Cytoplasmic Function Research Articles

Cytoplasmic functions of long noncoding RNAs.

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides found throughout the cell that lack protein-coding function. Their functions are closely linked to their interaction with RNA-binding proteins (RBPs) and nucleic acids. Nuclear lncRNAs have been studied extensively, revealing complexes with structural and regulatory roles that enable gene organization and control transcription. Cytoplasmic lncRNAs are less well understood, but accumulating evidence indicates that they also form complexes with diverse structural and regulatory functions. Here, we review our current knowledge of cytoplasmic lncRNAs and the different levels of gene regulation controlled by cytoplasmic lncRNA complexes, including mRNA turnover, translation, protein stability, sponging of cytosolic factors, and modulation of signaling pathways. We conclude by discussing areas of future study needed to elucidate comprehensively the biology of lncRNAs, to further understand the impact of lncRNAs on physiology and design lncRNA-centered therapeutic strategies. This article is categorized under: RNA Export and Localization > RNA Localization Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Prothymosin α, a protein implicated in the proliferation and survival of lymphocytes

Prothymosin α (ProTα) is a 109-11 amino acid protein widely distributed in mammalian tissues and particularly abundant in lymphoid cells. Genomic and proteomic studies led to consider ProTα as a multifunctional protein implicated in nuclear and cytoplasmic functions. The nuclear function of ProTα is related to chromatin activity through its interaction with core histones and proteins involved in chromatin remodelling, whereas, processes related to the phosphorylation, the proteolytic processing to generate Thymosin α1, and the role as anti-apoptotic factor of ProTα, are linked to its cytoplasmic location. Affinity chromatography and co-immunoprecipitation experiments have demonstrated novel interactions of ProTα with acidic proteins such as SET, ANP32A, and ANP32B in the cytoplasm of proliferating lymphocytes. The stabilization of these interactions by chemical cross-linking with formaldehyde shows that they are formed through associations in six acidic complexes which correspond to selective interactions of SET and ANP32 proteins with ProTα. These ProTα-complexes also include cytoplasmic proteins implicated in membrane remodelling and in mitochondrial activity. In conclusion, these novel protein interactions of ProTα observed in proliferation activity and apoptosis studies, suggest that they might be related to mechanisms involved in the proliferation activity and the apoptotic control of lymphocytes.

Thyroid hormone receptor localization in target tissues.

The thyroid hormone receptors, TRα1, TRβ1 and other subtypes, are members of the nuclear receptor superfamily that mediate the action of thyroid hormone signaling in numerous tissues to regulate important physiological and developmental processes. Their most well-characterized role is as ligand-dependent transcription factors; TRs bind thyroid hormone response elements in the presence or absence of thyroid hormone to facilitate the expression of target genes. Although primarily residing in the nucleus, TRα1 and TRβ1 shuttle rapidly between the nucleus and cytoplasm. We have identified multiple nuclear localization signals and nuclear export signals within TRα1 and TRβ1 that interact with importins and exportins, respectively, to mediate translocation across the nuclear envelope. More recently, enigmatic cytoplasmic functions have been ascribed to other TR subtypes, expanding the diversity of the cellular response to thyroid hormone. By integrating data on localization signal motifs, this review provides an overview of the complex interplay between TR's dynamic transport pathways and thyroid hormone signaling activities. We examine the variation in TR subtype response to thyroid hormone signaling, and what is currently known about regulation of the variety of tissue-specific localization patterns, including targeting to the nucleus, the mitochondria and the inner surface of the plasma membrane.

