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1.
Immunity ; 51(1): 104-118.e7, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31128961

RESUMO

Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction and the development of specialized ILC subsets. We generated "5x polychromILC" transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, RORγt, and RORα) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified precursors with potential to generate all ILC subsets and natural killer (NK) cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single-cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1, ILC3, and NK precursor cell cluster. This diversity might facilitate greater lineage potential upon progenitor recruitment to peripheral tissues.


Assuntos
Medula Óssea/imunologia , Subpopulações de Linfócitos/fisiologia , Linfócitos/fisiologia , Células Progenitoras Linfoides/fisiologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Imunidade Inata , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Análise de Célula Única , Fatores de Transcrição/genética
2.
Immunity ; 48(6): 1195-1207.e6, 2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29907525

RESUMO

The local regulation of type 2 immunity relies on dialog between the epithelium and the innate and adaptive immune cells. Here we found that alarmin-induced expression of the co-stimulatory molecule OX40L on group 2 innate lymphoid cells (ILC2s) provided tissue-restricted T cell co-stimulation that was indispensable for Th2 and regulatory T (Treg) cell responses in the lung and adipose tissue. Interleukin (IL)-33 administration resulted in organ-specific surface expression of OX40L on ILC2s and the concomitant expansion of Th2 and Treg cells, which was abolished upon deletion of OX40L on ILC2s (Il7raCre/+Tnfsf4fl/fl mice). Moreover, Il7raCre/+Tnfsf4fl/fl mice failed to mount effective Th2 and Treg cell responses and corresponding adaptive type 2 pulmonary inflammation arising from Nippostrongylus brasiliensis infection or allergen exposure. Thus, the increased expression of OX40L in response to IL-33 acts as a licensing signal in the orchestration of tissue-specific adaptive type 2 immunity, without which this response fails to establish.


Assuntos
Imunidade Adaptativa/imunologia , Imunidade Inata/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T Reguladores/imunologia , Células Th2/imunologia , Fatores de Necrose Tumoral/imunologia , Animais , Diferenciação Celular/imunologia , Interleucina-33/imunologia , Ativação Linfocitária/imunologia , Linfócitos/imunologia , Camundongos , Ligante OX40
3.
Nucleic Acids Res ; 51(D1): D1003-D1009, 2023 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-36243972

RESUMO

The HUGO Gene Nomenclature Committee (HGNC) assigns unique symbols and names to human genes. The HGNC database (www.genenames.org) currently contains over 43 000 approved gene symbols, over 19 200 of which are assigned to protein-coding genes, 14 000 to pseudogenes and nearly 9000 to non-coding RNA genes. The public website, www.genenames.org, displays all approved nomenclature within Symbol Reports that contain data curated by HGNC nomenclature advisors and links to related genomic, clinical, and proteomic information. Here, we describe updates to our resource, including improvements to our search facility and new download features.


Assuntos
Bases de Dados Genéticas , Humanos , Genoma , Genômica , Proteômica , Pseudogenes , Terminologia como Assunto
4.
Hum Genomics ; 16(1): 58, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36380364

RESUMO

The HUGO Gene Nomenclature Committee (HGNC) has been providing standardized symbols and names for human genes since the late 1970s. As funding agencies change their priorities, finding financial support for critical biomedical resources such as the HGNC becomes ever more challenging. In this article, we outline the key roles the HGNC currently plays in aiding communication and the need for these activities to be maintained.


Assuntos
Bases de Dados Genéticas , Genômica , Humanos
5.
Genome Res ; 27(11): 1795-1806, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29030468

RESUMO

By profiling the transcriptomes of individual cells, single-cell RNA sequencing provides unparalleled resolution to study cellular heterogeneity. However, this comes at the cost of high technical noise, including cell-specific biases in capture efficiency and library generation. One strategy for removing these biases is to add a constant amount of spike-in RNA to each cell and to scale the observed expression values so that the coverage of spike-in transcripts is constant across cells. This approach has previously been criticized as its accuracy depends on the precise addition of spike-in RNA to each sample. Here, we perform mixture experiments using two different sets of spike-in RNA to quantify the variance in the amount of spike-in RNA added to each well in a plate-based protocol. We also obtain an upper bound on the variance due to differences in behavior between the two spike-in sets. We demonstrate that both factors are small contributors to the total technical variance and have only minor effects on downstream analyses, such as detection of highly variable genes and clustering. Our results suggest that scaling normalization using spike-in transcripts is reliable enough for routine use in single-cell RNA sequencing data analyses.


