Nuclear RNA-related processes modulate the assembly of cytoplasmic RNA granules.

Stress granules (SGs) are cytoplasmic assemblies formed under various stress conditions as a consequence of translation arrest. SGs contain RNA-binding proteins, ribosomal subunits and messenger RNAs (mRNAs). It is well known that mRNAs contribute to SG formation; however, the connection between SG assembly and nuclear processes that involve mRNAs is not well established. Here, we examine the effects of inhibiting mRNA transcription, splicing and export on the assembly of SGs and the related cytoplasmic P body (PB). We demonstrate that inhibition of mRNA transcription, splicing and export reduces the formation of canonical SGs in a eukaryotic initiation factor 2α phosphorylation-independent manner, and alters PB size and quantity. We find that the splicing inhibitor madrasin promotes the assembly of stress-like granules. We show that the addition of synthetic mRNAs directly to the cytoplasm is sufficient for SG assembly, and that the assembly of these SGs requires the activation of stress-associated protein synthesis pathways. Moreover, we show that adding an excess of mRNA to cells that do not have active splicing, and therefore have low levels of cytoplasmic mRNAs, promotes SG formation under stress conditions. These findings emphasize the importance of the cytoplasmic abundance of newly transcribed mRNAs in the assembly of SGs.

Nucleic acid hybridization-based detection of pathogenic RNA using microscale thermophoresis.

Infectious diseases are one of the world's leading causes of morbidity. Their rapid spread emphasizes the need for accurate and fast diagnostic methods for large-scale screening. Here, we describe a robust method for the detection of pathogens based on microscale thermophoresis (MST). The method involves the hybridization of a fluorescently labeled DNA probe to a target RNA and the assessment of thermophoretic migration of the resulting complex in solution within a 2 to 30-time window. We found that the thermophoretic migration of the nucleic acid-based probes is primarily determined by the fluorescent molecule used, rather than the nucleic acid sequence of the probe. Furthermore, a panel of uniformly labeled probes that bind to the same target RNA yields a more responsive detection pattern than a single probe, and moreover, can be used for the detection of specific pathogen variants. In addition, intercalating agents (ICA) can be used to alter migration directionality to improve detection sensitivity and resolving power by several orders of magnitude. We show that this approach can rapidly diagnose viral SARS-CoV2, influenza H1N1, artificial pathogen targets, and bacterial infections. Furthermore, it can be used for anti-microbial resistance testing within 2 h, demonstrating its diagnostic potential for early pathogen detection.

Spatial and temporal organization of the genome: Current state and future aims of the 4D nucleome project.

The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.

Utilizing flow cytometry sorting signal width to enrich for cells positive to endogenous gene integration of fluorescent proteins.

Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.

Utilizing flow cytometry sorting signal width to enrich for cells positive to endogenous gene integration of fluorescent proteins.

Endogenous gene knock-in using CRIPSR is becoming the standard for fluorescent tagging of endogenous proteins. Some protocols, particularly those that utilize insert cassettes that carry a fluorescent protein tag, can yield many types of cells with off-target insertions that have diffuse fluorescent signal throughout the whole cell in addition to scarce cells with on-target gene insertions that show the correct sub-cellular localization of the tagged protein. As such, when searching for cells with on-target integration using flow cytometry, the off-target fluorescent cells yield a high percentage of false positives. Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select for even minuscule percentages of correct subcellular signal, and these parameters were validated by fluorescence microscopy. This method is a powerful tool to rapidly enhance the generation of cell-lines with correctly integrated gene knock-ins encoding endogenous fluorescent proteins.

A seven-transmembrane protein-TM7SF3, resides in nuclear speckles and regulates alternative splicing.