Ubiquitination of MBNL1 Is Required for Its Cytoplasmic Localization and Function in Promoting Neurite Outgrowth

The Muscleblind-like protein family (MBNL) plays an important role in regulating the transition between differentiation and pluripotency and in the pathogenesis of myotonic dystrophy type 1 (DM1), a CTGexpansion disorder. How different MBNL isoforms contribute to the differentiation and are affected in DM1 has not been investigated. Here, we show that the MBNL1 cytoplasmic, but not nuclear, isoform promotes neurite morphogenesis and reverses the morphological defects caused by expanded CUG RNA. Cytoplasmic MBNL1 is polyubiquitinated by lysine 63 (K63). Reduced cytoplasmic MBNL1 in theDM1 mouse brain is consistent with the reduced extent of K63 ubiquitination. Expanded CUG RNAinduced the deubiqutination of cytoplasmic MBNL1, which resulted in nuclear translocation and morphological impairment that could be ameliorated by inhibiting K63-linked polyubiquitin chain degradation. Our results suggest that K63-linked ubiquitination of MBNL1 is required for its cytoplasmic localization and that deubiquitination of cytoplasmic MBNL1 is pathogenic in the DM1 brain.

Cytoplasm Function

Cytoplasm Function

Mechanisms Associated with TDP-43 Neurotoxicity in ALS/FTLD.

The discovery of TDP-43 as a major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) was first made in 2006. Prior to 2006 there were only 11 publications related to TDP-43, now there are over 2000, indicating the importance of TDP-43 to unraveling the complex molecular mechanisms that underpin the pathogenesis of ALS/FTLD. Subsequent to this discovery, TDP-43 pathology was also found in other neurodegenerative diseases, including Alzheimer's disease, the significance of which is still in the early stages of exploration. TDP-43 is a predominantly nuclear DNA/RNA-binding protein, one of a number of RNA-binding proteins that are now known to be linked with ALS/FTLD, including Fused in Sarcoma (FUS), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). However, what sets TDP-43 apart is the vast number of cases in which TDP-43 pathology is present, providing a point of convergence, the understanding of which could lead to broadly applicable therapeutics. Here we will focus on TDP-43 in ALS/FTLD, its nuclear and cytoplasmic functions, and consequences should these functions go awry.

PKCε Controls Mitotic Progression by Regulating Centrosome Migration and Mitotic Spindle Assembly.

To form a proper mitotic spindle, centrosomes must be duplicated and driven poleward in a timely and controlled fashion. Improper timing of centrosome separation and errors in mitotic spindle assembly may lead to chromosome instability, a hallmark of cancer. Protein kinase C epsilon (PKCε) has recently emerged as a regulator of several cell-cycle processes associated with the resolution of mitotic catenation during the metaphase-anaphase transition and in regulating the abscission checkpoint. However, an engagement of PKCε in earlier (pre)mitotic events has not been addressed. Here, we now establish that PKCε controls prophase-to-metaphase progression by coordinating centrosome migration and mitotic spindle assembly in transformed cells. This control is exerted through cytoplasmic dynein function. Importantly, it is also demonstrated that the PKCε dependency of mitotic spindle organization is correlated with the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a characteristic of many transformed cells. Thus, PKCε appears to become specifically engaged in a programme of controls that are required to support cell-cycle progression in transformed cells, advocating for PKCε as a potential cancer therapeutic target.Implications: The close relationship between PKCε dependency for mitotic spindle organization and the nonfunctionality of the TOPO2A-dependent G2 checkpoint, a hallmark of transformed cells, strongly suggests PKCε as a therapeutic target in cancer. Mol Cancer Res; 16(1); 3-15. ©2017 AACR.

Remote Ischemic Preconditioning Ameliorates Acute Kidney Injury due to Contrast Exposure in Rats through Augmented O-GlcNAcylation.

Remote ischemic preconditioning (RIPC) is an adaptive response, manifesting when local short-term ischemic preconditioning reduces damage to adjacent or distant tissues or organs. O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of intracellular proteins denotes a type of posttranslational modification that influences multiple cytoplasmic and nuclear protein functions. Growing evidence indicates that stress can induce an acute increase in O-GlcNAc levels, which can be cytoprotective. The current study aimed to determine whether RIPC can provide renoprotection against contrast-induced acute kidney injury (CI-AKI) by augmenting O-GlcNAc signaling. We established a stable model of CI-AKI using 5/6 nephrectomized rats exposed to dehydration followed by iohexol injection via the tail vein. We found that RIPC increased UDP-GlcNAc levels through the hexosamine biosynthetic pathway as well as global renal O-GlcNAcylation. RIPC-induced elevation of O-GlcNAc signaling ameliorated CI-AKI based on the presence of less tubular damage and apoptosis and the amount of reactive oxygen species. In addition, the use of alloxan, an O-GlcNAc transferase inhibitor, and azaserine, a glutamine fructose-6-phosphate amidotransferase inhibitor, neutralized the protective effect of RIPC against oxidative stress and tubular apoptosis. In conclusion, RIPC attenuates local oxidative stress and tubular apoptosis induced by contrast exposure by enhancing O-GlcNAc glycosylation levels; this can be a potentially useful approach for lowering the risk of CI-AKI.

