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1.
Proc Natl Acad Sci U S A ; 119(32): e2201453119, 2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-35914138

RESUMO

Because multipartite viruses package their genome segments in different viral particles, they face a potentially huge cost if the entire genomic information, i.e., all genome segments, needs to be present concomitantly for the infection to function. Previous work with the octapartite faba bean necrotic stunt virus (FBNSV; family Nanoviridae, genus Nanovirus) showed that this issue can be resolved at the within-host level through a supracellular functioning; all viral segments do not need to be present within the same host cell but may complement each other through intercellular trafficking of their products (protein or messenger RNA [mRNA]). Here, we report on whether FBNSV can as well decrease the genomic integrity cost during between-host transmission. Using viable infections lacking nonessential virus segments, we show that full-genome infections can be reconstituted and function through separate acquisition and/or inoculation of complementary sets of genome segments in recipient hosts. This separate acquisition/inoculation can occur either through the transmission of different segment sets by different individual aphid vectors or by the sequential acquisition by the same aphid of complementary sets of segments from different hosts. The possibility of a separate between-host transmission of different genome segments thus offers a way to at least partially resolve the genomic maintenance problem faced by multipartite viruses.


Assuntos
Afídeos , Genoma Viral , Interações entre Hospedeiro e Microrganismos , Insetos Vetores , Nanovirus , Vicia faba , Animais , Afídeos/virologia , Genoma Viral/genética , Insetos Vetores/virologia , Nanovirus/genética , Doenças das Plantas/virologia , Transporte Proteico , Transporte de RNA , RNA Viral/genética , RNA Viral/metabolismo , Vicia faba/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
2.
Cell Mol Life Sci ; 79(9): 481, 2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-35962235

RESUMO

Although 5-methylcytosine (m5C) has been identified as a novel and abundant mRNA modification and associated with energy metabolism, its regulation function in adipose tissue and skeletal muscle is still limited. This study aimed at investigating the effect of mRNA m5C on adipogenesis and myogenesis using Jinhua pigs (J), Yorkshire pigs (Y) and their hybrids Yorkshire-Jinhua pigs (YJ). We found that Y grow faster than J and YJ, while fatness-related characteristics observed in Y were lower than those of J and YJ. Besides, total mRNA m5C levels and expression rates of NSUN2 were higher both in backfat layer (BL) and longissimus dorsi muscle (LDM) of Y compared to J and YJ, suggesting that higher mRNA m5C levels positively correlate with lower fat and higher muscle mass. RNA bisulfite sequencing profiling of m5C revealed tissue-specific and dynamic features in pigs. Functionally, hyper-methylated m5C-containing genes were enriched in pathways linked to impaired adipogenesis and enhanced myogenesis. In in vitro, m5C inhibited lipid accumulation and promoted myogenic differentiation. Furthermore, YBX2 and SMO were identified as m5C targets. Mechanistically, YBX2 and SMO mRNAs with m5C modification were recognized and exported into the cytoplasm from the nucleus by ALYREF, thus leading to increased YBX2 and SMO protein expression and thereby inhibiting adipogenesis and promoting myogenesis, respectively. Our work uncovered the critical role of mRNA m5C in regulating adipogenesis and myogenesis via ALYREF-m5C-YBX2 and ALYREF-m5C-SMO manners, providing a potential therapeutic target in the prevention and treatment of obesity, skeletal muscle dysfunction and metabolic disorder diseases.


