The mRNA-capping enzyme localizes to stress granules in the cytoplasm and maintains cap homeostasis of target mRNAs.

The model of RNA stability has undergone a transformative shift with the revelation of a cytoplasmic capping activity that means a subset of transcripts are recapped autonomously of their nuclear counterparts. The present study demonstrates nucleo-cytoplasmic shuttling of the mRNA-capping enzyme (CE, also known as RNA guanylyltransferase and 5'-phosphatase; RNGTT), traditionally acknowledged for its nuclear localization and functions, elucidating its contribution to cytoplasmic capping activities. A unique nuclear export sequence in CE mediates XPO1-dependent nuclear export of CE. Notably, during sodium arsenite-induced oxidative stress, cytoplasmic CE (cCE) congregates within stress granules (SGs). Through an integrated approach involving molecular docking and subsequent co-immunoprecipitation, we identify eIF3b, a constituent of SGs, as an interactive associate of CE, implying that it has a potential role in guiding cCE to SGs. We measured the cap status of specific mRNA transcripts from U2OS cells that were non-stressed, stressed and recovered from stress, which indicated that cCE-target transcripts lost their caps during stress but remarkably regained cap stability during the recovery phase. This comprehensive study thus uncovers a novel facet of cytoplasmic CE, which facilitates cellular recovery from stress by maintaining cap homeostasis of target mRNAs.

Increase in primary cilia number and length upon VDAC1 depletion contributes to attenuated proliferation of cancer cells.

Primary cilia (PCs) that are present in most human cells and perform sensory function or signal transduction are lost in many solid tumors. Previously, we identified VDAC1, best known to regulate mitochondrial bioenergetics, to negatively regulate ciliogenesis. Here, we show that downregulation of VDAC1 in pancreatic cancer-derived Panc1 and glioblastoma-derived U-87MG cells significantly increased ciliation. Those PCs were significantly longer than the control cells. Such increased ciliation possibly inhibited cell cycle, which contributed to reduced proliferation of these cells. VDAC1-depletion also led to longer PCs in quiescent RPE1 cells. Therefore, serum-induced PC disassembly was slower in VDAC1-depleted RPE1 cells. Overall, this study reiterates the importance of VDAC1 in modulating tumorigenesis, due to its novel role in regulating PC disassembly and cilia length.

Long noncoding RNAs are substrates for cytoplasmic capping enzyme.

Cytoplasmic capping returns a cap to specific mRNAs, thus protecting uncapped RNAs from decay. Prior to the identification of cytoplasmic capping, uncapped mRNAs were thought to be degraded. Here, we test whether long noncoding RNAs (lncRNAs) are substrates of the cytoplasmic capping enzyme (cCE). The subcellular localisation of 14 lncRNAs associated with sarcomas were examined in U2OS osteosarcoma cells. We used 5' rapid amplification of cDNA ends (RACE) to assay uncapped forms of these lncRNAs. Inhibiting cytoplasmic capping elevated uncapped forms of selected lncRNAs indicating a plausible role of cCE in targeting them. Analysis of published cap analysis of gene expression (CAGE) data shows increased prevalence of certain 5'-RACE cloned sequences, suggesting that these uncapped lncRNAs are targets of cytoplasmic capping.

Molecular regulation of hypoxia through the lenses of noncoding RNAs and epitranscriptome.

Cells maintain homeostasis in response to environmental stress through specific cell stress responses. Hypoxic stress, well known to be associated with diverse solid tumors, is one of the main reasons for cancer-related mortality. Although cells can balance themselves well during hypoxic stress, the underlying molecular mechanisms are not well understood. The enhanced appreciation of diverse roles played by noncoding transcriptome and epigenome in recent years has brought to light the involvement of noncoding RNAs and epigenetic modifiers in hypoxic regulation. The emergence of techniques like deep sequencing has facilitated the identification of large numbers of long noncoding RNAs (lncRNAs) that are differentially regulated in various cancers. Similarly, proteomic studies have identified diverse epigenetic modifiers such as HATs, HDACs, DNMTs, polycomb groups of proteins, and their possible roles in the regulation of hypoxia. The crosstalk between lncRNAs and epigenetic modifiers play a pivotal role in hypoxia-induced cancer initiation and progression. Besides the lncRNAs, several other noncoding RNAs like circular RNAs, miRNAs, and so forth are also expressed during hypoxic conditions. Hypoxia has a profound effect on the expression of noncoding RNAs and epigenetic modifiers. Conversely, noncoding RNAs/epigenetic modifies can regulate the hypoxia signaling axis by modulating the stability of the hypoxia-inducible factors (HIFs). The focus of this review is to illustrate the molecular orchestration underlying hypoxia biology, especially in cancers, which can help in identifying promising therapeutic targets in hypoxia-induced cancers. This article is categorized under: RNA Turnover and Surveillance > Regulation of RNA Stability RNA in Disease and Development > RNA in Disease RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry.

Germ cell ribonucleoprotein granules in different clades of life: From insects to mammals.

