (Jady et al., 2004).

The nucleolus is the site of ribosome biosynthesis, but is now known to have other functions as well. In the present study we have investigated how the distribution of signal recognition particle (SRP) RNA within the nucleolus relates to the known sites of ribosomal RNA synthesis, processing, and nascent ribosome assembly (i.e., the fibrillar centers, the dense fibrillar component (DFC), and the granular component). Very little SRP RNA was detected in fibrillar centers or the DFC of the nucleolus, as defined by the RNA polymerase I-specific upstream binding factor and the protein fibrillarin, respectively. Some SRP RNA was present in the granular component, as marked by the protein B23, indicating a possible interaction with ribosomal subunits at a later stage of maturation. However, a substantial portion of SRP RNA was also detected in regions of the nucleolus where neither B23, UBF, or fibrillarin were concentrated. Dual probe in situ hybridization experiments confirmed that a significant fraction of nucleolar SRP RNA was not spatially coincident with 28S ribosomal RNA. These results demonstrate that SRP RNA concentrates in an intranucleolar location other than the classical stations of ribosome biosynthesis, suggesting that there may be nucleolar regions that are specialized for other functions.

The dynamic intra-nuclear localization of MRP RNA, the RNA component of the ribonucleoprotein enzyme RNase MRP, was examined in living cells by the method of fluorescent RNA cytochemistry (Wang, J., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1991. Proc. Natl. Acad. Sci. USA. 88:7391-7395). MRP RNA very rapidly accumulated in nucleoli after nuclear microinjection of normal rat kidney (NRK) epithelial cells. Localization was specifically in the dense fibrillar component of the nucleolus, as revealed by immunocytochemistry with a monoclonal antibody against fibrillarin, a known dense fibrillar component protein, as well as by digital optical sectioning microscopy and 3-D stereo reconstruction. When MRP RNA was injected into the cytoplasm it was not imported into the nucleus. Nuclear microinjection of mutant MRP RNAs revealed that nucleolar localization requires a sequence element (nucleotides 23-62) previously implicated as a binding site for a nucleolar protein, the To antigen. These results demonstrate the dynamic localization of MRP RNA in the nucleus and provide important insights into the nucleolar targeting of MRP RNA.

The processing and ribonucleoprotein assembly of U4 small nuclear RNA has been investigated in HeLa cells. After a 45-min pulse label with [3H]uridine, a set of apparently cytoplasmic RNAs was observed migrating just behind the gel electrophoretic position of mature U4 RNA. These molecules were estimated to be one to at least seven nucleotides longer than mature U4 RNA. They reacted with Sm autoimmune patient sera and a monoclonal Sm antibody, indicating their association with proteins characteristic of small nuclear ribonucleoprotein complexes. The same set of RNAs was identified by hybrid selection of pulse-labeled RNA with cloned U4 DNA, confirming that these are U4 RNA sequences. No larger nuclear precursors of these RNAs were detected. Pulse-chase experiments revealed a progressive decrease in the radioactivity of the U4 precursor RNAs coincident with an accumulation of labeled mature U4 RNA, confirming a precursor-product relationship.

Eucaryotic messenger RNA precursors are processed in nuclear ribonucleoprotein particles (hnRNP). Here recent work on the structure of hnRNP is reviewed, with emphasis on function. Detailed analysis of a specific case, the altered assembly of hnRNP in heat-shocked Drosophila and mammalian cells, leads to a general hypothesis linking hnRNP structure and messenger RNA processing.

The small nuclear RNAs are known to be complexed with proteins in the cell (snRNP). To learn more about these proteins, we developed an in vitro system for studying their interactions with individual small nuclear RNA species. Translation of HeLa cell poly(A)+ mRNA in an exogenous message-dependent reticulocyte lysate results in the synthesis of snRNP proteins. Addition of human small nuclear RNA U1 to the translation products leads to the formation of a U1 RNA-protein complex that is recognized by a human autoimmune antibody specific for U1 snRNP. This antibody does not react with free U1 RNA. Moreover, addition of a 10- to 20-fold molar excess of transfer RNA instead of U1 RNA does not lead to the formation of an antibody-recognized RNP. The proteins forming the specific complex with U1 RNA correspond to the A, B1, and B2 species (32,000, 27,000, and 26,000 mol wt, respectively) observed in previous studies with U1 snRNP obtained by antibody-precipitation of nuclear extracts. The availability of this in vitro system now permits, for the first time, direct analysis of snRNA-protein binding interactions and, in addition, provides useful information on the mRNAs for snRNP proteins.

The processing of heterogeneous nuclear RNA into messenger RNA takes place in special nuclear ribonucleoprotein particles known as hnRNP. We report here the identification of proteins tightly complexed with poly(A)+ hnRNA in intact HeLa cells, as revealed by a novel in situ RNA-protein cross-linking technique. The set of cross-linked proteins includes the A, B, and C "core" hnRNP proteins, as well as the greater than 42,000 mol wt species previously identified in noncross-linked hnRNP. These proteins are shown to be cross-linked by virtue of remaining bound to the poly(A)+ hnRNA in the presence of 0.5% sodium dodecyl sulfate, 0.5 M NaCl, and 60% formamide, during subsequent oligo(dT)-cellulose chromatography, and in isopycnic banding in Cs2SO4 density gradients. These results establish that poly(A)+ hnRNA is in direct contact with a moderately complex set of nuclear proteins in vivo. This not only eliminates earlier models of hnRNP structure that were based upon the concept of a single protein component but also suggests that these proteins actively participate in modulating hnRNA structure and processing in the cell.

We have investigated the intracellular site and posttranscriptional immediacy of U1 small nuclear RNA processing and ribonucleoprotein (RNP) assembly in HeLa cells. After 30 or 45 min of labeling with [3H]uridine, a large amount of U1-related RNA radioactivity in the cytoplasm was found by using either hypotonic or isotonic homogenization buffers. The pulse-labeled cytoplasmic U1 RNA was resolved as a ladder of closely spaced bands running just behind mature-size U1 (165 nucleotides) on RNA sequencing gels, corresponding to a series of molecules between one and at least eight nucleotides longer than mature U1. They were further identified as U1 RNA sequences by gel blot hybridization with cloned U1 DNA. The ladder of cytoplasmic U1 RNA bands reacted with both RNP and Sm autoimmune sera and with a monoclonal Sm antibody, indicating a cytoplasmic assembly of these U1 RNA-related molecules into complexes containing the same antigens as nuclear U1 RNP particles. The cytoplasmic molecules behave as precursors to mature nuclear U1 RNA in both pulse-chase and continuous labeling experiments. While not excluding earlier or subsequent nuclear stages, these results suggest that the cytoplasm is a site of significant U1 RNA processing and RNP assembly. This raises the possibility that nuclear-transcribed eucaryotic RNAs are always processed in the cell compartment other than that in which they ultimately function, which suggests a set of precise signals regulating RNA and ribonucleoprotein traffic between nucleus and cytoplasm.

Interphase HeLa cells manifest a stepwise shutoff of RNA synthesis when the tonicity of the extracellular medium is gradually increased. Synthesis of heterogeneous nuclear RNA is most sensitive and is selectively inhibited at 1.5 times isotonicity (450 milliosmols/liter), while 45S ribosomal RNA synthesis is not affected significantly below 2.0 times isotonicity. Transfer RNA synthesis is least sensitive to increased osmolarity and is not completely inhibited until the electrolyte concentration of the medium is elevated to 2.8 times isotonicity. Although the transcription and methylation of 45S ribosomal precursor is unaffected at 1.5 times isotonicity, there is pronounced impairment of its processing into 32S and 18S RNA. Using a refined cell synchronization technique, we have been able to compare these effects of hypertonicity with the shutoff of RNA synthesis which occurs during the G(2)-prophase interval of the cell division cycle. In this case, as with random cells in hypertonic medium, a selective inhibition of heterogeneous nuclear RNA synthesis and slowed processing of 45S ribosomal RNA were found, whereas synthesis of 45S and transfer RNA continued unabated throughout G(2)-prophase. While it is known that RNA synthesis essentially ceases during metaphase, we have noted that transfer RNA synthesis continues in metaphase at 10-15% of the interphase rate, which is of particular interest in view of the relative resistance of this species to hypertonicity. The close correlation between the patterns of cessation of RNA synthesis at mitosis and during exposure to hypertonic medium supports our earlier contention that alteration of intracellular electrolyte levels provides a useful model for studying the mechanism of mitosis.

Interphase HeLa cells exposed to solutions that are 1.6 x isotonic manifest a series of morphological transformations, several of which grossly resemble those which occur when untreated cells enter prophase. These include chromosome condensation with preferential localization at the nuclear envelope and nucleolus, ruffling of the nuclear envelope, and polyribosome breakdown. The nucleolus loses its fibrous component and appears diffusely granular. At 2.8 x isotonicity the nuclear envelope is selectively dispersed although other membranes show morphological alterations also. The characteristic transitions of the lysosomes, Golgi complex, and microtubules seen in normal mitosis do not occur during hypertonic treatment. All the changes induced with hypertonic solutions are rapidly reversible, and the nucleus particularly goes through a recovery phase which bears some similarity to that of the telophase nucleus. The prophase-like condensation of the chromatin following exposure of the intact cell to hypertonic medium cannot be reproduced on an ultrastructural level in the isolated nucleus with any known variation in salt concentration, suggesting significant modifications of the nuclear contents during isolation. In addition to these morphological responses, hypertonic solutions also markedly and reversibly depress macromolecular synthesis. The polyribosome disaggregation that results from exposure to hypertonic solutions may be partially prevented by prior exposure to elevated Mg(++) concentrations; this same ion is also partially effective in preventing the polyribosome breakdown which normally occurs as cells enter mitosis.

The cell-free synthesis of histone-like polypeptides has been achieved using a selected class of small polyribosomes as the only particulate fraction. This synthesis is prevented if the deoxyribonucleic acid (DNA) inhibitor, cytosine arabinoside, is added to the cells prior to disruption, and it is not detected when the cytoplasm used is derived from postmitotic (G(1)) cells. When the 100,000 g supernate from pure metaphase populations was compared with that from S phase cells, the cell-free synthesis of histone-like polypeptides in the presence of S phase polyribosomes remained unchanged. These data suggest that, except for the histone messenger RNA-ribosome complex, the cytoplasmic factors requisite for histone synthesis are present throughout the cycle, and that the shut-off of this synthesis is not under translational control.

Measurements of actinomycin-(3)H binding in synchronized HeLa cells reveal that the binding capacity of chromatin decreases progressively during the S phase despite a doubling of nuclear DNA content, reaches a minimal level during G(2) and mitosis, and then increases gradually throughout the subsequent G(1) interval. Since this pattern was evident in experiments with living cells, ethanol-fixed cells, and isolated nuclei, but not with purified DNA, the actinomycin binding profile may reflect changes in the degree of association between DNA and chromosomal proteins at different stages of the cell cycle.