Assuntos
Adipogenia , Proteínas de Ligação a RNA , Adipogenia/genética , Animais , Desenvolvimento Muscular/genética , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Suínos
3.
Curr Opin Struct Biol ; 75: 102431, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35930970

RESUMO

In eukaryotes, the expression of genetic information begins in the cell nucleus with precursor messenger RNA (pre-mRNA) transcription and processing into mature mRNA. The mRNA is subsequently recognized and packaged by proteins into an mRNA ribonucleoprotein complex (mRNP) and exported to the cytoplasm for translation. Each of the nuclear mRNA maturation steps is carried out by a dedicated molecular machine. Here, we highlight recent structural and mechanistic insights into how these machines function, including the capping enzyme, the spliceosome, the 3'-end processing machinery, and the transcription-export complex. While we increasingly understand individual steps of nuclear gene expression, many questions remain. For example, we are only beginning to reveal how mature mRNAs are recognized and packaged for nuclear export and how mRNA maturation events are coupled to transcription and to each other. Advances in the preparation of recombinant and endogenous protein-nucleic acid complexes, cryo-electron microscopy, and machine learning promise exciting insights into the mechanisms of nuclear gene expression and its spatial organization.


Assuntos
Núcleo Celular , Transporte de RNA , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Microscopia Crioeletrônica , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Cell Mol Life Sci ; 79(7): 391, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35776213

RESUMO

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1, as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1.


Assuntos
Neoplasias da Mama , Proteínas Nucleares , RNA Longo não Codificante , Proteínas de Ligação a RNA , Fatores de Transcrição , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica , Feminino , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo
5.
Science ; 376(6598): eabm9129, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35679405

RESUMO

INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y­shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment­specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease­associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell­based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled­coil hub that tethers two separate mRNP­remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan­specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N­terminal S­shaped α­helical solenoid followed by a coiled­coil oligomerization element, numerous Ran­interacting domains, an E3 ligase domain, and a C­terminal prolyl­isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N­terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell­based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo­ET density matched the dimensions of the CFNC coiled­coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled­coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo­ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near­atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].


Assuntos
Citoplasma , Proteínas Fúngicas , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Transporte de RNA , RNA Mensageiro , Chaetomium , Microscopia Crioeletrônica , Citoplasma/química , Proteínas Fúngicas/química , Humanos , Chaperonas Moleculares/química , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , RNA Mensageiro/metabolismo
6.
Bioessays ; 44(8): e2200027, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35754154

RESUMO

The nuclear export of mRNA through the nuclear pore complex (NPC) is a process required for the healthy functioning of human cells, making it a critical area of research. However, the geometries of mRNA and the NPC are well below the diffraction limit of light microscopy, thereby presenting significant challenges in evaluating the discrete interactions and dynamics involved in mRNA nuclear export through the native NPC. Recent advances in biotechnology and single-molecule super-resolution light microscopy have enabled researchers to gain granular insight into the specific contributions made by discrete nucleoporins in the nuclear basket of the NPC to the export of mRNA. Specifically, by expanding upon the docking step facilitated by the protein TPR in the nuclear basket as well as identifying NUP153 as being the primary nuclear basket protein initiating export through the central channel of the NPC.


Assuntos
Poro Nuclear , Transporte de RNA , Transporte Ativo do Núcleo Celular , Células HeLa , Humanos , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Nucleus ; 13(1): 170-193, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35593254

RESUMO

The Nuclear Pore Complex (NPC) represents a critical passage through the nuclear envelope for nuclear import and export that impacts nearly every cellular process at some level. Recent technological advances in the form of Auxin Inducible Degron (AID) strategies and Single-Point Edge-Excitation sub-Diffraction (SPEED) microscopy have enabled us to provide new insight into the distinct functions and roles of nuclear basket nucleoporins (Nups) upon nuclear docking and export for mRNAs. In this paper, we provide a review of our recent findings as well as an assessment of new techniques, updated models, and future perspectives in the studies of mRNA's nuclear export.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Transporte Ativo do Núcleo Celular , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
8.
Int J Mol Sci ; 23(10)2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35628261

RESUMO

The relationship between transcription and aging is one that has been studied intensively and experimentally with diverse attempts. However, the impact of the nuclear mRNA export on the aging process following its transcription is still poorly understood, although the nuclear events after transcription are coupled closely with the transcription pathway because the essential factors required for mRNA transport, namely TREX, TREX-2, and nuclear pore complex (NPC), physically and functionally interact with various transcription factors, including the activator/repressor and pre-mRNA processing factors. Dysregulation of the mediating factors for mRNA export from the nucleus generally leads to the aberrant accumulation of nuclear mRNA and further impairment in the vegetative growth and normal lifespan and the pathogenesis of neurodegenerative diseases. The optimal stoichiometry and density of NPC are destroyed during the process of cellular aging, and their damage triggers a defect of function in the nuclear permeability barrier. This review describes recent findings regarding the role of the nuclear mRNA export in cellular aging and age-related neurodegenerative disorders.


