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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.

The nucleolus is a multifunctional nuclear body. To tease out the roles of nucleolar structure without resorting to the use of multi-action drugs, we knocked down the RNA polymerase I subunit RPA194 in HeLa cells by siRNA. Loss of RPA194 resulted in nucleolar-structural segregation and effects on both nucleolus-proximal and distal-nuclear components. The perinucleolar compartment was disrupted, centromere clustering around nucleoli was significantly reduced, and the intranuclear locations of specific genomic loci were altered. Moreover, Cajal bodies, distal from nucleoli, underwent morphological and some compositional changes. In comparison, when the preribosomal RNA-processing factor, UTP4, was knocked down, neither nucleolar segregation nor the intranuclear effects were observed, demonstrating that the changes of nucleolar proximal and distal nuclear domains in RPA194 knockdown cells unlikely arise from a cessation of ribosome synthesis, rather from the consequence of nucleolar-structure alteration. These findings point to a commutative system that links nucleolar structure to the maintenance and spatial organization of certain nuclear domains and genomic loci.

Recent advances in our understanding of how the genome is folded within the nucleus have included cases in which this positioning correlates with gene expression, either positively or negatively. But is the 3-D location of a gene a cause or an effect of its expression? In this Perspective I articulate the problem and then cite as guideposts recent cases where causation has indeed been arguably established. The hope is to critically illuminate this issue for continued consideration in this important, evolving field.

This report summarizes an international conference on molecular machines convened at New York University, Abu Dhabi by Piergiorgio Percipalle, George Shubeita and Serdal Kirmizialtin. The meeting was conceived around the epistemological question of what do we understand, or not understand (if we have open minds), about the degree to which cells operate by the individual actions of single enzymes or non-catalytic protein effectors, versus combinations of these in which their heterotypic association creates an entity that is more finely tuned and efficient - a machine. This theme was explored through a vivid series of talks, summarizing the latest findings on macromolecular complexes that operate in the nucleus or cytoplasm., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

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.)

DNA lesions induce recruitment and accumulation of various repair factors, resulting in formation of discrete nuclear foci. Using superresolution fluorescence microscopy as well as live cell and quantitative imaging, we demonstrate that X-ray repair cross-complementing protein 1 (XRCC1), a key factor in single-strand break and base excision repair, is recruited into nuclear bodies formed in response to replication-related single-strand breaks. Intriguingly, these bodies are assembled immediately in the vicinity of these breaks and never fully colocalize with replication foci. They are structurally organized, containing canonical promyelocytic leukemia (PML) nuclear body protein SP100 concentrated in a peripheral layer, and XRCC1 in the center. They also contain other factors, including PML, poly(ADP-ribose) polymerase 1 (PARP1), ligase IIIα, and origin recognition complex subunit 5. The breast cancer 1 and -2 C terminus domains of XRCC1 are essential for formation of these repair foci. These results reveal that XRCC1-contaning foci constitute newly recognized PML-like nuclear bodies that accrete and locally deliver essential factors for repair of single-strand DNA breaks in replication regions.-Kordon, M. M., Szczurek, A., Berniak, K., Szelest, O., Solarczyk, K., Tworzydło, M., Wachsmann-Hogiu, S., Vaahtokari, A., Cremer, C., Pederson, T., Dobrucki, J. W. PML-like subnuclear bodies, containing XRCC1, juxtaposed to DNA replication-based single-strand breaks.

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.)

We address several processes and domains in the nucleus wherein holding the perspective of physics either reveals a conundrum or is likely to enable progress., (© 2015 Pederson et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The nucleus is physically distinct from the cytoplasm in ways that suggest new ideas and approaches for interrogating the operation of this organelle. Chemical bond formation and breakage underlie the lives of cells, but as this special issue of Molecular Biology of the Cell attests, the nonchemical aspects of cell nuclei present a new frontier to biologists and biophysicists., (© 2014 Pederson and Marko. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Three recent papers, published just weeks apart, describe the use of fluorescent TALEs to tag specific DNA sequences in live cells and, in one case, also in fixed cells, the latter with potential clinical applications.