Ribonucleoprotein (RNP) granules are no newcomers in biology. Found in all life forms, ranging across taxa, these membrane-less "organelles" have been classified into different categories based on their composition, structure, behavior, function, and localization. Broadly, they can be listed as stress granules (SGs), processing bodies (PBs), neuronal granules (NGs), and germ cell granules (GCGs). Keeping in line with the topic of this review, RNP granules present in the germ cells have been implicated in a wide range of cellular functions including cellular specification, differentiation, proliferation, and so forth. The mechanisms used by them can be diverse and many of them remain partly obscure and active areas of research. GCGs can be of different types in different organisms and at different stages of development, with multiple types coexisting in the same cell. In this review, the different known subcategories of GCGs have been studied with respect to five distinct model organisms, namely, Drosophila, Caenorhabditis elegans, Xenopus, Zebrafish, and mammals. Of them, the cytoplasmic polar granules in Drosophila, P granules in C. elegans, balbiani body in Xenopus and Zebrafish, and chromatoid bodies in mammals have been specifically emphasized upon. A descriptive account of the same has been provided along with insights into our current understanding of their functional significance with respect to cellular events relating to different developmental and reproductive processes. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes RNA Export and Localization > RNA Localization RNA in Disease and Development > RNA in Disease.

Clinical pharmacogenomics: patient perspectives of pharmacogenomic testing and the incidence of actionable test results in a chronic disease cohort.

AIM: This study aimed to examine pharmacogenomic test results and patient perspectives at an academic cardiovascular medicine clinic. PATIENTS & METHODS: Test results for three common cardiovascular drug-gene tests (warfarin-CYP2C9-VKORC1, clopidogrel-CYP2C19 and simvastatin-SLCO1B1) of 208 patients in the Ohio State University-Coriell Personalized Medicine Collaborative were examined to determine the incidence of potentially actionable test results. A post-hoc, anonymous, patient survey was also conducted. RESULTS: Potentially actionable test results for at least one of the three drug-gene tests were determined in 170 (82%) patients. Survey responses (n = 134) suggested that patients generally considered their test results to be important (median of 7.5 on a 10-point scale of importance) and were interested (median of 7.3 on a 10-point scale of interest) in a Clinical Pharmacogenomic Service. CONCLUSION: Attitudes toward pharmacogenomic testing were generally favorable, and potentially actionable test results were not uncommon in this cardiovascular medicine cohort.

RNA guanine-7 methyltransferase catalyzes the methylation of cytoplasmically recapped RNAs.

Cap homeostasis is a cyclical process of decapping and recapping that impacts a portion of the mRNA transcriptome. The metastable uncapped forms of recapping targets redistribute from polysomes to non-translating mRNPs, and recapping is all that is needed for their return to the translating pool. Previous work identified a cytoplasmic capping metabolon consisting of capping enzyme (CE) and a 5'-monophosphate kinase bound to adjacent domains of Nck1. The current study identifies the canonical cap methyltransferase (RNMT) as the enzyme responsible for guanine-N7 methylation of recapped mRNAs. RNMT binds directly to CE, and its presence in the cytoplasmic capping complex was demonstrated by pulldown assays, gel filtration and proximity-dependent biotinylation. The latter also identified the RNMT cofactor RAM, whose presence is required for cytoplasmic cap methyltransferase activity. These findings guided development of an inhibitor of cytoplasmic cap methylation whose action resulted in a selective decrease in levels of recapped mRNAs.

Cap homeostasis is independent of poly(A) tail length.

Cap homeostasis is a cyclical process of decapping and recapping that maintains the cap on a subset of the cytoplasmic transcriptome. Interfering with cytoplasmic capping results in the redistribution of target transcripts from polysomes to non-translating mRNPs, where they accumulate in an uncapped but nonetheless stable form. It is generally thought that decapping is preceded by shortening of the poly(A) tail to a length that can no longer support translation. Therefore recapped target transcripts would either have to undergo cytoplasmic polyadenylation or retain a reasonably long poly(A) tail if they are to return to the translating pool. In cells that are inhibited for cytoplasmic capping there is no change in the overall distribution of poly(A) lengths or in the elution profile of oligo(dT)-bound targets. Poly(A) tail lengths were similar for target mRNAs on polysomes or in non-translating mRNPs, and the presence of polyadenylated uncapped mRNA in mRNPs was confirmed by separation into capped and uncapped pools prior to assay. Finally, in silico analysis of cytoplasmic capping targets revealed significant correlations with genes encoding transcripts with uridylated or multiply modified 3' ends, and genes possessing multiple 3'-untranslated regions (UTRs) generated by alternative cleavage and polyadenylation.

The cytoplasmic capping complex assembles on adapter protein nck1 bound to the proline-rich C-terminus of Mammalian capping enzyme.