The seven-transmembrane superfamily member 3 protein (TM7SF3) is a p53-regulated homeostatic factor that attenuates cellular stress and the unfolded protein response. Here we show that TM7SF3 localizes to nuclear speckles; eukaryotic nuclear bodies enriched in splicing factors. This unexpected location for a trans -membranal protein enables formation of stable complexes between TM7SF3 and pre-mRNA splicing factors including DHX15, LARP7, HNRNPU, RBM14, and HNRNPK. Indeed, TM7SF3 regulates alternative splicing of >330 genes, mainly at the 3'end of introns by directly modulating the activity of splicing factors such as HNRNPK. These effects are observed both in cell lines and primary human pancreatic islets. Accordingly, silencing of TM7SF3 results in differential expression of 1465 genes (about 7% of the human genome); with 844 and 621 genes being up- or down-regulated, respectively. Our findings implicate TM7SF3, as a resident protein of nuclear speckles and suggest a role for seven-transmembrane proteins as regulators of alternative splicing.

RNA export through the nuclear pore complex is directional.

The changes occurring in mRNA organization during nucleo-cytoplasmic transport and export, are not well understood. Moreover, directionality of mRNA passage through the nuclear pore complex (NPC) has not been examined within individual NPCs. Here we find that an mRNP is compact during nucleoplasmic travels compared to a more open structure after transcription and at the nuclear periphery. Compaction levels of nuclear transcripts can be modulated by varying levels of SR proteins and by changing genome organization. Nuclear mRNPs are mostly rod-shaped with distant 5'/3'-ends, although for some, the ends are in proximity. The latter is more abundant in the cytoplasm and can be modified by translation inhibition. mRNAs and lncRNAs exiting the NPC exhibit predominant 5'-first export. In some cases, several adjacent NPCs are engaged in export of the same mRNA suggesting 'gene gating'. Altogether, we show that the mRNP is a flexible structure during travels, with 5'-directionality during export.

Nuclear speckles - a driving force in gene expression.

Nuclear speckles are dynamic membraneless bodies located in the cell nucleus. They harbor RNAs and proteins, many of which are splicing factors, that together display complex biophysical properties dictating nuclear speckle formation and maintenance. Although these nuclear bodies were discovered decades ago, only recently has in-depth genomic analysis begun to unravel their essential functions in modulation of gene activity. Major advancements in genomic mapping techniques combined with microscopy approaches have enabled insights into the roles nuclear speckles may play in enhancing gene expression, and how gene positioning to specific nuclear landmarks can regulate gene expression and RNA processing. Some studies have drawn a link between nuclear speckles and disease. Certain maladies either involve nuclear speckles directly or dictate the localization and reorganization of many nuclear speckle factors. This is most striking during viral infection, as viruses alter the entire nuclear architecture and highjack host machinery. As discussed in this Review, nuclear speckles represent a fascinating target of study not only to reveal the links between gene positioning, genome subcompartments and gene activity, but also as a potential target for therapeutics.

The Association of MEG3 lncRNA with Nuclear Speckles in Living Cells.

Nuclear speckles are nuclear bodies containing RNA-binding proteins as well as RNAs including long non-coding RNAs (lncRNAs). Maternally expressed gene 3 (MEG3) is a nuclear retained lncRNA found to associate with nuclear speckles. To understand the association dynamics of MEG3 lncRNA with nuclear speckles in living cells, we generated a fluorescently tagged MEG3 transcript that could be detected in real time. Under regular conditions, transient association of MEG3 with nuclear speckles was observed, including a nucleoplasmic fraction. Transcription or splicing inactivation conditions, known to affect nuclear speckle structure, showed prominent and increased association of MEG3 lncRNA with the nuclear speckles, specifically forming a ring-like structure around the nuclear speckles. This contrasted with metastasis-associated lung adenocarcinoma (MALAT1) lncRNA that is normally highly associated with nuclear speckles, which was released and dispersed in the nucleoplasm. Under normal conditions, MEG3 dynamically associated with the periphery of the nuclear speckles, but under transcription or splicing inhibition, MEG3 could also enter the center of the nuclear speckle. Altogether, using live-cell imaging approaches, we find that MEG3 lncRNA is a transient resident of nuclear speckles and that its association with this nuclear body is modulated by the levels of transcription and splicing activities in the cell.