This volume brings together a number of perspectives on how certain physical phenomena contribute to the functional design and operation of the nucleus. This collection could not be more timely, resonating with an increasing awareness of the opportunities that lie at the interface of cell biology and the physical sciences. For example, this was a major theme in the 2012 and 2013 annual meetings of the American Society for Cell Biology, and one that the Society aims to emphasize even further going forward. In addition, the emerging canonical relevance of the physical sciences to cell biology has in recent summers made a most conspicuous appearance in the curriculum (lectures and intense labs) of the famed Physiology Course at the Marine Biological Laboratory in Woods Hole. So, much credit is due to Ronald Hancock and Kwang Jeon, the coeditors of this volume, and all the authors for creating a work that is so au courant., (© 2014 Elsevier Inc. All rights reserved.)

The movement of polyadenylated RNA transcripts (poly(A) RNA) through speckles in the nucleus can be detected and studied using fluorescence correlation microscopy (FCM) and photoactivation RNA tracking techniques. Speckles, sometimes called interchromatin granule clusters, are nuclear bodies that contain pre-mRNA splicing factors and poly(A) RNA. In the methods described here, speckles are marked in live cells using monomeric red fluorescent protein fused to SC35, a splicing protein that is a common speckle component. Endogenous poly(A) RNAs are tagged by in vivo hybridization with fluorescein-labeled oligo(dT) and FCM is performed at the marked speckles and in the nucleoplasm to measure the mobility of the tagged poly(A) RNA. The majority of the nuclear poly(A) RNA population diffuses rapidly throughout the nucleoplasm, and thus this method allows one to ask whether poly(A) RNA that is located in speckles at a given time is undergoing a dynamic transit or is, in contrast, a more immobile, perhaps structural, component. To visualize the movement of poly(A) RNA away from speckles, poly(A) RNA is tagged with caged-fluorescein-labeled oligo(dT) and speckle-associated poly(A) RNAs are specifically photoactivated using a laser beam directed through a pinhole in a rapid digital imaging microscopy system. The spatial distribution of the now-fluorescent RNA as it moves from the speckle photoactivation site is then recorded over time. Temperature and/or ATP levels can also be varied to test whether movement or localization of the poly(A) RNA is dependent on metabolic energy.

Now is an opportune moment to address the confluence of cell biological form and function that is the nucleus. Its arrival is especially timely because the recognition that the nucleus is extremely dynamic has now been solidly established as a paradigm shift over the past two decades, and also because we now see on the horizon numerous ways in which organization itself, including gene location and possibly self-organizing bodies, underlies nuclear functions.

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.

A number of recent studies have indicated that the location of a given mammalian chromosome within the interphase nucleus is related to its size, whereas other work has implicated a chromosome's gene density as a factor. Recent investigations of the degree to which an ordered arrangement of mitotic chromosomes on the metaphase plate is inherited and perpetuated during successive cell cycles have also yielded somewhat controversial results. The arrangement of chromosomes in the nucleus also has been investigated by the analysis of chromosomal translocations, with some surprising recent findings.

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 alpha-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.

Biophysical, chemical, and nanoscience approaches to the study of nuclear structure and activity have been developing recently and hold considerable promise. A selection of fundamental problems in genome organization and function are reviewed and discussed in the context of these new perspectives and approaches. Advancing these concepts will require coordinated networks of physicists, chemists, and materials scientists collaborating with cell, developmental, and genome biologists.