Cytoplasmic capping is catalyzed by a complex that contains capping enzyme (CE) and a kinase that converts RNA with a 5'-monophosphate end to a 5' diphosphate for subsequent addition of guanylic acid (GMP). We identify the proline-rich C-terminus as a new domain of CE that is required for its participation in cytoplasmic capping, and show the cytoplasmic capping complex assembles on Nck1, an adapter protein with functions in translation and tyrosine kinase signaling. Binding is specific to Nck1 and is independent of RNA. We show by sedimentation and gel filtration that Nck1 and CE are together in a larger complex, that the complex can assemble in vitro on recombinant Nck1, and Nck1 knockdown disrupts the integrity of the complex. CE and the 5' kinase are juxtaposed by binding to the adjacent domains of Nck1, and cap homeostasis is inhibited by Nck1 with inactivating mutations in each of these domains. These results identify a new domain of CE that is specific to its function in cytoplasmic capping, and a new role for Nck1 in regulating gene expression through its role as the scaffold for assembly of the cytoplasmic capping complex.

Identification of cytoplasmic capping targets reveals a role for cap homeostasis in translation and mRNA stability.

The notion that decapping leads irreversibly to messenger RNA (mRNA) decay was contradicted by the identification of capped transcripts missing portions of their 5' ends and a cytoplasmic complex that can restore the cap on uncapped mRNAs. In this study, we used accumulation of uncapped transcripts in cells inhibited for cytoplasmic capping to identify the targets of this pathway. Inhibition of cytoplasmic capping results in the destabilization of some transcripts and the redistribution of others from polysomes to nontranslating messenger ribonucleoproteins, where they accumulate in an uncapped state. Only a portion of the mRNA transcriptome is affected by cytoplasmic capping, and its targets encode proteins involved in nucleotide binding, RNA and protein localization, and the mitotic cell cycle. The 3' untranslated regions of recapping targets are enriched for AU-rich elements and microRNA binding sites, both of which function in cap-dependent mRNA silencing. These findings identify a cyclical process of decapping and recapping that we term cap homeostasis.

Inter-cellular variation in DNA content of Entamoeba histolytica originates from temporal and spatial uncoupling of cytokinesis from the nuclear cycle.

Accumulation of multiple copies of the genome in a single nucleus and several nuclei in a single cell has previously been noted in Entamoeba histolytica, contributing to the genetic heterogeneity of this unicellular eukaryote. In this study, we demonstrate that this genetic heterogeneity is an inherent feature of the cell cycle of this organism. Chromosome segregation occurs on a variety of novel microtubular assemblies including multi-polar spindles. Cytokinesis in E. histolytica is completed by the mechanical severing of a thin cytoplasmic bridge, either independently or with the help of neighboring cells. Importantly, cytokinesis is uncoupled from the nuclear division cycle, both temporally and spatially, leading to the formation of unequal daughter cells. Sorting of euploid and polyploid cells showed that each of these sub-populations acquired heterogeneous DNA content upon further growth. Our study conclusively demonstrates that genetic heterogeneity originates from the unique mode of cell division events in this protist.

Entamoeba shows reversible variation in ploidy under different growth conditions and between life cycle phases.

Under axenic growth conditions, trophozoites of Entamoeba histolytica contain heterogenous amounts of DNA due to the presence of both multiple nuclei and different amounts of DNA in individual nuclei. In order to establish if the DNA content and the observed heterogeneity is maintained during different growth conditions, we have compared E. histolytica cells growing in xenic and axenic cultures. Our results show that the nuclear DNA content of E. histolytica trophozoites growing in axenic cultures is at least 10 fold higher than in xenic cultures. Re-association of axenic cultures with their bacterial flora led to a reduction of DNA content to the original xenic values. Thus switching between xenic and axenic growth conditions was accompanied by significant changes in the nuclear DNA content of this parasite. Changes in DNA content during encystation-excystation were studied in the related reptilian parasite E. invadens. During excystation of E. invadens cysts, it was observed that the nuclear DNA content increased approximately 40 fold following emergence of trophozoites in axenic cultures. Based on the observed large changes in nuclear size and DNA content, and the minor differences in relative abundance of representative protein coding sequences, rDNA and tRNA sequences, it appears that gain or loss of whole genome copies may be occurring during changes in the growth conditions. Our studies demonstrate the inherent plasticity and dynamic nature of the Entamoeba genome in at least two species.

Genome re-duplication and irregular segregation occur during the cell cycle of Entamoeba histolytica.

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica.

The genome of the protist parasite Entamoeba histolytica.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Here we present the genome of E. histolytica, which reveals a variety of metabolic adaptations shared with two other amitochondrial protist pathogens: Giardia lamblia and Trichomonas vaginalis. These adaptations include reduction or elimination of most mitochondrial metabolic pathways and the use of oxidative stress enzymes generally associated with anaerobic prokaryotes. Phylogenomic analysis identifies evidence for lateral gene transfer of bacterial genes into the E. histolytica genome, the effects of which centre on expanding aspects of E. histolytica's metabolic repertoire. The presence of these genes and the potential for novel metabolic pathways in E. histolytica may allow for the development of new chemotherapeutic agents. The genome encodes a large number of novel receptor kinases and contains expansions of a variety of gene families, including those associated with virulence. Additional genome features include an abundance of tandemly repeated transfer-RNA-containing arrays, which may have a structural function in the genome. Analysis of the genome provides new insights into the workings and genome evolution of a major human pathogen.

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica.

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times without nuclear or cellular division. The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica.