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Dissecting the regulatory mechanisms controlling mammalian transcripts from production to degradation requires quantitative measurements of mRNA flow across the cell. We developed subcellular TimeLapse-seq to measure the rates at which RNAs are released from chromatin, exported from the nucleus, loaded onto polysomes, and degraded within the nucleus and cytoplasm in human and mouse cells. These rates varied substantially, yet transcripts from genes with related functions or targeted by the same transcription factors and RNA-binding proteins flowed across subcellular compartments with similar kinetics. Verifying these associations uncovered a link between DDX3X and nuclear export. For hundreds of RNA metabolism genes, most transcripts with retained introns were degraded by the nuclear exosome, while the remaining molecules were exported with stable cytoplasmic lifespans. Transcripts residing on chromatin for longer had extended poly(A) tails, whereas the reverse was observed for cytoplasmic mRNAs. Finally, machine learning identified molecular features that predicted the diverse life cycles of mRNAs., Competing Interests: Declaration of interests R.I. is a founder, board member, and/or shareholder of Cellforma, unrelated to the present work., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Schaaf-Yang syndrome (SYS) is an ultra-rare neurodevelopmental disorder caused by truncating mutations in MAGEL2 Heterologous expression of wild-type (WT) or a truncated (p.Gln638*) C-terminal HA-tagged MAGEL2 revealed a shift from a primarily cytoplasmic to a more nuclear localisation for the truncated protein variant. We now extend this analysis to six additional SYS mutations on a N-terminal FLAG-tagged MAGEL2. Our results replicate and extend our previous findings, showing that all the truncated MAGEL2 proteins consistently display a predominant nuclear localisation, irrespective of the C-terminal or N-terminal position and the chemistry of the tag. The variants associated with arthrogryposis multiplex congenita display a more pronounced nuclear retention phenotype, suggesting a correlation between clinical severity and the degree of nuclear mislocalisation. These results point to a neomorphic effect of truncated MAGEL2, which might contribute to the pathogenesis of SYS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

CircRNAs are an important class of RNAs with diverse cellular functions in human physiology and disease. A thorough knowledge of circRNAs including their biogenesis and subcellular distribution is important to understand their roles in a wide variety of processes. However, the analysis of circRNAs from total RNA sequencing data remains challenging. Therefore, we developed Calcifer, a versatile workflow for circRNA annotation. Using Calcifer, we analysed APEX-Seq data to compare circRNA occurrence between whole cells, nucleus and subnuclear compartments. We generally find that circRNAs show higher abundance in whole cells compared to nuclear samples, consistent with their accumulation in the cytoplasm. The notable exception is the single-exon circRNA circCANX(9), which is unexpectedly enriched in the nucleus. In addition, we observe that circFIRRE prevails over the linear lncRNA FIRRE in both the cytoplasm and the nucleus. Zooming in on the subnuclear compartments, we show that circRNAs are strongly depleted from nuclear speckles, indicating that excess splicing factors in this compartment counteract back-splicing. Our results thereby provide valuable insights into the subnuclear distribution of circRNAs. Regarding circRNA function, we surprisingly find that the majority of all detected circRNAs possess complete open reading frames with potential for cap-independent translation. Overall, we show that Calcifer is an easy-to-use, versatile and sustainable workflow for the annotation of circRNAs which expands the repertoire of circRNA tools and allows to gain new insights into circRNA distribution and function.

In eukaryotes, RNA is synthesised in the nucleus, spliced, and exported to the cytoplasm where it is translated and finally degraded. Any of these steps could be subject to temporal regulation during the circadian cycle, resulting in daily fluctuations of RNA accumulation and affecting the distribution of transcripts in different subcellular compartments. Our study analysed the nuclear and cytoplasmic, poly(A) and total transcriptomes of mouse livers collected over the course of a day. These data provide a genome-wide temporal inventory of enrichment in subcellular RNA, and revealed specific signatures of splicing, nuclear export and cytoplasmic mRNA stability related to transcript and gene lengths. Combined with a mathematical model describing rhythmic RNA profiles, we could test the rhythmicity of export rates and cytoplasmic degradation rates of approximately 1400 genes. With nuclear export times usually much shorter than cytoplasmic half-lives, we found that nuclear export contributes to the modulation and generation of rhythmic profiles of 10% of the cycling nuclear mRNAs. This study contributes to a better understanding of the dynamic regulation of the transcriptome during the day-night cycle., Competing Interests: The authors have declared that no competing interests exist.

The poly(A)-tail appended to the 3'-end of most eukaryotic transcripts plays a key role in their stability, nuclear transport, and translation. These roles are largely mediated by Poly(A) Binding Proteins (PABPs) that coat poly(A)-tails and interact with various proteins involved in the biogenesis and function of RNA. While it is well-established that the nuclear PABP (PABPN) binds newly synthesized poly(A)-tails and is replaced by the cytoplasmic PABP (PABPC) on transcripts exported to the cytoplasm, the distribution of transcripts for different genes or isoforms of the same gene on these PABPs has not been investigated on a genome-wide scale. Here, we analyzed the identity, splicing status, poly(A)-tail size, and translation status of RNAs co-immunoprecipitated with endogenous PABPN or PABPC in human cells. At steady state, many protein-coding and non-coding RNAs exhibit strong bias for association with PABPN or PABPC. While PABPN-enriched transcripts more often were incompletely spliced and harbored longer poly(A)-tails and PABPC-enriched RNAs had longer half-lives and higher translation efficiency, there are curious outliers. Overall, our study reveals the landscape of RNAs bound by PABPN and PABPC, providing new details that support and advance the current understanding of the roles these proteins play in poly(A)-tail synthesis, maintenance, and function., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Subcellular localization of transcripts is highly associated with regulation of gene expression, synthesis of protein, and also the development of the human brain cortex. Although many mechanisms are prevalent in the occurrence of neuroinflammation, the mechanisms based on differences in subcellular localization of transcripts have not been explored. To characterize the dynamic profile of nuclear and cytoplasmic transcripts during the progress of haemorrhage-induced neuroinflammation, we isolated nucleo-cytoplasmic RNA fractions of oxyhaemoglobin (oxy-Hb) treated microglia cells and sequenced both fractions. We discovered that cytoplasmic retained genes were the major forces to maintain the neuroinflammatory microenvironment with 10 hub genes and 40 conserved genes were identified. Moreover, antisense RNA Gm44096 and lincRNA Gm47270, which co-expressed with a crowd of inflammatory genes in the cytoplasm, were discovered as regulatory strategies for sustaining the neuroinflammatory microenvironment. Thus, our study provides a new perspective on understanding haemorrhage-induced neuroinflammation and also reveals a mechanism of lncRNA responsible for maintaining the neuroinflammatory microenvironment.

The TREX-2 complex is essential for the general nuclear mRNA export in eukaryotes. TREX-2 interacts with the nuclear pore and transcriptional apparatus and links transcription to the mRNA export. However, it remains poorly understood how the TREX-2-dependent nuclear export is connected to the subsequent stages of mRNA trafficking. Here, we show that the PCID2 subunit of Drosophila TREX-2 is present in the cytoplasm of the cell. The cytoplasmic PCID2 directly interacts with the NudC protein and this interaction maintains its stability in the cytoplasm. Moreover, PCID2 is associated with the cytoplasmic mRNA and microtubules. The PCID2 knockdown blocks nuclear export of mRNA and also affects the general mRNA transport into the cytoplasm. These data suggest that PCID2 could be the link between the nuclear TREX-2-dependent export and the subsequent cytoplasmic trafficking of mRNA.

Pre-mRNA splicing is critical for cells, as defects in this process can lead to altered open reading frames and defective proteins, potentially causing neurodegenerative diseases and cancer. Introns are removed in the nucleus and splicing is documented by the addition of exon-junction-complexes (EJCs) at exon-exon boundaries. This "memory" of splicing events is important for the ribosome, which translates the RNAs in the cytoplasm. In case a stop codon was detected before an EJC, translation is blocked and the RNA is eliminated by the nonsense-mediated decay (NMD). In the model organism Saccharomyces cerevisiae , two guard proteins, Gbp2 and Hrb1, have been identified as nuclear quality control factors for splicing. In their absence, intron-containing mRNAs leak into the cytoplasm. Their presence retains transcripts until the process is completed and they release the mRNAs by recruitment of the export factor Mex67. On transcripts that experience splicing problems, these guard proteins recruit the nuclear RNA degradation machinery. Interestingly, they continue their quality control function on exported transcripts. They support NMD by inhibiting translation and recruiting the cytoplasmic degradation factors. In this way, they link the nuclear and cytoplasmic quality control systems. These discoveries are also intriguing for humans, as homologues of these guard proteins are present also in multicellular organisms. Here, we provide an overview of the quality control mechanisms of pre-mRNA splicing, and present Gbp2 and Hrb1, as well as their human counterparts, as important players in these pathways.

The most frequent genetic alteration in acute myeloid leukemia (AML) is the mutation of nucleophosmin 1 (NPM1). Yet, its downstream oncogenic routes are not fully understood. Here, we report the identification of one long noncoding RNA (lncRNA) overexpressed in NPM1-mutated AML patients (named LONA) whose intracellular localization inversely reflects that of NPM1. While NPM1 is nuclear and LONA cytoplasmic in wild-type NPM1 AML cells, LONA becomes nuclear as mutant NPM1 moves toward the cytoplasm. Gain or loss of function combined with a genome-wide RNA-seq search identified a set of LONA mRNA targets encoding proteins involved in myeloid cell differentiation (including THSB1, MAFB, and ASB2) and interaction with its microenvironment. Consistently, LONA overexpression in mutant NPM1 established cell lines and primary AML cells exerts an anti-myeloid differentiation effect, whilst it exerts an opposite pro-myeloid differentiation effect in a wild type NPM1 setting. In vivo, LONA overexpression acts as an oncogenic lncRNA reducing the survival of mice transplanted with AML cells and rendering AML tumors more resistant to AraC chemotherapy.These data indicate that mutation-dependent nuclear export of NPM1 leads to nuclear retention and consequent oncogenic functions of the overexpressed lncRNA LONA, thus uncovering a novel NPM1 mutation-dependent pathway in AML pathogenesis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Circular RNA, typically generated from backsplicing reaction, is a class of single-stranded and covalently linked RNA. Although most circular RNAs are lowly expressed, some of them are able to accumulate to high levels and even exceed their cognate mRNAs due to their longer half-lives. Once produced in the nucleus, the majority of circular RNAs are exported to the cytoplasm for their proper functions or degradation. In this review, we will summarize the biogenesis and classification of circular RNAs and highlight the recent advances in our understanding of circular RNA nuclear export and degradation.

Transport of mRNAs is an important step of gene expression, which brings the genetic message from the DNA in the nucleus to a precise cytoplasmic location in a regulated fashion. Perturbation of this process can lead to pathologies such as developmental and neurological disorders. In this review, we discuss recent advances in the field of mRNA transport made using single molecule fluorescent imaging approaches. We present an overview of these approaches in fixed and live cells and their input in understanding the key steps of mRNA journey: transport across the nucleoplasm, export through the nuclear pores and delivery to its final cytoplasmic location. This review puts a particular emphasis on the coupling of mRNA transport with translation, such as localization-dependent translational regulation and translation-dependent mRNA localization. We also highlight the recently discovered translation factories, and how cellular and viral RNAs can hijack membrane transport systems to travel in the cytoplasm.

