Name: utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes
Uploaded: 2020-11-09T15:45:00.000+00:00
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Description: Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Este trabalho foi apoiado por uma bolsa da FAPESP (15/06477-9).

1. Preparação total do extrato de levedura
<ol><li>Cresça as células de levedura expressando um dos fatores de emenda fundidos à tag TAP9 em 1 L YNB-glu (Base de Nitrogênio levedura suplementada com 2% m/v de glicose) com os aminoácidos apropriados ou bases nucleicas, neste caso, adenina (20 μg/mL), leucina (30 μg/mL), triptofano (30 μg/mL), até OD600 = 1,0.</li>
	<li>Colete as células da cultura 1 L por centrifugação em três garrafas centrífugas de centrífugas de 500 mL a 17.000 x g por 10 min a 4 °C e lave duas vezes com água estéril fria de 10 mL.</li>
	<li>Resuspenha as células de levedura coletadas em 1/10 do volume celular do tampão frio A (10 mmol L-1 HEPES pH = 7,9, 1,5 mmol L-1 MgCl2, 50 mmol L-1 KCl, 5% v/v glicerol, 0,5 mmol L-1 DTT, Coquetel Inibidor de Protease sem EDTA).</li>
	<li>Congele pequenas gotas de suspensão celular em nitrogênio líquido. As pequenas gotas podem ser obtidas com a pipetação da solução celular resuspended e a queda de 50 μL diretamente em nitrogênio líquido. ATENÇÃO: O nitrogênio líquido pode causar lesões em contato com a pele ou os olhos. Manuseie usando equipamentos de segurança apropriados. NOTA: O protocolo pode ser pausado aqui. As gotas congeladas de suspensão celular podem ser armazenadas a -80 °C.</li>
	<li>Prepare as células de levedura lysates moendo em um dispositivo Ball Mill através de seis ciclos a 20 Hz/s por 3 min. NOTA: Após cada ciclo, mergulhe o recipiente que abriga as células congeladas em nitrogênio líquido para evitar o derretimento. O protocolo pode ser pausado aqui. Os extratos congelados podem ser armazenados a -80 °C. Extratos de emenda à levedura também podem ser preparados usando homogeneizadores para liseste de esferoplastos10, ou usando argamassa e pilão11,12.</li>
	<li>Derreta o extrato colocando os tubos que os contêm na água à temperatura ambiente, tremendo ocasionalmente.</li>
	<li>Extratos de centrífuga a 45.000 x g por 1h a 4 °C.</li>
	<li>Quantifique o teor proteico do supernanato limpo pelo método BCA13.</li>
	<li>Prepare alíquotas dos extratos, congele-os rapidamente em nitrogênio líquido e armazene a -80 °C. NOTA: O protocolo pode ser pausado aqui.</li>
</ol>2. Preparação do gradiente de glicerol
<ol><li>Prepare duas soluções de glicerol no Buffer A, uma contendo 10% v/v e outra contendo 30% v/v de glicerol.</li>
	<li>Adicione o glutaraldeído do agente de crosslinking a 0,1% v/v na solução de glicerol v/v de 30% v/v e misture para homogeneizar. ATENÇÃO: Manuseie o glutaraldeído dentro do capô da fumaça usando equipamentos de segurança adequados.</li>
	<li>Adicione 6 mL da solução de glicerol de 10% v/v no fundo do tubo centrífuga de 12 mL (14 x 89 mm).</li>
	<li>Adicione 6 mL da solução de glicerol de 30% v/v a frio complementada com glutaraldeído com uma seringa presa a uma agulha longa fornecida com o dispositivo Gradiente Mestre na parte inferior do tubo, logo abaixo da solução de glicerol v/v de 10%. NOTA: Alternativamente, a solução de glicerol de 10% v/v pode ser cuidadosamente encarsa na parte superior da solução de glicerol/glutaraldeído de 30% v/v.</li>
	<li>Use o dispositivo Gradiente Mestre para gerar um gradiente de densidade contínua. NOTA: No dispositivo Gradiente Mestre, os tubos são colocados em um rack apropriado e girados brevemente, seguindo as recomendações do fabricante para determinar os parâmetros (tempo/ângulo/velocidade). Neste trabalho foram utilizados: 2:25 min/81.5°/11 rpm.</li>
	<li>Adicione cuidadosamente 200 μL de uma almofada de 7% v/v glicerol/tampão A na parte superior pouco antes da adição dos extratos celulares. NOTA: A concentração de glicerol da almofada deve ser inferior à solução menos concentrada de glicerol usada para criar o gradiente linear.</li>
</ol>3. Extrai centrifugação
<ol><li>Carregue aproximadamente 2 mg de proteína total no topo de cada 12 mL de gradiente linear de glicerol 10%-30% glutaraldeído com almofada.</li>
	<li>Coloque os tubos em um rotor pré-resfriado. NOTA: Manuseie os tubos suavemente para evitar a mistura antes da ultracentrifugação.</li>
	<li>Centrifugar a 194.000 x g para 16 h a 4 °C.</li>
	<li>Alíquota de 12 mL em 24 frações de 500 μL usando um EconoSystem adaptado ou por pipetação cuidadosa. NOTA: Um EconoSystem adaptado consiste em uma bomba peristáltica, o detector UV e o coletor de frações conectado ao dispositivo perfurante do tubo. O perfil de sedimentação pode ser monitorado pela medição da absorvância a 280 nm.</li>
	<li>Use uma solução de glicerol de 40% v/v através da bomba peristáltica para empurrar o glicerol de 10%-30% de gradiente do tubo para o coletor de frações. NOTA: O protocolo pode ser pausado aqui. Armazene as frações a -80 °C até o uso.</li>
	<li>Carregue 50 μL de cada fração diretamente em membranas de nitrocelulose em um dispositivo de mancha de ponto ou entalhe. Detecte proteína por imunoblot usando anticorpo contra CBP (1:6.000, para detectar a porção CBP da tag TAP) e anti-coelho IgG conjugado com Peroxidase de Rabanete (1:15.000) como o anticorpo secundário.</li>
</ol>