Autophagy-mediated upregulation of cytoplasmic claudin 1 stimulates the degradation of SQSTM1/p62 under starvation

Claudin 1, a major tight junction protein, is highly expressed in various types of tumors such as thyroid, breast, and colorectal cancers. Moreover, claudin 1 is frequently found in the cytoplasm in various types of tumor cells. However, the cytoplasmic function of claudin 1 in tumors still remains largely unknown. Here, we investigated the novel function of cytoplasmic claudin 1 in autophagy. The mRNA expression level of claudin 1 was higher in several types of tumors than in normal tissues. Furthermore, colon tumor tissues showed increased autophagy compared with the adjacent normal tissues. Both endogenous and exogenous claudin 1 showed a cytoplasmic punctate staining pattern and were co-stained with the lysosome-associated membrane protein 1 (LAMP1). Importantly, autophagy-induced conditions, including starvation, increased the protein stability of claudin 1. Moreover, the increased level of claudin 1 stimulated autophagy by decreasing the level of the autophagy substrate, sequestosome1/p62 (SQSTM1), under autophagy-inducing conditions; activation of AMP-activated protein kinase (AMPK) and inhibition of mammalian target of rapamycin (mTOR). Taken together, we demonstrate that the novel function of cytoplasmic claudin 1 is related to autophagy. This study is the first to show a cytoplasmic function of claudin 1 as an autophagy regulator and provides the evidence that claudin 1-mediated autophagy regulation is an integral part of the mechanism by which claudin 1 regulates cancer progression.

Abstract B039: KDM4B inhibition confers suseptibility to PI3K inhibitor in PTEN-deficient TNBC through overactivating UPR pathway

Abstract Triple-negative breast cancer (TNBC) is among the most lethal cancers in females. Up to 35% of TNBC show PTEN mutation or PTEN loss (PTEN deficiency). Although PTEN deficiency leads to aberrant activation of PI3K/AKT signaling, therapeutic targeting of PI3K/AKT pathway has yielded very limited clinical benefit. The effective therapeutic targets for PTEN-deficient cancers are in urgent need of identifying common vulnerabilities of this important pathway. To identify additional chemical drugs that target PTEN-deficient TNBC, we performed a drug screening together with a series of in vitro and in vivo validation. Here, we show that PTEN deficiency confers vulnerability to inhibition of histone demethylase KDM4B, resulting from perturbation of endoplasmic reticulum (ER) homeostasis through activating eIF2α/ATF4 unfolded protein response (UPR) pathway. Mechanistically, KDM4B suppresses UPR pathway by interacting with and inhibiting eIF2α activation, and this function requires its demethylase activity but is independent of histone modification. Consistent with the sensitivity, PTEN-deficient cells and samples from PTEN-deficient patients show constitutively activation of UPR, which usually confers survival advantage but induces cell death once it is further activated and prolonged by KDM4B inhibition. More importantly, targeting KDM4B can robustly synergize with PI3K inhibitor to induce cell death in PTEN-deficient TNBC. This synergistic killing is caused by "overactivating" the UPR pathway resulting from transcriptional activation of UPR genes by ATF4 and FoxO1. Collectively, our study thus identifies a novel cytoplasmic function of KDM4B in regulating cellular stress and provides a new therapeutic approach to treat PTEN-deficient TNBC. Citation Format: Wenyu Wang, Gokce Oguz, Puay Leng Lee, Siti Maryam Yatim, Qiang Yu. KDM4B inhibition confers suseptibility to PI3K inhibitor in PTEN-deficient TNBC through overactivating UPR pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B039.