The TREX-TAP pathway is vital for mRNA export. For spliced mRNA, the TREX complex is recruited during splicing; however, for intronless mRNA, recruitment is sequence dependent. However, the export of cytoplasmic long noncoding RNA (lncRNA) is poorly characterized. We report the identification of a cytoplasmic accumulation region (CAR-N) in the intronless lncRNA, NKILA. CAR-N removal led to strong nuclear retention of NKILA, and CAR-N insertion promoted the export of cDNA transcripts. In vitro RNP purification via CAR-N, mass spectrometry, and siRNA screening revealed that SRSF1 and SRSF7 were vital to NKILA export, and identified a cluster of SRSF1/7 binding sites within a 55 nucleotide sequence in CAR-N. Significant nuclear enrichment of NKILA was observed for NKILA lacking CAR-N or the cluster of binding sites in knock-in models. Depletion of TREX-TAP pathway components resulted in strong nuclear retention of NKILA. RNA and protein immunoprecipitation verified that SRSF1/7 were bound to NKILA and interacted with UAP56 and ALYREF. Moreover, NKILA lacking CAR-N was unable to inhibit breast cancer cell migration. We concluded that the binding of SRSF1/7 to clustered motifs in CAR-N facilitated TREX recruitment, promoting the export of NKILA, and confirmed the importance of NKILA localization to its function., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The oxidative phosphorylation (OXPHOS) system is the only structure in animal cells with components encoded by two genomes, maternally transmitted mitochondrial DNA (mtDNA), and biparentally transmitted nuclear DNA (nDNA). MtDNA-encoded genes have to physically assemble with their counterparts encoded in the nucleus to build together the functional respiratory complexes. Therefore, structural and functional matching requirements between the protein subunits of these molecular complexes are rigorous. The crosstalk between nDNA and mtDNA needs to overcome some challenges, as the nuclear-encoded factors have to be imported into the mitochondria in a correct quantity and match the high number of organelles and genomes per mitochondria that encode and synthesize their own components locally. The cell is able to sense the mito-nuclear match through changes in the activity of the OXPHOS system, modulation of the mitochondrial biogenesis, or reactive oxygen species production. This implies that a complex signaling cascade should optimize OXPHOS performance to the cellular-specific requirements, which will depend on cell type, environmental conditions, and life stage. Therefore, the mitochondria would function as a cellular metabolic information hub integrating critical information that would feedback the nucleus for it to respond accordingly. Here, we review the current understanding of the complex interaction between mtDNA and nDNA., (© 2020 The Authors. IUBMB Life published by Wiley Periodicals LLC on behalf of International Union of Biochemistry and Molecular Biology.)

Herpesviruses infect a majority of the human population, establishing lifelong latent infections for which there is no cure. Periodic viral reactivation spreads infection to new hosts while causing various disease states particularly detrimental in the immunocompromised. Efficient viral replication, and ultimately the spread of infection, is dependent on the nuclear egress complex (NEC), a conserved viral heterodimer that helps translocate viral capsids from the nucleus to the cytoplasm where they mature into infectious virions. Here, we have identified peptides, derived from the capsid protein UL25, that are capable of inhibiting the membrane-budding activity of the NEC from herpes simplex virus type 1 in vitro. We show that the inhibitory ability of the peptides depends on their length and the propensity to form an α-helix but not on the exact amino acid sequence. Current therapeutics that target viral DNA replication machinery are rendered ineffective by drug resistance due to viral mutations. Our results establish a basis for the development of an alternative class of inhibitors against nuclear egress, an essential step in herpesvirus replication, potentially expanding the current repertoire of available therapeutics.

Most mammalian cytoplasmic tRNAs contain ribothymidine (T) and pseudouridine (Ψ) at positions 54 and 55, respectively. However, some tRNAs contain Ψ at both positions. Several Ψ54-containing tRNAs function as primers in retroviral DNA synthesis. The Ψ54 of these tRNAs is produced by PUS10, which can also synthesize Ψ55. Two other enzymes, TRUB1 and TRUB2, can also produce Ψ55. By nearest-neighbor analyses of tRNAs treated with recombinant proteins and subcellular extracts of wild-type and specific Ψ55 synthase knockdown cells, we determined that while TRUB1, PUS10, and TRUB2 all have tRNA Ψ55 synthase activities, they have different tRNA structural requirements. Moreover, these activities are primarily present in the nucleus, cytoplasm, and mitochondria, respectively, suggesting a compartmentalization of Ψ55 synthase activity. TRUB1 produces the Ψ55 of most elongator tRNAs, but cytoplasmic PUS10 produces both Ψs of the tRNAs with Ψ54Ψ55. The nuclear isoform of PUS10 is catalytically inactive and specifically binds the unmodified U54U55 versions of Ψ54Ψ55-containing tRNAs, as well as the A54U55-containing tRNA iMet This binding inhibits TRUB1-mediated U55 to Ψ55 conversion in the nucleus. Consequently, the U54U55 of Ψ54Ψ55-containing tRNAs are modified by the cytoplasmic PUS10. Nuclear PUS10 does not bind the U55 versions of T54Ψ55- and A54Ψ55-containing elongator tRNAs. Therefore, TRUB1 is able to produce Ψ55 in these tRNAs. In summary, the tRNA Ψ55 synthase activities of TRUB1 and PUS10 are not redundant but rather are compartmentalized and act on different sets of tRNAs. The significance of this compartmentalization needs further study., (© 2021 Mukhopadhyay et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Nuclear pore complexes are multiprotein channels that span the nuclear envelope, which connects the nucleus to the cytoplasm. In addition to their main role in the regulation of nucleocytoplasmic molecule exchange, it has become evident that nuclear pore complexes and their components also have multiple transport-independent functions. In recent years, an increasing number of studies have reported the involvement of nuclear pore complex components in embryogenesis, cell differentiation and tissue-specific processes. Here, we review the findings that highlight the dynamic nature of nuclear pore complexes and their roles in many cell type-specific functions during development and tissue homeostasis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

