RESUMO
In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation.
Assuntos
Proteômica , Trypanosoma cruzi , Transporte Ativo do Núcleo Celular , RNA , Splicing de RNA , Transporte de RNARESUMO
MicroRNAs (miRNAs) are a class of small (20-24 nucleotides), highly conserved, non-coding RNA molecules whose main function is the post-transcriptional regulation of gene expression through sequence-specific manners, such as mRNA degradation or translational repression. Since these key regulatory molecules are implicated in several biological processes, their altered expression affects the preservation of cellular homeostasis and leads to the development of a wide range of pathologies. Over the last few years, relevant investigations have elucidated that miRNAs participate in different stages of bone growth and development. Moreover, the abnormal expression of these RNA molecules in bone cells and tissues has been significantly associated with the progression of numerous bone diseases, including osteoporosis, osteosarcoma, osteonecrosis and bone metastasis, among others. In fact, miRNAs regulate multiple pathological mechanisms, including altering either osteogenic or osteoblast differentiation, metastasis, osteosarcoma cell proliferation, and bone loss. Therefore, in this present review, aiming to impulse the research arena of the biological implications of miRNA transcriptome in bone diseases and to explore their potentiality as a theragnostic target, we summarize the recent findings associated with the clinical significance of miRNAs in these ailments.
Assuntos
Biomarcadores , Doenças Ósseas/etiologia , Doenças Ósseas/terapia , Suscetibilidade a Doenças , Regulação da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Animais , Doenças Ósseas/metabolismo , Doenças Ósseas/patologia , Gerenciamento Clínico , Exossomos , Humanos , Metástase Neoplásica , Osteogênese/genética , Osteonecrose/etiologia , Osteonecrose/metabolismo , Osteonecrose/patologia , Osteoporose/genética , Osteoporose/metabolismo , Osteoporose/patologia , Osteossarcoma/etiologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Transporte de RNA , Transdução de SinaisRESUMO
BACKGROUND: The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation. RESULTS: To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN interacts with mRNA and the Nuclear RNA Export Factor 1 (NXF1). CONCLUSIONS: Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.
Assuntos
Núcleo Celular/metabolismo , Neoplasias/metabolismo , RNA Mensageiro/genética , Helicase da Síndrome de Werner/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Células HeLa , Humanos , Redes e Vias Metabólicas/fisiologia , Oxirredução , Processamento Pós-Transcricional do RNA/fisiologia , Transporte de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , RecQ Helicases/genética , Síndrome de Werner/metabolismoRESUMO
RNA transport and localization are evolutionarily conserved processes that allow protein translation to occur at specific subcellular sites and thereby having fundamental roles in the determination of cell fates, embryonic patterning, asymmetric cell division, and cell polarity. In addition to localizing RNA molecules to specific subcellular sites, plants have the ability to exchange RNA molecules between cells through plasmodesmata (PD). Plant RNA viruses hijack the mechanisms of intracellular and intercellular RNA transport to establish localized replication centers within infected cells and then to disseminate their infectious genomes between cells and throughout the plant organism with the help of their movement proteins (MP). In this chapter, we describe the transient expression of the tobacco mosaic virus movement protein (TMV-MP) and the application of the MS2 system for the in vivo labeling of the MP-encoding mRNA. The MS2 method is based on the binding of the bacteriophage coat protein (CP) to its origin of assembly (OAS) in the phage RNA. Thus, to label a specific mRNA in vivo, a tandem repetition of a 19-nucleotide-long stem-loop (SL) sequence derived from the MS2 OAS sequence (MSL) is transcriptionally fused to the RNA under investigation. The RNA is detected by the co-expression of fluorescent protein-tagged MS2 CP (MCP), which binds to each of the MSL elements. In providing a detailed protocol for the in vivo visualization of TMV-MP mRNA tagged with the MS2 system in Nicotiana benthamiana epidermal cells, we describe (1) the specific DNA constructs, (2) Agrobacterium tumefaciens-mediated transfection for their transient expression in plants, and (3) imaging conditions required to obtain high-quality mRNA imaging data.
Assuntos
Agrobacterium tumefaciens/genética , Levivirus/metabolismo , Proteínas do Movimento Viral em Plantas/genética , Transporte de RNA/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , RNA Viral/genética , Vírus do Mosaico do Tabaco/metabolismo , Transporte Biológico , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/metabolismo , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Levivirus/genética , Proteínas Luminescentes , Microscopia de Fluorescência , Proteínas do Movimento Viral em Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plasmodesmos/metabolismo , RNA Mensageiro/genética , Nicotiana/genética , Nicotiana/metabolismo , Vírus do Mosaico do Tabaco/genéticaRESUMO
Transference of RNAs and ribosomes from Schwann cell-to-axon was demonstrated in normal and regenerating peripheral nerves. Previously, we have shown that RNAs transfer is dependent on F-actin cytoskeleton and Myosin Va. Here, we explored the contribution of microtubules to newly synthesized RNAs transport from Schwann cell nuclei up to nodal microvilli in sciatic nerves. Results using immunohistochemistry and quantitative confocal FRET analysis indicate that Schwann cell-derived RNAs co-localize with microtubules in Schwann cell cytoplasm. Additionally, transport of Schwann cell-derived RNAs is nocodazole and colchicine sensitive demonstrating its dependence on microtubule network integrity. Moreover, mRNAs codifying neuron-specific proteins are among Schwann cell newly synthesized RNAs population, and some of them are associated with KIF1B and KIF5B microtubules-based motors.
Assuntos
Axônios/metabolismo , Microtúbulos/metabolismo , RNA/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Masculino , Bainha de Mielina/metabolismo , Regeneração Nervosa , RNA/análise , Transporte de RNA , Ratos , Ratos Sprague-DawleyRESUMO
Fragmentation of tRNAs generates a family of small RNAs collectively known as tRNA-derived fragments. These fragments vary in sequence and size but have been shown to regulate many processes involved in cell homoeostasis and adaptations to stress. Additionally, the field of extracellular RNAs (exRNAs) is rapidly growing because exRNAs are a promising source of biomarkers in liquid biopsies, and because exRNAs seem to play key roles in intercellular and interspecies communication. Herein, we review recent descriptions of tRNA-derived fragments in the extracellular space in all domains of life, both in biofluids and in cell culture. The purpose of this review is to find consensus on which tRNA-derived fragments are more prominent in each extracellular fraction (including extracellular vesicles, lipoproteins and ribonucleoprotein complexes). We highlight what is becoming clear and what is still controversial in this field, in order to stimulate future hypothesis-driven studies which could clarify the role of full-length tRNAs and tRNA-derived fragments in the extracellular space.
Assuntos
Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , Animais , Biomarcadores , Ácidos Nucleicos Livres , Meios de Cultivo Condicionados , Espaço Extracelular , Vesículas Extracelulares/metabolismo , Humanos , Lipoproteínas/metabolismo , Transporte de RNA , RNA de Transferência/química , RNA de Transferência/classificaçãoRESUMO
Extracellular vesicles (EVs) are cell-derived nanoparticles that act as natural carriers of nucleic acids between cells. They offer advantages as delivery vehicles for therapeutic nucleic acids such as small RNAs. Loading of desired nucleic acids into EVs can be achieved by electroporation or transfection once purified. An attractive alternative is to transfect cells with the desired small RNAs and harness the cellular machinery for RNA sorting into the EVs. This possibility has been less explored because cells are believed to secrete only specific RNAs. However, we hypothesized that, even in the presence of selective secretion, concentration-driven RNA sorting to EVs would still be feasible. To show this, we transfected cells with glycine 5' tRNA halves, which we have previously shown to better resist RNases. We then measured their levels in EVs and in recipient cells and found that, in contrast to unstable RNAs of random sequence, these tRNA halves were present in vesicles and in recipient cells in amounts proportional to the concentration of RNA used for transfection. Similar efficiencies were obtained with other stable oligonucleotides of random sequence. Our results demonstrate that RNA stability is a key factor needed to maintain high intracellular concentrations, a prerequisite for efficient non-selective RNA sorting to EVs and delivery to cells. Given that glycine 5' tRNA halves belong to the group of stress-induced tRNA fragments frequently detected in extracellular space and biofluids, we propose that upregulation of extracellular tRNA fragments is consequential to cellular stress and might be involved in intercellular signalling.