Full-Length Isoforms of Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen Accumulate in the Cytoplasm of Cells Undergoing the Lytic Cycle of Replication.

The latency-associated nuclear antigen (LANA) of the Kaposi's sarcoma-associated herpesvirus (KSHV) performs a variety of functions to establish and maintain KSHV latency. During latency, LANA localizes to discrete punctate spots in the nucleus, where it tethers viral episomes to cellular chromatin and interacts with nuclear components to regulate cellular and viral gene expression. Using highly sensitive tyramide signal amplification, we determined that LANA localizes to the cytoplasm in different cell types undergoing the lytic cycle of replication after de novo primary infection and after spontaneous, tetradecanoyl phorbol acetate-, or open reading frame 50 (ORF50)/replication transactivator (RTA)-induced activation. We confirmed the presence of cytoplasmic LANA in a subset of cells in lytically active multicentric Castleman disease lesions. The induction of cellular migration by scratch-wounding confluent cell cultures, culturing under subconfluent conditions, or induction of cell differentiation in primary cultures upregulated the number of cells permissive for primary lytic KSHV infection. The induction of lytic replication was characterized by high-level expression of cytoplasmic LANA and nuclear ORF59, a marker of lytic replication. Subcellular fractionation studies revealed the presence of multiple isoforms of LANA in the cytoplasm of ORF50/RTA-activated Vero cells undergoing primary infection. Mass spectrometry analysis demonstrated that cytoplasmic LANA isoforms were full length, containing the N-terminal nuclear localization signal. These results suggest that trafficking of LANA to different subcellular locations is a regulated phenomenon, which allows LANA to interact with cellular components in different compartments during both the latent and the replicative stages of the KSHV life cycle.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes AIDS-related malignancies, including lymphomas and Kaposi's sarcoma. KSHV establishes lifelong infections using its latency-associated nuclear antigen (LANA). During latency, LANA localizes to the nucleus, where it connects viral and cellular DNA complexes and regulates gene expression, allowing the virus to maintain long-term infections. Our research shows that intact LANA traffics to the cytoplasm of cells undergoing permissive lytic infections and latently infected cells in which the virus is induced to replicate. This suggests that LANA plays important roles in the cytoplasm and nuclear compartments of the cell during different stages of the KSHV life cycle. Determining cytoplasmic function and mechanism for regulation of the nuclear localization of LANA will enhance our understanding of the biology of this virus, leading to therapeutic approaches to eliminate infection and block its pathological effects.

Polysomes, Stress Granules, and Processing Bodies: A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function.

Discoveries illuminate highly regulated dynamics of mRNA translation, sequestration, and degradation within the cytoplasm of plants.

DCF1 subcellular localization and its function in mitochondria

Dendritic cell factor 1 (DCF1) is a transmembrane protein that plays important roles in regulating neural stem cell differentiation and dendritic spine formation. Apart from its cytoplasmic functions, DCF1 plays a role in autophagy during the regulation of amyloid precursor proteins. However, the subcellular localization of DCF1 remains unknown. Therefore, in this study, DCF1 tagged with green fluorescent protein was transiently expression in HelaS3 and HEK293T cells. The results showed that DCF1 was widely expressed in different organelles, including the mitochondria, Golgi apparatus, endoplasmic reticulum, endosomes and lysosomes. An iodixanol step gradient further confirmed that DCF1 is localized to the mitochondria, endosomes, lysosomes, endoplasmic reticulum, and proteasome. Finally, functional analysis of the mitochondria revealed that DCF1 affected the expression and localization of MGST1. This study presents a comprehensive evaluation of the subcellular localization of DCF1, which provides important information on complex functions mediated by DCF1.