Assuntos
Análise de Sequência de RNA/normas , Análise de Célula Única/normas , Algoritmos , Animais , Linhagem Celular , Perfilação da Expressão Gênica/normas , Regulação da Expressão Gênica , Camundongos , Reprodutibilidade dos Testes
6.
RNA ; 17(8): 1551-65, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21705432

RESUMO

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with mitochondria in vivo. This supports earlier cell fractionation and microarray-based studies that proposed mMP association with the mitochondrial fraction. Interestingly, a number of mMPs showed a dependency on the mitochondrial Puf3 RNA-binding protein, as well as nonessential proteins of the translocase of the outer membrane (TOM) complex import machinery, for normal colocalization with mitochondria. We examined the specific determinants of ATP2 and OXA1 mRNA localization and found a mutual dependency on the 3' UTR, Puf3, Tom7, and Tom70, but not Tom20, for localization. Tom6 may facilitate the localization of specific mRNAs as OXA1, but not ATP2, mRNA was mislocalized in tom6Δ cells. Interestingly, a substantial fraction of OXA1 and ATP2 RNA granules colocalized with the endoplasmic reticulum (ER) and a deletion in MDM10, which mediates mitochondria-ER tethering, resulted in a significant loss of OXA1 mRNA localization with ER. Finally, neither ATP2 nor OXA1 mRNA targeting was affected by a block in translation initiation, indicating that translation may not be essential for mRNA anchoring. Thus, endogenously expressed mRNAs are targeted to the mitochondria in vivo, and multiple factors contribute to mMP localization.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Nucleares/metabolismo , ATPases Translocadoras de Prótons/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico , Complexo IV da Cadeia de Transporte de Elétrons/genética , Proteínas Mitocondriais/genética , Mutação , Proteínas Nucleares/genética , Biossíntese de Proteínas , ATPases Translocadoras de Prótons/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
7.
RNA ; 17(12): 2249-55, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22025736

RESUMO

Protein localization within cells can be achieved by the targeting and localized translation of mRNA. Yet, our understanding of the dynamics of mRNA targeting and protein localization, and of how general this phenomenon is, is not clear. Plasmid-based expression systems have been used to visualize exogenously expressed mRNAs and proteins; however, these methods typically produce them at levels greater than endogenous and can result in mislocalization. Hence, a method that allows for the simultaneous visualization of endogenous mRNAs and their translation products in living cells is needed. We previously developed a method (m-TAG) to localize endogenously expressed mRNAs in yeast by chromosomal insertion of the MS2 aptamer sequence between the open-reading frame (ORF) and 3' UTR of any gene. Upon coexpression with the MS2 RNA-binding coat protein (MS2-CP) fused with GFP, the aptamer-tagged mRNAs bearing their 3' UTRs are localized using fluorescence microscopy. Here we describe an advanced method (mp-TAG) that allows for the simultaneous visualization of both endogenously expressed mRNAs and their translation products in living yeast for the first time. Homologous recombination is used to insert the mCherry gene and MS2-CP binding sites downstream from any ORF, in order to localize protein and mRNA, respectively. As proof of the concept, we tagged ATP2 as a representative gene and demonstrated that endogenous ATP2 mRNA and protein localize to mitochondria, as shown previously. In addition, we demonstrate that tagged proteins like Hhf2, Vph1, and Yef3 localize to their expected subcellular location, while the localization of their mRNAs is revealed for the first time.


Assuntos
Proteínas Fúngicas/análise , Marcação de Genes/métodos , RNA Mensageiro/análise , Leveduras/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Histonas/genética , Histonas/metabolismo , Recombinação Homóloga , Espaço Intracelular/genética , Espaço Intracelular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Biossíntese de Proteínas , Transporte Proteico , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Leveduras/metabolismo
8.
Proc Natl Acad Sci U S A ; 106(47): 19848-53, 2009 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-19903887

RESUMO

Targeted mRNA trafficking and local translation may play a significant role in controlling protein localization. Here we examined for the first time the localization of all ( approximately 50) mRNAs encoding peroxisomal proteins (mPPs) involved in peroxisome biogenesis and function. By using the bacteriophage MS2-CP RNA-binding protein (RBP) fused to multiple copies of GFP, we demonstrated that >40 endogenously expressed mPPs tagged with the MS2 aptamer form fluorescent RNA granules in vivo. The use of different RFP-tagged organellar markers revealed 3 basic patterns of mPP granule localization: to peroxisomes, to the endoplasmic reticulum (ER), and nonperoxisomal. Twelve mPPs (i.e., PEX1, PEX5, PEX8, PEX11-15, DCI1, NPY1, PCS60, and POX1) had a high percentage (52%-80%) of mRNA colocalization with peroxisomes. Thirteen mPPs (i.e., AAT2, PEX6, MDH3, PEX28, etc.) showed a low percentage (30%-42%) of colocalization, and 1 mPP (PEX3) preferentially localized to the ER. The mPPs of the nonperoxisomal pattern (i.e., GPD1, PCD1, PEX7) showed <<30% colocalization. mPP association with the peroxisome or ER was verified using cell fractionation and RT-PCR analysis. A model mPP, PEX14 mRNA, was found to be in close association with peroxisomes throughout the cell cycle, with its localization depending in part on the 3'-UTR, initiation of translation, and the Puf5 RBP. The different patterns of mPP localization observed suggest that multiple mechanisms involved in mRNA localization and translation may play roles in the importation of protein into peroxisomes.