Assuntos
Vesículas Extracelulares/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Transporte Biológico , Comunicação Celular , Linhagem Celular Tumoral , Exossomos/metabolismo , Expressão Gênica , Humanos , Transporte de RNA , RNA de Transferência/química , RNA não Traduzido/genética , Transfecção , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Nucleo-cytoplasmic RNA export is an essential post-transcriptional step to control gene expression in eukaryotic cells and is poorly understood in apicomplexan parasites. With the exception of UAP56, a component of TREX (Transcription Export) complex, other components of mRNA export machinery are not well conserved in divergent supergroups. Here, we use Toxoplasma gondii as a model system to functionally characterize TgUAP56 and its potential interaction factors. We demonstrate that TgUAP56 is crucial for mRNA export and that functional interference leads to significant accumulation of mRNA in the nucleus. It was necessary to employ bioinformatics and phylogenetic analysis to identify orthologs related to mRNA export, which show a remarkable low level of conservation in T. gondii. We adapted a conditional Cas9/CRISPR system to carry out a genetic screen to verify if these factors were involved in mRNA export in T. gondii. Only the disruption of TgRRM_1330 caused accumulation of mRNA in the nucleus as found with TgUAP56. This protein is potentially a divergent partner of TgUAP56, and provides insight into a divergent mRNA export pathway in apicomplexans.
Assuntos
RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Biologia Computacional/métodos , Humanos , Proteínas Nucleares/genética , Transporte de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Toxoplasma/enzimologia , Fatores de Transcrição/metabolismoRESUMO
Intracellular trafficking and asymmetric localization of RNA molecules within cells are a prevalent process across phyla involved in developmental control and signaling and thus in the determination of cell fate. In addition to intracellular localization, plants support the trafficking of RNA molecules also between cells through plasmodesmata (PD), which has important roles in the cell-to-cell and systemic communication during plant growth and development. Viruses have developed strategies to exploit the underlying plant RNA transport mechanisms for the cell-to-cell and systemic dissemination of infection. In vivo RNA visualization methods have revolutionized the study of RNA dynamics in living cells. However, their application in plants is still in its infancy. To gain insights into the RNA transport mechanisms in plants, we study the localization and transport of Tobacco mosaic virus RNA using MS2 tagging. This technique involves the tagging of the RNA of interest with repeats of an RNA stem-loop (SL) that is derived from the origin of assembly of the bacteriophage MS2 and recruits the MS2 coat protein (MCP). Thus, expression of MCP fused to a fluorescent marker allows the specific visualization of the SL-carrying RNA. Here we describe a detailed protocol for Agrobacterium tumefaciens-mediated transient expression and in vivo visualization of MS2-tagged mRNAs in Nicotiana benthamiana leaves.
Assuntos
Nicotiana/virologia , Imagem Óptica/métodos , Folhas de Planta/virologia , RNA Viral/análise , Vírus do Mosaico do Tabaco/isolamento & purificação , Agrobacterium tumefaciens/genética , Proteínas do Capsídeo/genética , Expressão Gênica , Levivirus/genética , Microscopia de Fluorescência/métodos , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Transporte de RNA , RNA Viral/genética , RNA Viral/metabolismo , Nicotiana/genética , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/metabolismoRESUMO
Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, participate in intercellular communication, and particularly, in paracrine and endocrine signalling. The EVs and their specific contents have been considered hallmarks of different diseases. It has been recently discovered that EVs can co-transport nucleic acids such as DNAs, ribosomal RNAs, circular RNAs (circRNAs), long noncoding RNAs (lnRNAs) and microRNAs (miRNAs). miRNAs are important regulators of gene expression at the post-transcriptional level, although they may also play other roles. Recent evidence supports the hypothesis that miRNAs can activate Toll-like receptors (TLRs) under certain circumstances. TLRs belong to a multigene family of immune system receptors and have been recently described in the nervous system. In the immune system, TLRs are important for the recognition of the invading microorganisms, whereas in the nervous system, they recognise endogenous ligands released by undifferentiated or necrotic/injured cells. In the neuronal disease field, TLRs activity has been associated with amyotrophic lateral sclerosis (ALS), stroke, Alzheimer's and Parkinson's disease. Herein, we reviewed the current knowledge of the relationship between miRNA release by EVs and the inflammation signalling triggered by TLRs in neighbouring cells or during long-distance cell-to-cell communication. We highlight novel aspects of this communication mechanism, offering a valuable insight into such pathways in health and disease.