The notion that the interior of the nucleus is compartmentalized goes back to the discovery of the nucleolus in the 1830s. Today, we know that numerous, discrete domains related to gene expression exist within the interchromatin spaces of the interphase nucleus. These domains might arise from, and thus be positioned by, the transcriptional activity of the chromosomes, themselves tethered to the nuclear envelope, or they might assemble autonomously. Beyond their roles in gene expression or other nuclear functions, the dynamic behaviour of some of these interchromatin domains is providing clues to the modes of mass transport operating in the nucleus, as well as to the long-elusive deep structure of the nucleoplasm.

Actin is an abundant protein in most nonmuscle cells. It has often been observed in isolated nuclei, yet cytoplasmic contamination was of course initially regarded as the most plausible origin. Numerous studies on nuclear actin appeared in the 1970s and 1980s, but the picture remained rather muddy. The viewpoint at that time was that actin-shown to move freely between cytoplasm and nucleus-was a mere "thermodynamic wanderer," transiently occupying the nucleus. More recently, evidence has been mounting that actin's presence in the nucleus is not simply governed by the laws of diffusion. The same holds true for the finding of various actin-related proteins in the nucleus, and the case for nuclear myosin, specifically myosin I, is now quite convincing. Moreover, the first intimations of functional roles of nuclear actin are now emerging. Here we examine the overall subject from cell biological and chemical perspectives. The major issue is no longer the presence of actin in the nucleus but rather its supramolecular organization, intranuclear locations, and, of course, functions. These issues interface with recent findings that reveal a surprisingly diverse repertoire of actin conformations and oligomer and polymer forms beyond monomeric G-actin and polymeric F-actin. We present ideas for advancing the nuclear actin field and call for a renewed attack on this major problem in cell biology.

Although several systems are now available for the controlled expression of eukaryotic genes transcribed by RNA polymerase II, regulated expression has been more difficult to achieve in the case of genes transcribed by RNA polymerase III. In the present study the gene for bacteriophage T7 RNA polymerase, implanted with a eukaryotic nuclear localization signal, was linked to a 5'-flanking ecdysone-responsive promoter and stably transformed human cell lines were constructed in which the ecdysone promoter-T7 RNA polymerase gene had been integrated intact, as demonstrated by a polymerase chain reaction assay. Exposure of these cells to the ecdysone analog ponasterone A resulted in the appearance of a single protein having the expected size of T7 RNA polymerase in immunoblots of cell extracts probed with an affinity purified antibody raised against the C-terminus of T7 RNA polymerase. The induced T7 RNA polymerase was exclusively localized in the nucleus of induced cells and was undetectable in uninduced cells either by immunoblotting or immunofluorescence. The induced T7 RNA polymerase was present at numerous punctate foci dispersed throughout the nucleoplasmic regions of the nucleus and was also present in the nucleoli. Both of these observed intranuclear localizations have relevance to the potential applications of this system.

A cell fraction that would today be termed "the nuclear matrix" was first described and patented in 1948 by Russian investigators. In 1974 this fraction was rediscovered and promoted as a fundamental organizing principle of eukaryotic gene expression. Yet, convincing evidence for this functional role of the nuclear matrix has been elusive and has recently been further challenged. What do we really know about the nonchromatin elements (if any) of internal nuclear structure? Are there objective reasons (as opposed to thinly veiled disdain) to question experiments that use harsh nuclear extraction steps and precipitation-prone conditions? Are the known biophysical properties of the nucleoplasm in vivo consistent with the existence of an extensive network of anastomosing filaments coursing dendritically throughout the interchromatin space? To what extent may the genome itself contribute information for its own quarternary structure in the interphase nucleus? These questions and recent work that bears on the mystique of the nuclear matrix are addressed in this essay. The degree to which gene expression literally depends on nonchromatin nuclear structure as a facilitating organizational format remains an intriguing but unsolved issue in eukaryotic cell biology, and considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the in vivo situation.