Assuntos
Peroxissomos , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ciclo Celular/fisiologia , Retículo Endoplasmático/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Peroxinas , Peroxissomos/química , Peroxissomos/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
PLoS One ; 16(5): e0251233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34003838

RESUMO

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Macrófagos/imunologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Pneumonia/imunologia , Células Th2/imunologia , Animais , Antígenos de Helmintos/imunologia , Antígenos de Helmintos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Nippostrongylus/imunologia , Pneumonia/parasitologia , Pneumonia/patologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia
10.
Methods Mol Biol ; 1979: 177-183, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028638

RESUMO

Examining transcriptomics of populations at the single-cell level allows for higher resolution when studying functionality in development, differentiation, and physiology. Real-time quantitative PCR (qPCR) enables a sensitive detection of specific gene expression; however, processing a large number of samples for single-cell research involves a time-consuming process and high reagent costs. Here we describe a protocol for single-cell qPCR using nanofluidic chips. This method reduces the number of handling steps and volumes per reaction, allowing for more samples and genes to be measured.


Assuntos
Reação em Cadeia da Polimerase em Tempo Real/métodos , Análise de Célula Única/métodos , Animais , Desenho de Equipamento , Citometria de Fluxo/métodos , Perfilação da Expressão Gênica/economia , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Humanos , Dispositivos Lab-On-A-Chip , Reação em Cadeia da Polimerase em Tempo Real/economia , Reação em Cadeia da Polimerase em Tempo Real/instrumentação , Transcrição Reversa , Análise de Célula Única/economia , Análise de Célula Única/instrumentação
11.
Nat Struct Mol Biol ; 25(3): 279-288, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29434345

RESUMO

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.


Assuntos
Complexos Multiproteicos/química , Biossíntese de Proteínas , Multimerização Proteica , Subunidades Proteicas/biossíntese , Evolução Molecular , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Domínios Proteicos , Engenharia de Proteínas , Dobramento de Proteína , Subunidades Proteicas/química , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Solubilidade
12.
Genome Biol ; 17: 103, 2016 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-27176874

RESUMO

BACKGROUND: Differentiation of lymphocytes is frequently accompanied by cell cycle changes, interplay that is of central importance for immunity but is still incompletely understood. Here, we interrogate and quantitatively model how proliferation is linked to differentiation in CD4+ T cells. RESULTS: We perform ex vivo single-cell RNA-sequencing of CD4+ T cells during a mouse model of infection that elicits a type 2 immune response and infer that the differentiated, cytokine-producing cells cycle faster than early activated precursor cells. To dissect this phenomenon quantitatively, we determine expression profiles across consecutive generations of differentiated and undifferentiated cells during Th2 polarization in vitro. We predict three discrete cell states, which we verify by single-cell quantitative PCR. Based on these three states, we extract rates of death, division and differentiation with a branching state Markov model to describe the cell population dynamics. From this multi-scale modelling, we infer a significant acceleration in proliferation from the intermediate activated cell state to the mature cytokine-secreting effector state. We confirm this acceleration both by live imaging of single Th2 cells and in an ex vivo Th1 malaria model by single-cell RNA-sequencing. CONCLUSION: The link between cytokine secretion and proliferation rate holds both in Th1 and Th2 cells in vivo and in vitro, indicating that this is likely a general phenomenon in adaptive immunity.


Assuntos
Linfócitos T CD4-Positivos/citologia , Diferenciação Celular , Proliferação de Células , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Feminino , Malária/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transcriptoma
13.
Cell Rep ; 7(4): 1130-42, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24813893

RESUMO

T helper 2 (Th2) cells regulate helminth infections, allergic disorders, tumor immunity, and pregnancy by secreting various cytokines. It is likely that there are undiscovered Th2 signaling molecules. Although steroids are known to be immunoregulators, de novo steroid production from immune cells has not been previously characterized. Here, we demonstrate production of the steroid pregnenolone by Th2 cells in vitro and in vivo in a helminth infection model. Single-cell RNA sequencing and quantitative PCR analysis suggest that pregnenolone synthesis in Th2 cells is related to immunosuppression. In support of this, we show that pregnenolone inhibits Th cell proliferation and B cell immunoglobulin class switching. We also show that steroidogenic Th2 cells inhibit Th cell proliferation in a Cyp11a1 enzyme-dependent manner. We propose pregnenolone as a "lymphosteroid," a steroid produced by lymphocytes. We speculate that this de novo steroid production may be an intrinsic phenomenon of Th2-mediated immune responses to actively restore immune homeostasis.