A mammalian embryo experiences the first cell segregation at the blastocyst stage, in which cells giving form to the embryo are sorted into two lineages; trophectoderm (TE) and inner cell mass (ICM). This first cell segregation process is governed by cell position-dependent Hippo signaling, which is a phosphorylation cascade determining whether Yes-associated protein 1 (YAP1), one of the key components of the Hippo signaling pathway, localizes within the nucleus or cytoplasm. YAP1 localization determines the transcriptional on/off switch of a key gene, Cdx2, required for TE differentiation. However, the control mechanisms involved in YAP1 nucleocytoplasmic shuttling post blastocyst formation remain unknown. This study focused on the mechanisms involved in YAP1 release from TE nuclei after blastocoel contraction in bovine blastocysts. The blastocysts contracted by blastocoel fluid aspiration showed that the YAP1 translocation from nucleus to cytoplasm in the TE cells was concomitant with the protruded actin cytoskeleton. This YAP1 release from TE nuclei in the contracted blastocysts was prevented by actin disruption and stabilization. In contrast, Y27632, which is a potent inhibitor of Rho-associated coiled-coil containing protein kinase 1/2 (ROCK) activity, was found to promote YAP1 nuclear localization in the TE cells of contracted blastocysts. Meanwhile, lambda protein phosphatase (LPP) treatment inducing protein dephosphorylation could not prevent YAP1 release from TE nuclei in the contracted blastocysts, indicating that YAP1 release from TE nuclei does not depend on the Hippo signaling pathway. These results suggested that blastocyst contraction causes YAP1 release from TE nuclei through actin cytoskeleton remodeling in a Hippo signaling-independent manner. Thus, the present study raised the possibility that YAP1 subcellular localization is controlled by actin cytoskeletal organization after the blastocyst formation. Our results demonstrate diverse regulatory mechanisms for YAP1 nucleocytoplasmic shuttling in TE cells., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Tip60 lysine acetyltransferase is a tumor suppressor in most cancers but an oncogene in prostate and gastric cancer. Tip60 is commonly found in the nucleus, where it acetylates proteins involved in transcription, DNA repair, and chromatin; however, it has also been shown to acetylate cytoplasmic targets. In this study, we investigated the relationship between Tip60 localization and breast and lung cancer. In cell fractionation experiments, cancer-derived cell lines showed a shift from nuclear to cytoplasmic endogenous Tip60 compared with cell lines derived from normal cells. With immunofluorescence, we observed four different localization patterns of overexpressed Tip60 and found that cancer cells had increased cytoplasmic localization of Tip60 compared with HEK-293 cells. The addition of a nuclear localization signal (NLS) increased the number of cells containing nuclear Tip60, whereas mutation of a putative endogenous NLS increased the number of cells with cytoplasmic Tip60. Overexpression of Tip60 increased cancer cell line sensitivity to paclitaxel regardless of changes in localization. These results suggest that dysregulation of Tip60 in breast and lung cancer is not limited to reduced expression but may also involve subcellular localization.

Recent studies have revealed that global extrinsic noise arising from stochasticity in the intracellular biochemical environment plays a critical role in heterogeneous cell physiologies. However, it remains largely unclear how such extrinsic noise dynamically influences downstream reactions and whether it could be neutralized by cellular reactions. Here, using fluorescent protein (FP) maturation as a model biochemical reaction, we explored how cellular reactions might combat global extrinsic noise in mammalian cells. We developed a novel single-cell assay to systematically quantify the maturation rate and the associated noise for over a dozen FPs. By exploiting the variation in the maturation rate for different FPs, we inferred that global extrinsic noise could be temporally filtered by maturation reactions, and as a result, the noise levels for slow-maturing FPs are lower compared to fast-maturing FPs. This mechanism is validated by directly perturbing the maturation rates of specific FPs and measuring the resulting noise levels. Together, our results revealed a potentially general principle governing extrinsic noise propagation, where timescale separation allows cellular reactions to cope with dynamic global extrinsic noise., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Animal cloning technology has been developed to produce progenies genetically identical to a given donor cell. However, in nuclear transfer protocols, the recipient oocytes contribute a heritable mitochondrial genomic (mtDNA) background to the progeny. Additionally, a small amount of donor cell-derived mitochondria accompanies the transferred nucleus in the process; hence, the mtDNAs of two origins are mixed in the cytoplasm (heteroplasmy) of the reconstituted oocyte. Herein, I would like to introduce some of our previous results concerning five key considerations associated with animal cloning, including: mtDNA heteroplasmy in somatic cell nuclear transferred (SCNT) animals, the variation in the transmission of mtDNA heteroplasmy to subsequent generations SCNT cows and pigs, the influence of mtDNA sequence differences on mitochondrial proteins in SCNT cows and pigs, the effects of the introduction of mitochondria derived from somatic cells into recipient oocytes on embryonic development, and alterations of mtDNA heteroplasmy in inter/intraspecies nuclear transfer embryos.

Correlation between nuclear and cell size, the nucleocytoplasmic ratio, is a cellular phenomenon that has been reported throughout eukaryotes for more than a century but the mechanisms that achieve it are not well understood. Here, we review work that has shed light on the cellular processes involved in nuclear size control. These studies have implicated nucleocytoplasmic transport, LINC complexes, RNA processing, regulation of nuclear envelope expansion and partitioning of importin α in nuclear size control, moving us closer to a mechanistic understanding of this phenomenon.

Gynodioecy is a sexual polymorphism in angiosperms, where hermaphroditic and female individuals coexist. This is often caused by a cytoplasmic genetic element (CGE) that destroys male functions, which is called cytoplasmic male-sterility (CMS). On the other hand, nuclear genes tend to evolve the ability to restore male function. The coevolutionary process of CMS and the restoration has been studied theoretically. Recently, a theoretical study suggested that these coevolutionary dynamics could be influenced by the rate of selfing within populations, although it assumed that the selfing rate of a population was a fixed parameter. Accordingly, we theoretically study the coevolution of three traits in this paper: CMS, nuclear restorer and selfing rate, in which we hypothesize that selfing evolution can suppress CMS evolution under some conditions. The analysis indicates three significant properties of the system; (1) CMS-restorer evolution can result in bistability under a given selfing rate, (2) the coevolution of three traits can realize intermediate levels of selfing, and (3) the evolution of high levels of selfing is conditionally associated with no CMS and/or no restoration, which may support our hypothesis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain.)