A cytoplasmic COMPASS is necessary for cell survival and triple-negative breast cancer pathogenesis by regulating metabolism

Mutations and translocations within the COMPASS (complex of proteins associated with Set1) family of histone lysine methyltransferases are associated with a large number of human diseases, including cancer. Here we report that SET1B/COMPASS, which is essential for cell survival, surprisingly has a cytoplasmic variant. SET1B, but not its SET domain, is critical for maintaining cell viability, indicating a novel catalytic-independent role of SET1B/COMPASS. Loss of SET1B or its unique cytoplasmic-interacting protein, BOD1, leads to up-regulation of expression of numerous genes modulating fatty acid metabolism, including ADIPOR1 (adiponectin receptor 1), COX7C, SDC4, and COQ7 Our detailed molecular studies identify ADIPOR1 signaling, which is inactivated in both obesity and human cancers, as a key target of SET1B/COMPASS. Collectively, our study reveals a cytoplasmic function for a member of the COMPASS family, which could be harnessed for therapeutic regulation of signaling in human diseases, including cancer.

Increased expression of Myc-interacting zinc finger protein 1 in APP/PS1 mice.

Myc-interacting zinc-finger protein 1 (Miz1) is a member of the poxvirus and zinc-finger domain/zinc finger transcription factor family. Its transcription activation and repression functions in the nucleus are well elucidated; however its cytoplasmic inflammation function is poorly understood and may be associated with the pathogenesis of Alzheimer's disease (AD). The aim of the present study was to investigate the association between AD and Miz1 expression. In the present study, the expression and distribution of Miz1 in wild-type (WT) and amyloid precursor protein/presenelin-1 (AD) mice was studied using reverse transcription-quantitative polymerase chain reaction, western blot analysis, and immunohistochemical and immunofluorescence staining. The results indicated that Miz1 was significantly upregulated in the cortex of AD mice (P<0.05). Double immunofluorescence labeling revealed that Miz1 protein was predominantly expressed in neurons and astrocytes, as evidenced by co-localization with the dendritic markers microtubule associated protein 2 and glial fibrillary acidic protein, respectively. The results of the present study suggest that the expression of Miz1 in the brain tissue of AD mice may serve an important role in AD pathogenesis.

N-terminal splicing extensions of the human MYO1C gene fine-tune the kinetics of the three full-length myosin IC isoforms

The MYO1C gene produces three alternatively spliced isoforms, differing only in their N-terminal regions (NTRs). These isoforms, which exhibit both specific and overlapping nuclear and cytoplasmic functions, have different expression levels and nuclear-cytoplasmic partitioning. To investigate the effect of NTR extensions on the enzymatic behavior of individual isoforms, we overexpressed and purified the three full-length human isoforms from suspension-adapted HEK cells. MYO1CC favored the actomyosin closed state (AMC), MYO1C16 populated the actomyosin open state (AMO) and AMC equally, and MYO1C35 favored the AMO state. Moreover, the full-length constructs isomerized before ADP release, which has not been observed previously in truncated MYO1CC constructs. Furthermore, global numerical simulation analysis predicted that MYO1C35 populated the actomyosin·ADP closed state (AMDC) 5-fold more than the actomyosin·ADP open state (AMDO) and to a greater degree than MYO1CC and MYO1C16 (4- and 2-fold, respectively). On the basis of a homology model of the 35-amino acid NTR of MYO1C35 (NTR35) docked to the X-ray structure of MYO1CC, we predicted that MYO1C35 NTR residue Arg-21 would engage in a specific interaction with post-relay helix residue Glu-469, which affects the mechanics of the myosin power stroke. In addition, we found that adding the NTR35 peptide to MYO1CC yielded a protein that transiently mimics MYO1C35 kinetic behavior. By contrast, NTR35, which harbors the R21G mutation, was unable to confer MYO1C35-like kinetic behavior. Thus, the NTRs affect the specific nucleotide-binding properties of MYO1C isoforms, adding to their kinetic diversity. We propose that this level of fine-tuning within MYO1C broadens its adaptability within cells.