Assuntos
Núcleo Celular , Transporte de RNA , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/metabolismo , Poro Nuclear/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
9.
Genes Dev ; 36(9-10): 550-565, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35589130

RESUMO

Although splicing is a major driver of RNA nuclear export, many intronless RNAs are efficiently exported to the cytoplasm through poorly characterized mechanisms. For example, GC-rich sequences promote nuclear export in a splicing-independent manner, but how GC content is recognized and coupled to nuclear export is unknown. Here, we developed a genome-wide screening strategy to investigate the mechanism of export of NORAD, an intronless cytoplasmic long noncoding RNA (lncRNA). This screen revealed an RNA binding protein, RBM33, that directs the nuclear export of NORAD and numerous other transcripts. RBM33 directly binds substrate transcripts and recruits components of the TREX-NXF1/NXT1 RNA export pathway. Interestingly, high GC content emerged as the feature that specifies RBM33-dependent nuclear export. Accordingly, RBM33 directly binds GC-rich elements in target transcripts. These results provide a broadly applicable strategy for the genetic dissection of nuclear export mechanisms and reveal a long-sought nuclear export pathway for transcripts with GC-rich sequences.


Assuntos
Proteínas de Transporte Nucleocitoplasmático , RNA Viral , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA , RNA Viral/metabolismo
10.
Methods Mol Biol ; 2431: 49-69, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35412271

RESUMO

In neurons, specific mRNAs are transported into axons, where their local translation supports essential cellular functions. Over the years, our knowledge of the molecular mechanisms underlying axonal mRNA translation has rapidly expanded. However, tools to study mRNA localization and translation in real time with high spatial precision were not available until recently. Here, we present a live imaging approach to examine axonal mRNA trafficking and translation simultaneously in Xenopus retinal ganglion cells (RGCs), using in vitro synthesized fluorescently labeled mRNAs coupled with a genetically encoded protein tagging system to visualize synthesizing peptides at single-molecule resolution. We further describe the process of image analysis in detail, thus providing a methodology that can be used to investigate new research questions in the field.


Assuntos
Axônios , Transporte de RNA , Animais , Transporte Axonal/fisiologia , Axônios/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Ganglionares da Retina/metabolismo , Xenopus laevis/metabolismo
11.
Methods Mol Biol ; 2431: 225-237, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35412279

RESUMO

The use of fluorescent proteins has revolutionized the study of protein localization and transport. However, the visualization of other molecules and specifically RNA during live-cell imaging remains challenging. In this chapter, we provide guidance to the available methods, their advantages and drawbacks as well as provide a detailed protocol for the detection of RNA transport using the MS2/PP7-split-Venus system for background-free RNA imaging.


Assuntos
Neurônios , Transporte de RNA , Axônios/metabolismo , Neurônios/metabolismo , RNA/metabolismo , RNA Mensageiro/genética
12.
Methods Mol Biol ; 2502: 113-125, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35412235

RESUMO

Single molecule RNA fluorescence in situ hybridization (smRNA FISH) is a widely used method for examining cellular localization of RNA and assessing gene expression outputs. The Nuclear Pore Complex (NPC) is a nuclear macro-complex known to both mediate nucleocytoplasmic transport and influence transcription via interactions with chromatin. Consequently, depletion of NPC proteins can result in defects in either transcription or nuclear export of mRNA. To distinguish between these two different functions of NPC components, it is preferable to analyze transcription and mRNA export simultaneously or in the same cell. Here, we present a smRNA FISH protocol with downstream custom MATLAB image analysis for application in Drosophila larval salivary gland tissues. This method can detect both nuclear export and transcriptional phenotypes in the same cell and as a single assay, and can be adapted to many other cell types and organisms.


Assuntos
Transporte Ativo do Núcleo Celular , Drosophila , Hibridização in Situ Fluorescente , RNA , Imagem Individual de Molécula , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Drosophila/genética , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenótipo , RNA/metabolismo , Transporte de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Imagem Individual de Molécula/métodos
13.
Mol Cell Proteomics ; 21(3): 100208, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35091090

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 RNA
14.
RNA Biol ; 19(1): 206-220, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35067197

RESUMO

The majority of long noncoding RNAs (lncRNAs) contain transposable elements (TEs). PAHAL, a nuclear-retained lncRNA that is inserted by a Gypsy retrotransposon, has been shown to be a vital regulator of phenylalanine hydroxylase (PAH) gene expression that controls dopamine biosynthesis and behavioural aggregation in the migratory locust. However, the role of the Gypsy retrotransposon in the transcriptional regulation of PAHAL remains unknown. Here, we identified a Gypsy retrotransposon (named Gypsy element) as an inverted long terminal repeat located in the 3' end of PAHAL, representing a feature shared by many other lncRNAs in the locust genome. The embedded Gypsy element contains a RNA nuclear localization signal motif, which promotes the stable accumulation of PAHAL in the nucleus. The Gypsy element also provides high-affinity SRSF2 binding sites for PAHAL that induce the recruitment of SRSF2, resulting in the PAHAL-mediated transcriptional activation of PAH. Thus, our data demonstrate that TEs provide discrete functional domains for lncRNA organization and highlight the contribution of TEs to the regulatory significance of lncRNAs.


Assuntos
Regulação da Expressão Gênica , Gafanhotos/genética , RNA Longo não Codificante/genética , Retroelementos , Transcrição Genética , Animais , Linhagem Celular , Genoma , Genômica/métodos , Gafanhotos/metabolismo , Estabilidade de RNA , Transporte de RNA , Fatores de Processamento de Serina-Arginina/metabolismo
15.
Mol Cancer ; 21(1): 23, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-35042525

RESUMO

BACKGROUND: Functions of CircMET (hsa_circ_0082002) which is a circular RNA and derived from MET gene remain understood incompletely. In the present study, Xp11.2 translocation/NONO-TFE3 fusion renal cell carcinoma (NONO-TFE3 tRCC) with up-regulated CircMET was employed to investigate its mechanism in cancer progression and post-transcriptional regulation. METHODS: FISH and real-time PCR were performed to explore the expression and localization circMET in NONO-TFE3 tRCC tissues and cells. The functions of circMET in tRCC were investigated by proliferation analysis, EdU staining, colony and sphere formation assay. The regulatory mechanisms among circMET, CDKN2A and SMAD3 were investigated by luciferase assay, RNA immunoprecipitation, RNA pulldown and targeted RNA demethylation system. RESULTS: The expression of circMET was upregulated by NONO-TFE3 fusion in NONO-TFE3 tRCC tissues and cells, and overexpression of circMET significantly promoted the growth of NONO-TFE3 tRCC. Mechanistic studies revealed that circMET was delivered to cytosol by YTHDC1 in N6-methyladenosine (m6A)-depend manner. CircMET enhances mRNA decay of CDKN2A by direct interaction and recruitment of YTHDF2. Meanwhile, circMET competitively absorbed miR-1197 and prevented those from SMAD3 mRNA. CONCLUSIONS: CircMET promotes the development of NONO-TFE3 tRCC, and the regulation to both CDKN2A and SMAD3 of circMET was revealed. CircMET has the potential to serve as a novel target for the molecular therapy of NONO-TFE3 tRCC as well as the other cancer with high-expressing circMET.


Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-met/genética , Estabilidade de RNA , RNA Circular , Proteína Smad3/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proliferação de Células , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Xenoenxertos , Humanos , Metilação , Camundongos , Modelos Biológicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-met/metabolismo , Processamento Pós-Transcricional do RNA , Transporte de RNA , RNA Mensageiro/genética , Translocação Genética
16.
Nat Rev Genet ; 23(2): 73-88, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34545247

RESUMO

More than a century ago, August Weissman defined a distinction between the germline (responsible for propagating heritable information from generation to generation) and the perishable soma. A central motivation for this distinction was to argue against the inheritance of acquired characters, as the germline was partly defined by its protection from external conditions. However, recent decades have seen an explosion of studies documenting the intergenerational and transgenerational effects of environmental conditions, forcing a re-evaluation of how external signals are sensed by, or communicated to, the germline epigenome. Here, motivated by the centrality of small RNAs in paradigms of epigenetic inheritance, we review across species the myriad examples of intercellular RNA trafficking from nurse cells or somatic tissues to developing gametes.


Assuntos
Epigênese Genética/genética , Epigenômica , Regulação da Expressão Gênica/genética , Interação Gene-Ambiente , Células Germinativas/metabolismo , RNA/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Células Germinativas/citologia , Humanos , Modelos Genéticos , RNA/metabolismo , Transporte de RNA/genética
17.
Life Sci Alliance ; 5(2)2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34785537

RESUMO

The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.


Assuntos
DNA Intergênico/química , DNA Intergênico/genética , Quadruplex G , RNA/química , RNA/genética , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/genética , Regulação da Expressão Gênica , Humanos , Transporte de RNA , RNA Viral
18.
RNA ; 28(3): 433-446, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34949721

RESUMO

Detection of nucleic acids within subcellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics, which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization, and immunostaining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels that escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.


Assuntos
Imunofluorescência/métodos , Hibridização in Situ Fluorescente/métodos , RNA/metabolismo , Células Cultivadas , Fixadores/química , Corantes Fluorescentes/química , Células HeLa , Humanos , Nanopartículas/química , RNA/química , Processamento Pós-Transcricional do RNA , Transporte de RNA
19.
Cells ; 10(12)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34943863

RESUMO

In the last decade, an increasing number of studies have demonstrated that non-coding RNA (ncRNAs) cooperate in the gene regulatory networks with other biomolecules, including coding RNAs, DNAs and proteins. Among them, microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) are involved in transcriptional and translation regulation at different levels. Intriguingly, ncRNAs can be packed in vesicles, released in the extracellular space, and finally internalized by receiving cells, thus affecting gene expression also at distance. This review focuses on the mechanisms through which the ncRNAs can be selectively packaged into extracellular vesicles (EVs).


Assuntos
Vesículas Extracelulares/metabolismo , RNA/metabolismo , Animais , Humanos , Modelos Biológicos , Proteínas/metabolismo , RNA/genética , Transporte de RNA
20.
RNA Biol ; 18(sup2): 612-622, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34904931

RESUMO

Upregulation of utrophin, the autosomal homologue of dystrophin, can compensate dystrophin deficiency in Duchenne Muscular Dystrophy (DMD) although the therapeutic success is yet to be achieved. The present study has identified Poly (C) binding protein 2 (PCBP2) as a post-transcriptional suppresser for the expression of utrophin-A, the muscle-specific utrophin isoform. This study confirms nuclear retention of utrophin-A mRNA in C2C12 cells, which is mediated by PCBP2. Further investigation demonstrates PCBP2-dependent nuclear retention of follistatin mRNA as well. Its involvement in nuclear retention of mRNA sheds light on a novel function of PCBP2 that makes utrophin-A mRNA less available in cytosol. PCBP2, therefore, may be a target to de-repress utrophin-A expression in DMD.


Assuntos
Núcleo Celular/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Utrofina/genética , Regiões 5' não Traduzidas , Animais , Linhagem Celular , Núcleo Celular/genética , Camundongos , Imagem Molecular , Músculo Esquelético/metabolismo , Ligação Proteica , Processamento Pós-Transcricional do RNA , Transporte de RNA , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Utrofina/metabolismo
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