Maintaining a healthy cellular environment requires the constant control of proteostasis. E3 ubiquitin ligase complexes facilitate the post-translational addition of ubiquitin, which based on the quantity and specific lysine linkages, results in different outcomes. Our studies reveal the CUL4-DDB1 substrate receptor, WDR23, as both a positive and a negative regulator in cellular stress responses. These opposing roles are mediated by two distinct isoforms: WDR-23A in the cytoplasm and WDR-23B in the nucleus. C. elegans expressing only WDR-23A display activation of SKN-1 and enhanced survival to oxidative stress, whereas animals with restricted WDR-23B expression do not. Additionally, we identify GEN-1, a Holliday junction resolvase, as an evolutionarily conserved WDR-23 substrate and find that the nuclear and cytoplasmic isoforms of WDR-23 differentially affect double-strand break repair. Our results suggest that through differential ubiquitination, nuclear WDR-23B inhibits the activity of substrates, most likely by promoting protein turnover, while cytoplasmic WDR-23A performs a proteasome-independent role. Together, our results establish a cooperative role between two spatially distinct isoforms of WDR-23 in ensuring proper regulation of WDR-23 substrates.

Investigations of information dynamics in eukaryotic cells focus almost exclusively on heritable information in the genome. Gene networks are modeled as "central processors" that receive, analyze, and respond to intracellular and extracellular signals with the nucleus described as a cell's control center. Here, we present a model in which cellular information is a distributed system that includes non-genomic information processing in the cell membrane that may quantitatively exceed that of the genome. Within this model, the nucleus largely acts a source of macromolecules and processes information needed to synchronize their production with temporal variations in demand. However, the nucleus cannot produce microsecond responses to acute, life-threatening perturbations and cannot spatially resolve incoming signals or direct macromolecules to the cellular regions where they are needed. In contrast, the cell membrane, as the interface with its environment, can rapidly detect, process, and respond to external threats and opportunities through the large amounts of potential information encoded within the transmembrane ion gradient. Our model proposes environmental information is detected by specialized protein gates within ion-specific transmembrane channels. When the gate receives a specific environmental signal, the ion channel opens and the received information is communicated into the cell via flow of a specific ion species (i.e., K + , Na + , Cl - , Ca 2+ , Mg 2+ ) along electrochemical gradients. The fluctuation of an ion concentration within the cytoplasm adjacent to the membrane channel can elicit an immediate, local response by altering the location and function of peripheral membrane proteins. Signals that affect a larger surface area of the cell membrane and/or persist over a prolonged time period will produce similarly cytoplasmic changes on larger spatial and time scales. We propose that as the amplitude, spatial extent, and duration of changes in cytoplasmic ion concentrations increase, the information can be communicated to the nucleus and other intracellular structure through ion flows along elements of the cytoskeleton to the centrosome (via microtubules) or proteins in the nuclear membrane (via microfilaments). These dynamics add spatial and temporal context to the more well-recognized information communication from the cell membrane to the nucleus following ligand binding to membrane receptors. Here, the signal is transmitted and amplified through transduction by the canonical molecular (e.g., Mitogen Activated Protein Kinases (MAPK) pathways. Cytoplasmic diffusion allows this information to be broadly distributed to intracellular organelles but at the cost of loss of spatial and temporal information also contained in ligand binding.

Localization of RNAs to various subcellular destinations has emerged as a widely used mechanism that regulates a large proportion of transcripts in polarized cells. A number of methodologies have been developed that allow detection and imaging of RNAs at single-molecule resolution. However, methodologies to quantitatively describe RNA distributions are limited. Such approaches usually rely on the identification of cytoplasmic and nuclear boundaries which are used as reference points. Here, we describe an automated, interactive image analysis program that facilitates the accurate generation of cellular outlines from single cells and the subsequent calculation of metrics that quantify how a population of RNA molecules is distributed in the cell cytoplasm. We apply this analysis to mRNAs in mouse and human cells to demonstrate how these metrics can highlight differences in the distribution patterns of distinct RNA species. We further discuss considerations for the practical use of this tool. This program provides a way to facilitate and expedite the analysis of subcellular RNA localization for mechanistic and functional studies.

Reproductive isolation is an important component of species differentiation. The plastid accD gene coding for the acetyl-CoA carboxylase subunit and the nuclear bccp gene coding for the biotin carboxyl carrier protein were identified as candidate genes governing nuclear-cytoplasmic incompatibility in peas. We examined the allelic diversity in a set of 195 geographically diverse samples of both cultivated ( Pisum sativum , P. abyssinicum ) and wild ( P. fulvum and P. elatius ) peas. Based on deduced protein sequences, we identified 34 accD and 31 bccp alleles that are partially geographically and genetically structured. The accD is highly variable due to insertions of tandem repeats. P. fulvum and P. abyssinicum have unique alleles and combinations of both genes. On the other hand, partial overlap was observed between P. sativum and P. elatius . Mapping of protein sequence polymorphisms to 3D structures revealed that most of the repeat and indel polymorphisms map to sequence regions that could not be modeled, consistent with this part of the protein being less constrained by requirements for precise folding than the enzymatically active domains. The results of this study are important not only from an evolutionary point of view but are also relevant for pea breeding when using more distant wild relatives.

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The sequestration of DNA within the membrane-bound nucleus is a defining characteristic of eukaryotic cells. Replication and transcription are therefore restricted to the nucleus, however, the regulation of these events relies on cytoplasmic processes including protein synthesis and signal transduction pathways. Because a variety of cellular activities depend on nuclear transport, researchers from diverse fields have found it useful to examine the nuclear localization of proteins of interest. Here we present some important technical considerations for studying nuclear and cytoplasmic localization, and provide guidance for quantifying protein levels using fluorescence microscopy and ImageJ software. We include discussion of the use of regions of interest and image segmentation for quantification of protein localization. Nucleocytoplasmic transport is fundamentally important for controlling protein levels and activity in the nucleus or cytoplasm, and quantitative analysis can provide insight into how biological output is achieved., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The localization of long noncoding RNAs (lncRNAs) within the cell is the primary determinant of their molecular functions. LncRNAs are often thought of as chromatin-restricted regulators of gene transcription and chromatin structure. However, a rich population of cytoplasmic lncRNAs has come to light, with diverse roles including translational regulation, signaling, and respiration. RNA maps of increasing resolution and scope are revealing a subcellular world of highly specific localization patterns and hint at sequence-based address codes specifying lncRNA fates. We propose a new framework for analyzing sequencing-based data, which suggests that numbers of cytoplasmic lncRNA molecules rival those in the nucleus. New techniques promise to create high-resolution, transcriptome-wide maps associated with all organelles of the mammalian cell. Given its intimate link to molecular roles, subcellular localization provides a means of unlocking the mystery of lncRNA functions., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Nuclear size correlates with cell size, but the mechanism by which this scaling is achieved is not known. Here we screen fission yeast gene deletion mutants to identify essential factors involved in this process. Our screen has identified 25 essential factors that alter nuclear size, and our analysis has implicated RNA processing and LINC complexes in nuclear size control. This study has revealed lower and more extreme higher nuclear size phenotypes and has identified global cellular processes and specific structural nuclear components important for nuclear size control., Competing Interests: The authors have declared that no competing interests exist.