Assuntos
Pregnenolona/biossíntese , RNA/metabolismo , Células Th1/imunologia , Células Th2/imunologia , Animais , Homeostase/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pregnenolona/genética , Pregnenolona/imunologia , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Células Th1/metabolismo , Células Th2/metabolismo , Transcriptoma
14.
Mol Biol Cell ; 24(19): 3069-84, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23904265

RESUMO

mRNAs encoding secreted/membrane proteins (mSMPs) are believed to reach the endoplasmic reticulum (ER) in a translation-dependent manner to confer protein translocation. Evidence exists, however, for translation- and signal recognition particle (SRP)-independent mRNA localization to the ER, suggesting that there are alternate paths for RNA delivery. We localized endogenously expressed mSMPs in yeast using an aptamer-based RNA-tagging procedure and fluorescence microscopy. Unlike mRNAs encoding polarity and secretion factors that colocalize with cortical ER at the bud tip, mSMPs and mRNAs encoding soluble, nonsecreted, nonpolarized proteins localized mainly to ER peripheral to the nucleus (nER). Synthetic nontranslatable uracil-rich mRNAs were also demonstrated to colocalize with nER in yeast. This mRNA-ER association was verified by subcellular fractionation and reverse transcription-PCR, single-molecule fluorescence in situ hybridization, and was not inhibited upon SRP inactivation. To better understand mSMP targeting, we examined aptamer-tagged USE1, which encodes a tail-anchored membrane protein, and SUC2, which encodes a soluble secreted enzyme. USE1 and SUC2 mRNA targeting was not abolished by the inhibition of translation or removal of elements involved in translational control. Overall we show that mSMP targeting to the ER is both translation- and SRP-independent, and regulated by cis elements contained within the message and trans-acting RNA-binding proteins (e.g., She2, Puf2).


Assuntos
Retículo Endoplasmático/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplasmático/ultraestrutura , Microscopia de Fluorescência , Transporte Proteico , Proteínas Qc-SNARE/metabolismo , RNA Mensageiro/ultraestrutura , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , beta-Frutofuranosidase/metabolismo
15.
Methods Mol Biol ; 714: 237-47, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431745

RESUMO

Localized mRNA translation is involved in cell-fate determination, polarization, and morphogenesis in eukaryotes. While various tools are available to examine mRNA localization, no easy and quick method has allowed for the visualization of endogenously expressed mRNAs in vivo. We describe a simple method (m-TAG) for PCR-based chromosomal gene tagging that uses homologous recombination to insert binding sites for the RNA-binding MS2 coat protein (MS2-CP) between the coding region and 3'-untranslated region of any yeast gene. Upon co-expression of MS2-CP fused with GFP, specific endogenously expressed mRNAs can be visualized in vivo for the first time. This method allows for the easy examination of mRNA localization using fluorescence microscopy and leaves the yeast cells amenable for further genetic analysis.


Assuntos
Aptâmeros de Nucleotídeos/genética , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Sondas Moleculares/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Saccharomyces cerevisiae/metabolismo , Células Imobilizadas/metabolismo , Precipitação Química , DNA/genética , DNA/isolamento & purificação , Integrases/metabolismo , Sondas Moleculares/genética , RNA Fúngico/análise , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Transformação Genética
17.
Nat Protoc ; 4(9): 1274-84, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19680241

RESUMO

This protocol describes m-TAG, a novel method for the visualization of endogenously expressed mRNAs in live yeast (Saccharomyces cerevisiae). First, a gene of interest is tagged with multiple binding sites for the RNA-binding MS2 coat protein (MS2-CP), using a PCR-based genomic-tagging strategy and homologous recombination. Next, MS2-CP fused to GFP(x3) is expressed in cells; binding of this fusion protein to the tagged mRNA yields an RNA granule that can be visualized by fluorescence microscopy. While existing methods necessitate cell fixation (for in situ hybridization) or the detection of exogenously expressed mRNAs (from plasmids), or give transient signals (i.e., with fluorescent hybridization probes), m-TAG allows for the robust and stable visualization of endogenously expressed mRNAs in vivo and facilitates the study of mRNA dynamics under different growth conditions. The m-TAG procedure is simple, easy to perform and takes <3 weeks to yield cultured yeast strains for mRNA visualization.


Assuntos
Reação em Cadeia da Polimerase/métodos , RNA Fúngico/análise , RNA Mensageiro/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Genoma Fúngico , RNA Fúngico/genética , RNA Mensageiro/genética
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