The movement of various RNAs from their sites of chromosomal synthesis to their functional locations in the cell is an important step in eukaryotic gene readout, though one less well understood than the transcription, RNA processing, and various functions of RNA. The segregation of the many classes of RNA out into to their appropriate sites in the cell is, from a physical chemical point of view, a remarkable phenomenon. This paper summarizes investigations my colleagues and I have undertaken over the past 7 years to describe the intracellular traffic and localization of RNA in living cells. One approach we have developed is to glass-needle microinject approximately 0.01 pl of fluorescent RNA solutions into the nucleus or cytoplasm of cultured mammalian cells. This 'fluorescent RNA cytochemistry' approach has resolved intranuclear sites ('speCkles') for which premessenger RNAs (pre-mRNA) have high affinity and has revealed very rapid movements of certain other RNAs from their nucleoplasmic injection sites to the nucleoli. One of these rapidly trafficking nucleolar RNAs is the signal recognition particle (SRP) RNA, and further results indicate that the nucleolus is a site of SRP RNA processing or ribonucleoprotein assembly prior to export to the cytoplasm. In these fluorescent RNA microinjection studies, we have also used mutant RNA molecules to identify specific nucleotide sequences that function as targeting elements for the localization of RNAs at their respective intranuclear sites. In a second approach, we have used fluorescent correlation spectroscopy (FCS), a classical biophysical method for measuring molecular motion in vitro, coupled with confocal fluorescence microscopy to measure the movement of poly(A) RNA in the nucleus, with the interesting finding that these RNAs appear to move about inside the nucleus at rates comparable to diffusion in aqueous solution. Parallel experiments using the method of fluorescence recovery after photobleaching (FRAP) revealed a diffusion coefficient for intranuclear poly(A) RNA close to that measured by FCS. These results bear on the structure of the nucleoplasmic ground substance-an extremely controversial and unsolved problem in cell biology (29). The methods we have developed and these initial results represent the first major step toward a comprehensive understanding of RNA traffic in the cell nucleus.

Background: Messenger RNA (mRNA) is transcribed and processed in the nucleus of eucaryotic cells and then exported to the cytoplasm through nuclear pores. It is not known whether the movement of mRNA from its site of synthesis to the nuclear pore is directed or random. Directed movement would suggest that there is an energy-requiring step in addition to the step required for active transport through the pore, whereas random movement would indicate that mRNAs can make their way to the nuclear envelope by diffusion., Results: We devised a method to visualize movement of endogenous polymerase II transcripts in the nuclei of living cells. Oligo(dT) labeled with chemically masked (caged) fluorescein was allowed to penetrate cells and hybridize to nuclear poly(A) RNA. Laser spot photolysis then uncaged the oligo(dT) at a given intranuclear site and the resultant fluorescent, hybridized oligo(dT) was tracked using high-speed imaging microscopy. Poly(A) RNA moved away from the uncaging spot in all directions with a mean square displacement that varied linearly with time, and the same apparent diffusion coefficient was measured for the movement at both 37 degrees C and 23 degrees C. These properties are characteristic of a random diffusive process. High resolution three-dimensional imaging of live cells containing both Hoechst-labeled chromosomes and uncaged oligo(dT) showed that, excluding nucleoli, the poly(A) RNA could access most, if not all, of the non-chromosomal space in the nucleus., Conclusions: Poly(A) RNA can move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion.

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence 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 x 10(-7) cm2/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 (

The ribonucleoprotein enzyme RNase P catalyzes the 5' processing of pre-transfer RNA, and has also recently been implicated in pre-ribosomal RNA processing. In the present investigation, in situ hybridization revealed that RNase P RNA is present throughout the nucleus of mammalian cells. However, rhodamine-labeled human RNase P RNA microinjected into the nucleus of rat kidney (NRK) epithelial cells or human (HeLa) cells initially localized in nucleoli, and subsequently became more evenly distributed throughout the nucleus, similar to the steadystate distribution of endogenous RNase P RNA. Parallel microinjection and immunocytochemical experiments revealed that initially nucleus-microinjected RNase P RNA localized specifically in the dense fibrillar component of the nucleolus, the site of pre-rRNA processing. A mutant RNase P RNA lacking the To antigen binding domain (nucleotides 25-75) did not localize in nucleoli after nuclear microinjection. In contrast, a truncated RNase P RNA containing the To binding domain but lacking nucleotides 89-341 became rapidly localized in nucleoli following nuclear microinjection. However, unlike the full-length RNase P RNA, this 3' truncated RNA remained stably associated with the nucleoli and did not translocate to the nucleoplasm. These results suggest a nucleolar phase in the maturation, ribonucleoprotein assembly or function of RNase P RNA, mediated at least in part by the nucleolar To antigen. These and other recent findings raise the intriguing possibility of a bifunctional role of RNase P in the nucleus: catalyzing pre-ribosomal RNA processing in the nucleolus and pre-transfer RNA processing in the nucleoplasm.

We have studied the nuclear localization of rhodamine-labeled pre-mRNA after microinjection into nuclei of cultured rat kidney epithelial cells. Intranuclear localization of the injected RNA was followed in the living cells by fluorescence microscopy and digital image processing. Injected human beta-globin pre-mRNA became localized in 30-60 discrete nuclear sites that were coincident with loci defined by monoclonal antibodies against small nuclear ribonucleoproteins (Sm) or another spliceosome component (SC-35) in parallel immunocytochemical studies on the same nuclei. Similar patterns of nuclear localization were observed with a rat proenkephalin pre-mRNA. Nuclear microinjection of an intronlacking beta-globin RNA, a splicing-defective beta-globin mutant pre-mRNA, or an antisense beta-globin pre-mRNA did not result in localization at discrete sites. These results indicate that pre-mRNA binds preferentially to nuclear Sm and SC-35 antibody-reactive sites in vivo and that the binding requires intron sequences.

Oligodeoxynucleotides containing phosphodiester or modified internucleoside linkages were investigated with respect to their ability to be acted on by ribonuclease H activities present in a HeLa cell nuclear extract after hybridization with complementary sequences in RNA. Oligodeoxynucleotides complementary to nucleotides 2-14 of human U1 small nuclear RNA were investigated. Extensive cleavage of U1 RNA was observed with the unmodified oligodeoxynucleotide and with the phosphorothioate analogue but not with U1-complementary oligodeoxynucleotides containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate internucleoside linkages. Additional experiments using a 514-nucleotide-long RNA substrate demonstrated the capacity of complementary phosphodiester- and phosphorothioate-linked oligodeoxynucleotides (but not ones containing methylphosphonate, phosphoro-N-morpholidate, or phosphoro-N-butylamidate linkages) to serve as RNase H targets when hybridized to an internal RNA site. Detailed comparisons revealed phosphodiester-linked oligodeoxynucleotides to be more efficient than the comparable phosphorothioate-linked oligomers with respect to RNase H action. Various pentadecamer oligodeoxynucleotides complementary to the 514-nucleotide-long test RNA and containing 2-6 consecutive phosphodiester- or phosphorothioate-linked nucleotides flanked by RNase H-resistant methylphosphonate linkages afforded precise "site-directed" RNase H excision within the DNA.RNA hybrid. These results serve to assort modified oligodeoxynucleotide-containing hybrids into RNase H-sensitive and -resistant classes and also provide clues as to how RNase H makes contact with the DNA strand in a DNA.RNA hybrid.