The serine-threonine kinase AKT/PKB is a critical regulator of various essential cellular processes, and dysregulation of AKT has been implicated in many diseases, including cancer. Despite AKT action is known to function mainly in the cytoplasm, AKT has been reported to translocate to the nucleus. However, very little is known about the mechanism required for the nuclear import of AKT as well as its function in this cellular compartment. In the present study, we characterized the presence of endogenous nuclear AKT in human melanoma cells and addressed the possible role of AKT by exploring its potential association with key interaction nuclear partners. Confocal and Western blot analyses showed that both phosphorylated and non-phosphorylated forms of AKT are present in melanoma cells nuclei. Using mass spectrometry in combination with protein-crosslinking and co-immunoprecipitation, we identified a series of putative protein partners of nuclear AKT, including heterogeneous nuclear ribonucleoprotein (hnRNP), cytoskeleton proteins β-actin, γ-actin, β-actin-like 2 and vimentin. Confocal microscopy and biochemical analyses validated β-actin as a new nuclear AKT-interacting partner. Cofilin and active RNA Polymerase II, two proteins that have been described to interact and work in concert with nuclear actin in transcription regulation, were also found associated with nuclear AKT. Overall, the present study uncovered a yet unrecognized nuclear coupling of AKT and provides insights into the involvement of AKT in the interaction network of nuclear actin., (© 2019 The Author(s).)

The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.

Pathogen co-evolution with plants involves selection for evasion of host surveillance systems. The oomycete Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis, and race-specific interactions between Arabidopsis accessions and Hpa isolates fit the gene-for-gene model in which host resistance or susceptibility are determined by matching pairs of plant Resistance (R) genes and pathogen Avirulence (AVR) genes. Arabidopsis Col-0 carries R gene RPP4 that confers resistance to Hpa isolates Emoy2 and Emwa1, but its cognate recognized effector(s) were unknown. We report here the identification of the Emoy2 AVR effector gene recognized by RPP4 and show resistance-breaking isolates of Hpa on RPP4-containing Arabidopsis carry the alleles that either are not expressed, or show cytoplasmic instead of nuclear subcellular localization.

Eukaryotes thought to have evolved clonally for millions of years are referred to as ancient asexuals. The oldest group among these are the arbuscular mycorrhizal fungi (AMF), which are plant symbionts harboring hundreds of nuclei within one continuous cytoplasm. Some AMF strains (dikaryons) harbor two co-existing nucleotypes but there is no direct evidence that such nuclei recombine in this life-stage, as is expected for sexual fungi. Here, we show that AMF nuclei with distinct genotypes can undergo recombination. Inter-nuclear genetic exchange varies in frequency among strains, and despite recombination all nuclear genomes have an average similarity of at least 99.8%. The present study demonstrates that AMF can generate genetic diversity via meiotic-like processes in the absence of observable mating. The AMF dikaryotic life-stage is a primary source of nuclear variability in these organisms, highlighting its potential for strain enhancement of these symbionts., Competing Interests: EC, SM, AH, KS, MP, AP, SN, JR, SD, JF, AB, NC No competing interests declared, (© 2018, Chen et al.)

While actin was discovered in the nucleus over 50 years ago, research lagged for decades due to strong skepticism. The revitalization of research into nuclear actin occurred after it was found that cellular stresses induce the nuclear localization and alter the structure of actin. These studies provided the first hints that actin has a nuclear function. Subsequently, it was established that the nuclear import and export of actin is highly regulated. While the structures of nuclear actin remain unclear, it can function as monomers, polymers, and even rods. Furthermore, even within a given structure, distinct pools of nuclear actin that can be differentially labeled have been identified. Numerous mechanistic studies have uncovered an array of functions for nuclear actin. It regulates the activity of RNA polymerases, as well as specific transcription factors. Actin also modulates the activity of several chromatin remodeling complexes and histone deacetylases, to ultimately impinge on transcriptional programing and DNA damage repair. Further, nuclear actin mediates chromatin movement and organization. It has roles in meiosis and mitosis, and these functions may be functionally conserved from ancient bacterial actin homologs. The structure and integrity of the nuclear envelope and sub-nuclear compartments are also regulated by nuclear actin. Furthermore, nuclear actin contributes to human diseases like cancer, neurodegeneration, and myopathies. Here, we explore the early discovery of actin in the nucleus and discuss the forms and functions of nuclear actin in both normal and disease contexts. Anat Rec, 301:1999-2013, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

We have demonstrated that the loss of programmed cell death 4 (Pdcd4), a translation inhibitor, induces apoptosis; however, when, where, and how Pdcd4 decreases in response to apoptotic stimuli and, conversely, exerts the anti-apoptotic function within normal cells are incompletely understood. Endogenous Pdcd4 was present in both the cytoplasm and nucleus of cells that survived. In cells that had committed to die by apoptotic stimuli, cytoplasmic Pdcd4 was lost more slowly than was nuclear Pdcd4; eventually, Pdcd4 remaining in the cytoplasm was lost and then apoptotic events were induced. Treatment with leptomycin B led to blocked nuclear export of Pdcd4 in cells exposed to apoptotic stimuli, assuming its translocation from the nucleus to the cytoplasm in the early phase of apoptotic processes. In cells overexpressing Pdcd4, the protein localized exclusively cytoplasmic. Overexpression of Pdcd4 resulted in reduced incidence of apoptosis in cells exposed to apoptotic stimuli compared to control cells. In addition, the expression of Procaspase-3, which is translated from the mRNA targeted by Pdcd4, was suppressed in cells overexpressing Pdcd4. Thus, the localization of Pdcd4 to the cytoplasm may be responsible for the suppression of the target mRNA translation and apoptosis.

The partitioning of genetic material between the nucleus and cytoplasmic (mitochondrial and plastid) genomes within eukaryotic cells necessitates coordinated integration between these genomic compartments, with important evolutionary and biomedical implications. Classic questions persist about the pervasive reduction of cytoplasmic genomes via a combination of gene loss, transfer and functional replacement - and yet why they are almost always retained in some minimal form. One striking consequence of cytonuclear integration is the existence of 'chimeric' enzyme complexes composed of subunits encoded in two different genomes. Advances in structural biology and comparative genomics are yielding important insights into the evolution of such complexes, including correlated sequence changes and recruitment of novel subunits. Thus, chimeric cytonuclear complexes provide a powerful window into the mechanisms of molecular co-evolution.

Importin 13 (IPO13) is a key member of the importin β superfamily, which can transport cargoes both into and out of the nucleus to contribute to a variety of important cellular processes. IPO13 is known to undergo phosphorylation, but the impact of this on function has not been investigated. Here, we show for the first time that IPO13 is phosphorylated by cAMP-dependent protein kinase A specifically at serine 193. Results from fluorescence recovery after photobleaching and fluorescence loss in photobleaching approaches establish that negative charge at serine 193 through phosphorylation or point mutation both reduces IPO13 nuclear import and increases its nuclear export. Importantly, phosphorylation also appears to enhance cargo interaction on the part of IPO13, with significant impact on localization, as shown for the Pax6 homeobox-containing transcription partner. This is the first report that IPO13 can be phosphorylated at Ser193 and that this modification regulates IPO13 subcellular localization and nucleocytoplasmic transport function, with important implications for IPO13's role in development and other processes., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Most intronic RNAs are degraded within seconds or minutes after their excision from newly formed transcripts. However, stable intronic sequence RNAs (sisRNAs) have been described from oocytes of the frog Xenopus , from Drosophila embryos, and from human cell lines. In Xenopus oocytes, sisRNAs are abundant in both the nucleus and cytoplasm, they occur in the form of lariats, and they are stable for days. In this study we demonstrate that cytoplasmic sisRNAs are also found in human, mouse, chicken, and zebrafish cells. They exist as circular (lariat) molecules, mostly 100-500 nucleotides in length, and are derived from many housekeeping genes. They tend to have an unusual cytosine branchpoint (with the exception of those from the frog). Stable lariats are exported from the nucleus to the cytoplasm by the NXF1/NXT1 system, demonstrating that their presence in the cytoplasm is not due to passive diffusion. Lariats in the cytoplasm are not associated with transcripts of the genes from which they are derived. The biological significance of cytoplasmic sisRNAs remains obscure., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Melanoma antigen family A4 (MAGEA4), a cancer/testis antigen, is overexpressed and is thus an immunotherapy target in various malignant tumors, including non-small cell lung cancer. However, whether MAGEA4 induces or inhibits the apoptosis of lung cancer cells remains controversial, as is its prognostic significance, particularly since there is no reliable method with which to detect MAGEA4 specifically. In this study, we optimized assay conditions to detect MAGEA4 based on cells transiently transfected with MAGEA genes, and found that MAGEA4 was expressed in four of eight non-small cell lung cancer cell lines, and in 25.4% of clinical lung cancer specimens. We also found that MAGEA4 overexpression decreased apoptosis, as measured by the levels of cleaved caspase-3 in stably transfected 293F cells. Notably, patients with nuclear MAGEA4, but not p53 expression exhibited a significantly poorer survival than those expressing both nuclear MAGEA4 and p53. Indeed, multivariate analysis identified nuclear MAGEA4 as an independent prognostic factor (P=0.0042), albeit only in the absence of p53. In this study, to the best of our knowledge, we are the first to demonstrate that the function and prognostic value of MAGEA4 depends on its subcellular localization and on the p53 status.

Nuclear retention of incompletely spliced or mature mRNAs emerges as a novel, previously underappreciated layer of gene regulation, which enables the cell to rapidly respond to stress, viral infection, differentiation cues or changing environmental conditions. Focusing on mammalian cells, we discuss recent insights into the mechanisms and functions of nuclear retention, describe retention-promoting features in protein-coding transcripts and propose mechanisms for their regulated release into the cytoplasm. Moreover, we discuss examples of how aberrant nuclear retention of mRNAs may lead to human diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We report a microfluidic system that physically separates nuclear RNA (nucRNA) and cytoplasmic RNA (cytRNA) from a single cell and enables single-cell integrated nucRNA and cytRNA-sequencing (SINC-seq). SINC-seq constructs two individual RNA-seq libraries, nucRNA and cytRNA, per cell, quantifies gene expression in the subcellular compartments, and combines them to create novel single-cell RNA-seq data. Leveraging SINC-seq, we discover distinct natures of correlation among cytRNA and nucRNA that reflect the transient physiological state of single cells. These data provide unique insights into the regulatory network of messenger RNA from the nucleus toward the cytoplasm at the single-cell level.

The MiT-TFE family of basic helix-loop-helix leucine-zipper transcription factors includes four members: TFEB, TFE3, TFEC, and MITF Originally described as oncogenes, these factors play a major role as regulators of lysosome biogenesis, cellular energy homeostasis, and autophagy. An important mechanism by which these transcription factors are regulated involves their shuttling between the surface of lysosomes, the cytoplasm, and the nucleus. Such dynamic changes in subcellular localization occur in response to nutrient fluctuations and various forms of cell stress and are mediated by changes in the phosphorylation of multiple conserved amino acids. Major kinases responsible for MiT-TFE protein phosphorylation include mTOR, ERK, GSK3, and AKT In addition, calcineurin de-phosphorylates MiT-TFE proteins in response to lysosomal calcium release. Thus, through changes in the phosphorylation state of MiT-TFE proteins, lysosome function is coordinated with the cellular metabolic state and cellular demands. This review summarizes the evidence supporting MiT-TFE regulation by phosphorylation at multiple key sites. Elucidation of such regulatory mechanisms is of fundamental importance to understand how these transcription factors contribute to both health and disease., (© 2018 The Authors.)