Glucocorticoids enhance chemotherapy-driven stress granule assembly and impair granule dynamics, leading to cell death.

Stress granules (SGs) can assemble in cancer cells upon chemotoxic stress. Glucocorticoids function during stress responses and are administered with chemotherapies. The roles of glucocorticoids in SG assembly and disassembly pathways are unknown. We examined whether combining glucocorticoids such as cortisone with chemotherapies from the vinca alkaloid family, which dismantle the microtubule network, affects SG assembly and disassembly pathways and influences cell viability in cancer cells and human-derived organoids. Cortisone augmented SG formation when combined with vinorelbine (VRB). Live-cell imaging showed that cortisone increased SG assembly rates but reduced SG clearance rates after stress, by increasing protein residence times within the SGs. Mechanistically, VRB and cortisone signaled through the integrated stress response mediated by eIF2α (also known as EIF2S1), yet induced different kinases, with cortisone activating the GCN2 kinase (also known as EIF2AK4). Cortisone increased VRB-induced cell death and reduced the population of cells trapped in mitotic catastrophe. These effects were mediated by the core SG proteins G3BP1 and G3BP2. In conclusion, glucocorticoids induce SG assembly and cell death when administered with chemotherapies, suggesting that combining glucocorticoids with chemotherapies can enhance cancer cell chemosensitivity.

Gene architecture directs splicing outcome in separate nuclear spatial regions.

How the splicing machinery defines exons or introns as the spliced unit has remained a puzzle for 30 years. Here, we demonstrate that peripheral and central regions of the nucleus harbor genes with two distinct exon-intron GC content architectures that differ in the splicing outcome. Genes with low GC content exons, flanked by long introns with lower GC content, are localized in the periphery, and the exons are defined as the spliced unit. Alternative splicing of these genes results in exon skipping. In contrast, the nuclear center contains genes with a high GC content in the exons and short flanking introns. Most splicing of these genes occurs via intron definition, and aberrant splicing leads to intron retention. We demonstrate that the nuclear periphery and center generate different environments for the regulation of alternative splicing and that two sets of splicing factors form discrete regulatory subnetworks for the two gene architectures. Our study connects 3D genome organization and splicing, thus demonstrating that exon and intron definition modes of splicing occur in different nuclear regions.

Peroxisome function relies on organelle-associated mRNA translation.

Crucial metabolic functions of peroxisomes rely on a variety of peroxisomal membrane proteins (PMPs). While mRNA transcripts of PMPs were shown to be colocalized with peroxisomes, the process by which PMPs efficiently couple translation with targeting to the peroxisomal membrane remained elusive. Here, we combine quantitative electron microscopy with proximity-specific ribosome profiling and reveal that translation of specific PMPs occurs on the surface of peroxisomes in the yeast Saccharomyces cerevisiae. This places peroxisomes alongside chloroplasts, mitochondria, and the endoplasmic reticulum as organelles that use localized translation for ensuring correct insertion of hydrophobic proteins into their membranes. Moreover, the correct targeting of these transcripts to peroxisomes is crucial for peroxisomal and cellular function, emphasizing the importance of localized translation for cellular physiology.

The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores.

Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection 'dead end'.

The Sub-Nuclear Localization of RNA-Binding Proteins in KSHV-Infected Cells.