To explore the relationships between transcription, messenger RNA (mRNA) processing, and nuclear structure, ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNP) have been purified from globin-producing mouse Friend erythroleukemia cells. These nuclear hnRNP particles sediment at 50S-200S and contain, in addition to high molecular weight hnRNA, a specific set of nuclear proteins predominated by a major component of approximately 38,000 mol wt. The hnRNP particles are free of histones and ribosomal structural proteins, indicating their purification from the two other major nucleoprotein components of the nucleus: chromatin and nucleolar ribosomal precursor RNP particles. Th authenticity of the Friend cell hnRNP particles is demonstrated by the results of reconstruction experiments with deproteinized hnRNA, and by the resistance of the articles to dissociation during isopycnic banding in Cs2SO4 gradients without prior aldehyde fixation. Hybridization analysis with cloned mouse beta-globin DNA demonstrates that hnRNP particles from induced Friend cells contain newly synthesized transcripts of the beta-globin gene. Agarose gel electrophoresis of hnRNP particle-derived RNA denatured in glyoxal followed by "Northern" transfer to diazobenzyloxymethyl paper and hybridization with 32P-labeled cloned mouse beta-globin DNA reveals the presence in hnRNP of two size classes of beta-globin gene transcripts, the larger of which corresponds to the pre-spliced 15S beta-globin mRNA precursor previously identified in whole nuclear RNA, and the smaller of which corresponds to completely processed 9S beta-globin mRNA. These results establish, for the first time, that the nuclear transcripts of a specific, well-defined eukaryotic structural gene can be isolated in an RNP particle form, and that their RNP structure persists throughout mRNA splicing.

Contacts between heterogeneous nuclear RNA (hnRNA) and protein in nuclear ribonucleoprotein particles have been photochemically crosslinked in intact HeLa or Friend erythroleukemia cell by irradiation with 254-nm light at doses of 10(1) to 10(5) ergs/mm2 (1 to 10(4) microJ/mm2). The resulting crosslinked particles were isolated and compared with conventional hnRNA . protein (hnRNP) preparations. By the criteria of nuclear fractionation behavior, sedimentation coefficients, nuclease digestion profiles, and RNA-to-protein ratio measured by banding in Cs2SO4 density gradients, the hnRNP particles crosslinked in vivo are identical to nonirradiated particles. Gel blot hybridization of RNA from Friend cell hnRNP crosslinked in vivo reveals that beta-globin RNA sequences remain both intact and hybridizable after the irradiation procedure. The crosslinked hnRNA--protein bonds are stable in 8 M urea/0.5% sodium dodecyl sulfate and withstand centrifugation in Cs2SO4 gradients of initial density 1.50 g/cm3. These results establish that hnRNA is tightly complexed with nuclear proteins in vivo and that hnRNP particles isolated by nuclear fractionation represent native structures.

Heterogeneous nuclear RNA (hnRNA) from HeLa cells contains intramolecular duplexes. Since hnRNA is associated with protein in vivo, it is possible that the double-stranded regions observed in deproteinized hnRNA form spontaneously upon the release of protein from single-stranded but potentially complementary sequences. We show here that this is not the case for a class of double-stranded sequences that is defined by resistance to RNases A + T(1) at high ionic strength. Exposure of HeLa hnRNA.ribonucleoprotein (hnRNP) particles to Escherichia coli RNase III, a double-strand-specific endoribonuclease, destroys most of the sequences resistant to RNases A + T(1). This effect is completely blocked when hnRNP is exposed to RNase III in the presence of an excess of purified double-stranded RNA. In addition, we show that there exist two classes of double-stranded RNA in hnRNP at a salt concentration of 0.13 M. These are distinguished by their relative resistance to RNases A + T(1). The more stable double-stranded sequences, which are resistant to RNases A + T(1) at 0.13 M, comprise 1.0-1.1% of the nucleotides in hnRNP. The less stable double-stranded sequences comprise an additional 1.5-2.0% of the nucleotides in hnRNP. These are sensitive to RNase III at 0.13 M, but are not resistant to RNases A + T(1) unless the salt concentration is raised to 0.63 M. The demonstration that double-stranded sequences resistant to RNases A + T(1) exist in native ribonucleoprotein and are not artifacts of deproteinization now makes it appropriate to seriously consider their possible functional role in hnRNA metabolism, perhaps as binding sites for regulatory proteins involved in mRNA processing.