The X-linked histone demethylase UTX has a pivotal role in cellular and developmental processes including embryogenesis, hematopoiesis and cancer. UTX removes di- and trimethyl groups on histone H3 lysine 27, thereby regulating gene expression. But there is growing evidence that UTX displays biological functions independent of its histone demethylase activity. To elucidate these novel functions, it is of great interest to define subcellular localizations of UTX. Here we show for the first time that native UTX is primarily localized in the cytoplasm whereas ectopic GFP and Flag-tagged UTX display nuclear and cytoplasmic localization. While its epigenetic function is exerted in the nucleus, its cytoplasmic localization points to a novel function., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Herpesviruses replicate and package their genomes into capsids in replication compartments within the nuclear interior. Capsids then move to the inner nuclear membrane for envelopment and release into the cytoplasm in a process called nuclear egress. We previously found that nuclear F-actin is induced upon infection with the betaherpesvirus human cytomegalovirus (HCMV) and is important for nuclear egress and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Despite these and related findings, it has not been shown that any specific motor protein is involved in herpesvirus nuclear egress. In this study, we have investigated whether the host motor protein, myosin Va, could be fulfilling this role. Using immunofluorescence microscopy and coimmunoprecipitation, we observed associations between a nuclear population of myosin Va and the viral major capsid protein, with both concentrating at the periphery of replication compartments. Immunoelectron microscopy showed that nearly 40% of assembled nuclear capsids associate with myosin Va. We also found that myosin Va and major capsid protein colocalize with nuclear F-actin. Importantly, antagonism of myosin Va with RNA interference or a dominant negative mutant revealed that myosin Va is important for the efficient production of infectious virus, capsid accumulation in the cytoplasm, and capsid localization away from replication compartment-like inclusions toward the nuclear rim. Our results lead us to suggest a working model whereby human cytomegalovirus capsids associate with myosin Va for movement from replication compartments to the nuclear periphery during nuclear egress. IMPORTANCE Little is known regarding how newly assembled and packaged herpesvirus capsids move from the nuclear interior to the periphery during nuclear egress. While it has been proposed that an actomyosin-based mechanism facilitates intranuclear movement of alphaherpesvirus capsids, a functional role for any specific myosin in nuclear egress has not been reported. Furthermore, the notion that an actomyosin-based mechanism facilitates intranuclear capsid movement is controversial. Here we show that human cytomegalovirus capsids associate with nuclear myosin Va and F-actin and that antagonism of myosin Va impairs capsid localization toward the nuclear rim and nuclear egress. Together with our previous results showing that nuclear F-actin is induced upon HCMV infection and is also important for these processes, our results lend support to the hypothesis that nascent human cytomegalovirus capsids migrate to the nuclear periphery via actomyosin-based movement. These results shed light on a poorly understood viral process and the cellular machinery involved., (Copyright © 2018 American Society for Microbiology.)

MicroRNAs (miRNAs) are endogenous non-coding RNAs that contain approximately 22 nucleotides. They serve as key regulators in various biological processes and their dysregulation is implicated in many diseases including cancer and autoimmune disorders. It has been well established that the maturation of miRNAs occurs in the cytoplasm and miRNAs exert post-transcriptional gene silencing (PTGS) via RNA-induced silencing complex (RISC) pathway in the cytoplasm. However, numerous studies reaffirm the existence of mature miRNA in the nucleus, and nucleus-cytoplasm transport mechanism has also been illustrated. Moreover, active regulatory functions of nuclear miRNAs were found including PTGS, transcriptional gene silencing (TGS), and transcriptional gene activation (TGA), in which miRNAs bind nascent RNA transcripts, gene promoter regions or enhancer regions and exert further effects via epigenetic pathways. Based on existing interaction rules, some miRNA binding sites prediction software tools are developed, which are evaluated in this article. In addition, we attempt to explore and review the nuclear functions of miRNA in immunity, tumorigenesis and invasiveness of tumor. As a non-canonical aspect of miRNA action, nuclear miRNAs supplement miRNA regulatory networks and could be applied in miRNA based therapies.

Confocal and electron microscopy images, and WB analysis of cellular fractions revealed that HP1γ is in the nucleus but also in the cytoplasm of C2C12 myoblasts, myotubes, skeletal and cardiac muscles, N2a, HeLa and HEK293T cells. Signal specificity was tested with different antibodies and by HP1γ knockdown. Leptomycin B treatment of myoblasts increased nuclear HP1γ, suggesting that its nuclear export is Crm-1-dependent. HP1γ exhibited a filamentous pattern of staining partially co-localizing with actin in the cytoplasm of myotubes and myofibrils. Immunoelectron microscopic analysis showed high-density immunogold particles that correspond to HP1γ localized to the Z-disk and A-band of the sarcomere of skeletal muscle. HP1γ partially co-localized with actin in C2C12 myotubes and murine myofibrils. Importantly, actin co-immunoprecipitated with HP1γ in the nuclear and cytosolic fractions of myoblasts. Actin co-immunoprecipitated with HP1γ in myoblasts incubated in the absence or presence of the actin depolymerizing agent cytochalasin D, suggesting that HP1γ may interact with G-and F-actin. In the cytoplasm, HP1γ was associated to the perinuclear actin cap that controls nuclear shape and position. In the nucleus, re-ChIP assays showed that HP1γ-actin associates to the promoter and transcribed regions of the house keeping gene GAPDH, suggesting that HP1γ may function as a scaffold protein for the recruitment of actin to control gene expression. When HP1γ was knocked-down, myoblasts were unable to differentiate or originated thin myotubes. In summary, HP1γ is present in the nucleus and the cytoplasm interacting with actin, a protein complex that may exert different functions depending on its subcellular localization., (Copyright © 2017 Elsevier B.V. All rights reserved.)