RNA-binding proteins, particularly splicing factors, localize to sub-nuclear domains termed nuclear speckles. During certain viral infections, as the nucleus fills up with replicating virus compartments, host cell chromatin distribution changes, ending up condensed at the nuclear periphery. In this study we wished to determine the fate of nucleoplasmic RNA-binding proteins and nuclear speckles during the lytic cycle of the Kaposi's sarcoma associated herpesvirus (KSHV). We found that nuclear speckles became fewer and dramatically larger, localizing at the nuclear periphery, adjacent to the marginalized chromatin. Enlarged nuclear speckles contained splicing factors, whereas other proteins were nucleoplasmically dispersed. Polyadenylated RNA, typically found in nuclear speckles under regular conditions, was also found in foci separated from nuclear speckles in infected cells. Poly(A) foci did not contain lncRNAs known to colocalize with nuclear speckles but contained the poly(A)-binding protein PABPN1. Examination of the localization of spliced viral RNAs revealed that some spliced transcripts could be detected within the nuclear speckles. Since splicing is required for the maturation of certain KSHV transcripts, we suggest that the infected cell does not dismantle nuclear speckles but rearranges their components at the nuclear periphery to possibly serve in splicing and transport of viral RNAs into the cytoplasm.

Speculating on the Roles of Nuclear Speckles: How RNA-Protein Nuclear Assemblies Affect Gene Expression.

Nuclear speckles are eukaryotic nuclear bodies enriched in splicing factors. Their exact purpose has been a matter of debate. The different proposed roles of nuclear speckles are reviewed and an additional layer of function is put forward, suggesting that by accumulating splicing factors within them, nuclear speckles can buffer the nucleoplasmic levels of splicing factors available for splicing and thereby modulate splicing rates. These findings build on the already established model that nuclear speckles function as a storage/recycling site for splicing factors. Many studies have demonstrated proximity between nuclear speckles and sites of active transcription, suggesting that this juxtaposition can enhance the rates of gene expression. It is found that nuclear speckle disassembly increases splicing factor availability in the nucleoplasm, leading to an increase in splicing rates and faster release of nascent transcripts from the gene after transcription. Altogether, this era in which genomic and imaging approaches are applied to study nuclear organization has expanded the outlook on the possible roles of nuclear speckles.

Dynamic Supraspliceosomes Are Assembled on Different Transcripts Regardless of Their Intron Number and Splicing State.

Splicing and alternative splicing of pre-mRNA are key sources in the formation of diversity in the human proteome. These processes have a central role in the regulation of the gene expression pathway. Yet, how spliceosomes are assembled on a multi-intronic pre-mRNA is at present not well understood. To study the spliceosomes assembled in vivo on transcripts with variable number of introns, we examined a series of three related transcripts derived from the ß-globin gene, where two transcript types contained increasing number of introns, while one had only an exon. Each transcript had multiple MS2 sequence repeats that can be bound by the MS2 coat protein. Using our protocol for isolation of endogenous spliceosomes under native conditions from cell nuclei, we show that all three transcripts are found in supraspliceosomes - 21 MDa dynamic complexes, sedimenting at 200S in glycerol gradients, and composed of four native spliceosomes connected by the transcript. Affinity purification of complexes assembled on the transcript with most introns (termed E6), using the MS2 tag, confirmed the assembly of E6 in supraspliceosomes with components such as Sm proteins and PSF. Furthermore, splicing inhibition by spliceostatin A did not inhibit the assembly of supraspliceosomes on the E6 transcript, yet increased the percentage of E6 pre-mRNA supraspliceosomes. These findings were corroborated in intact cells, using RNA FISH to detect the MS2-tagged E6 mRNA, together with GFP-tagged splicing factors, showing the assembly of splicing factors SRSF2, U1-70K, and PRP8 onto the E6 transcripts under normal conditions and also when splicing was inhibited. This study shows that different transcripts with different number of introns, or lacking an intron, are assembled in supraspliceosomes even when splicing is inhibited. This assembly starts at the site of transcription and can continue during the life of the transcript in the nucleoplasm. This study further confirms the dynamic and universal nature of supraspliceosomes that package RNA polymerase II transcribed pre-mRNAs into complexes composed of four native spliceosomes connected by the transcript, independent of their length, number of introns, or splicing state.

Specific, Sensitive, and Quantitative Detection of HER-2 mRNA Breast Cancer Marker by Fluorescent Light-Up Hybridization Probes.