Interrogation of \u03b3-tubulin alleles using high-resolution fitness measurements reveals a distinct cytoplasmic function in spindle alignment

γ-Tubulin has a well-established role in nucleating the assembly of microtubules, yet how phosphorylation regulates its activity remains unclear. Here, we use a time-resolved, fitness-based SGA approach to compare two γ-tubulin alleles, and find that the genetic interaction profile of γtub-Y362E is enriched in spindle positioning and cell polarity genes relative to that of γtub-Y445D, which is enriched in genes involved in spindle assembly and stability. In γtub-Y362E cells, we find a defect in spindle alignment and an increase in the number of astral microtubules at both spindle poles. Our results suggest that the γtub-Y362E allele is a separation-of-function mutation that reveals a role for γ-tubulin phospho-regulation in spindle alignment. We propose that phosphorylation of the evolutionarily conserved Y362 residue of budding yeast γ-tubulin contributes to regulating the number of astral microtubules associated with spindle poles, and promoting efficient pre-anaphase spindle alignment.

BRCA1 gene: function and deficiency.

The BRCA1 protein, a hereditary breast and ovarian cancer-causing gene product, is known as a multifunctional protein that performs various functions in cells. It is well known, along with BRCA 2, to cause hereditary breast and ovarian cancer, but here we will specifically focus on BRCA1. We introduce the mechanism and the latest report on homologous recombination repair, replication, involvement in checkpoint regulation, transcription, chromatin remodeling, and cytoplasmic function (centrosome regulation, apoptosis, selective autophagy), and consider the possibility of carcinogenesis from inhibition of the intracellular functions in each. We also consider the possibility of drug development based on each function. Finally, we will explain, from data obtained through basic research, that an appropriate regimen is important for raising the response rate for poly (ADP)-ribose polymerase inhibitors, in the case of low susceptibility, iatrogenic toxicity, tolerance, etc.

Cytoplasmic and Nuclear TAZ Exert Distinct Functions in Regulating PrimedPluripotency.

SummaryMouse epiblast stem cells (mEpiSCs) and human embryonic stem cells (hESCs) are primed pluripotent stem cells whose self-renewal can be maintained through cytoplasmic stabilization and retention of β-catenin. The underlying mechanism, however, remains largely unknown. Here, we show that cytoplasmic β-catenin interacts with and retains TAZ, a Hippo pathway effector, in the cytoplasm. Cytoplasmic retention of TAZ promotes mEpiSC self-renewal in the absence of nuclear β-catenin, whereas nuclear translocation of TAZ induces mEpiSC differentiation. TAZ is dispensable for naive mouse embryonic stem cell (mESC) self-renewal but required for the proper conversion of mESCs to mEpiSCs. The self-renewal of hESCs, like that of mEpiSCs, can also be maintained through the cytoplasmic retention of β-catenin and TAZ. Our study indicates that how TAZ regulates cell fate depends on not only the cell type but also its subcellular localization.

MiRNA Enriched in Human Neuroblast Nuclei Bind the MAZ Transcription Factor and Their Precursors Contain the MAZ Consensus Motif.

While the cytoplasmic function of microRNA (miRNA) as post-transcriptional regulators of mRNA has been the subject of significant research effort, their activity in the nucleus is less well characterized. Here we use a human neuronal cell model to show that some mature miRNA are preferentially enriched in the nucleus. These molecules were predominantly primate-specific and contained a sequence motif with homology to the consensus MAZ transcription factor binding element. Precursor miRNA containing this motif were shown to have affinity for MAZ protein in nuclear extract. We then used Ago1/2 RIP-Seq to explore nuclear miRNA-associated mRNA targets. Interestingly, the genes for Ago2-associated transcripts were also significantly enriched with MAZ binding sites and neural function, whereas Ago1-transcripts were associated with general metabolic processes and localized with SC35 spliceosomes. These findings suggest the MAZ transcription factor is associated with miRNA in the nucleus and may influence the regulation of neuronal development through Ago2-associated miRNA induced silencing complexes. The MAZ transcription factor may therefore be important for organizing higher order integration of transcriptional and post-transcriptional processes in primate neurons.