Currently, there is demand for fluorescent oligonucleotide probes for diagnostic purposes. To address this necessity, we developed nucleosides containing a flexible spacer with an intercalating moiety at its end (NIC molecules). The intercalator is based on 4-hydroxybenzylidene imidazolinone (HBI), found in the Green Fluorescent Protein. We synthesized 20-mer oligonucleotides, ON1-ON4, incorporating the DMTr phosphorodiamidite monomer of dUHBI, 2, and the corresponding dUDFHBI, 5b, monomer. ON1-ON4 target the HER-2 mRNA breast cancer marker for the diagnostics of breast cancer subtype. Hybridization of ON1/ON2 and ON3/ON4 with complementary 2'-OMe-RNA resulted in emission at 462 and 481 nm, respectively, and up to 46-fold increase in fluorescence intensity. CD and 19F-NMR data indicated that HBI and DFHBI fluorophores bind as intercalators and stabilize the duplexes (up to ΔTm 6 °C). Furthermore, addition of ON1-ON4 to total RNA extracted from cancer cells that overexpress HER-2 mRNA, resulted in a significant fluorescence enhancement of ON3 and ON4. The latter sensitively detected low concentrations of the target mRNA (at total RNA 30 ng/µL). These probes were photostable for 200 min. Using a dilution curve, we quantified the number of HER-2 transcripts in a cell. In conclusion, ON3 and ON4 are promising diagnostic probes for an easy, instantaneous, specific, and sensitive detection of levels of oncogenes. Importantly, the NIC concept, demonstrated here for diagnostics of breast cancer, is universal and may be applied not only in a clinical setting but also for the detection of any RNA.

Into the basket and beyond: the journey of mRNA through the nuclear pore complex.

The genetic information encoded in nuclear mRNA destined to reach the cytoplasm requires the interaction of the mRNA molecule with the nuclear pore complex (NPC) for the process of mRNA export. Numerous proteins have important roles in the transport of mRNA out of the nucleus. The NPC embedded in the nuclear envelope is the port of exit for mRNA and is composed of ∼30 unique proteins, nucleoporins, forming the distinct structures of the nuclear basket, the pore channel and cytoplasmic filaments. Together, they serve as a rather stationary complex engaged in mRNA export, while a variety of soluble protein factors dynamically assemble on the mRNA and mediate the interactions of the mRNA with the NPC. mRNA export factors are recruited to and dissociate from the mRNA at the site of transcription on the gene, during the journey through the nucleoplasm and at the nuclear pore at the final stages of export. In this review, we present the current knowledge derived from biochemical, molecular, structural and imaging studies, to develop a high-resolution picture of the many events that culminate in the successful passage of the mRNA out of the nucleus.

Availability of splicing factors in the nucleoplasm can regulate the release of mRNA from the gene after transcription.

Gene expression dynamics can be measured in single living cells. Using a detectable transcriptionally active gene in living cells, we previously found that an mRNA undergoing several splicing events was retained at this gene after transcription until completion of mRNA processing. To determine the reason for this delay in release and whether mRNA retention on the gene might depend on splicing factor availability, we modulated the levels of splicing factors in the nucleus. Increasing the abundance of the diffusing fraction of splicing factors by their overexpression or by Clk1 kinase overexpression to disassemble nuclear speckles, led to a reduction in splicing factor residence times on the active gene, and the retained mRNA was rapidly released from the gene. Other treatments such as overexpression of a mutant inactive Clk1, the downregulation of MALAT1 lncRNA or of the Son protein, or the overexpression of the splicing factor import factor TNPO3, did not affect the dynamics of mRNA release from the gene. We found that the faster release of the mRNA from the gene mediated by increased availability of splicing factors, was dependent on the RS domain of the splicing factors and its phosphorylation state. We propose that the relative abundancies of splicing factors in the nucleoplasm can affect their availability for the splicing events taking place, and regulate the kinetics of mRNA release from the gene after processing.

Imaging within single NPCs reveals NXF1's role in mRNA export on the cytoplasmic side of the pore.

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein-protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.