<ol>
	<li>Kastner, B., Will, C. L., Stark, H., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+nuclear+pre-mRNA+splicing+in+higher+eukaryotes.">Structural insights into nuclear pre-mRNA splicing in higher eukaryotes.</a> Cold Spring Harbor Perspectives in Biology. 11 (11), 032417 (2019).</li><li>Yan, C., Wan, R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+pre-mRNA+splicing+through+structural+biology+of+the+spliceosome.">Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome.</a> Cold Spring Harbor Perspectives in Biology. 11 (1), 032409 (2019).</li><li>Cordin, O., Hahn, D., Beggs, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+regulation+of+spliceosomal+RNA+helicases.">Structure, function and regulation of spliceosomal RNA helicases.</a> Current Opinion in Cell Biology. 24, 431-438 (2012).</li><li>Kastner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GraFix:+sample+preparation+for+single-particle+electron+cryomicroscopy.">GraFix: sample preparation for single-particle electron cryomicroscopy.</a> Nature Methods. 5 (1), 53-55 (2008).</li><li>Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafix:+stabilization+of+fragile+macromolecular+complexes+for+single+particle+cryo-EM.">Grafix: stabilization of fragile macromolecular complexes for single particle cryo-EM.</a> Methods in Enzymology. 481, 109-126 (2010).</li><li>Yan, C., Wan, R., Bai, R., Huang, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+yeast+activated+spliceosome+at+3.5+&#197;+resolution.">Structure of a yeast activated spliceosome at 3.5 &#197; resolution.</a> Science. 353 (6302), 895-904 (2016).</li><li>Ohi, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+analysis+revelas+stable+multiprotein+complexes+in+both+fission+and+budding+yeasts+containing+Myb-related+Cdc5p/Cef1p,+novel+pre-mRNA+splicing+factors,+and+snRNAs.">Proteomics analysis revelas stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.</a> Molecular and Cellular Biology. 22 (7), 2011-2024 (2002).</li><li>Lourenco, R. F., Leme, A. F. P., Oliveira, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+yeast+mutant+RNA+exosome+complexes.">Proteomic analysis of yeast mutant RNA exosome complexes.</a> Journal of Proteome Research. 12 (12), 5912-5922 (2013).</li><li>Rigaut, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterizatioin+and+proteome+exploration.">A generic protein purification method for protein complex characterizatioin and proteome exploration.</a> Nature Biotechnology. 17, 1030-1032 (1999).</li><li>Cheng, S. C., Newman, A., Lin, R. J., McFarland, G. D., Abelson, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+fractionation+of+yeast+splicing+extract.">Preparation and fractionation of yeast splicing extract.</a> Methods in Enzymology. 181, 89-96 (1990).</li><li>Umen, J. G., Guthrie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+a+U5+snRNP+protein+in+3'+splice+site+selection.">A novel role for a U5 snRNP protein in 3' splice site selection.</a> Genes &amp; Development. 9, 855-868 (1995).</li><li>Dunn, E. A., Rader, S. D., Hertel, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+yeast+whole+cell+splicing+extract.">Preparation of yeast whole cell splicing extract.</a> Spliceosomal Pre-mRNA Splicing: Methods and Protocols. Methods in Molecular Biology. 1126, 123-135 (2014).</li><li>Hill, H. D., Straka, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+determination+using+bicinchoninic+acid+in+the+presence+of+sulfhydryl+reagents.">Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents.</a> Analalytical Biochemistry. 170 (1), 203-208 (1988).</li><li>Cepeda, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+assembly+factor+Nop53+controls+association+of+the+RNA+exosome+with+pre-60S+particles+in+yeast.">The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.</a> The Journal of Biological Chemistry. 294 (50), 19365-19380 (2019).</li><li>Lin, R. J., Newman, A. J., Cheng, S. C., Abelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mRNA+splicing+in+vitro.">Yeast mRNA splicing in vitro.</a> The Journal of Biological Chemistry. 260 (27), 14780-14792 (1985).</li></ol>

Os autores não têm conflitos de interesse.

Interações proteína-proteína e ácidos ribonucleicos-proteínas podem ser estabilizadas usando agentes de crosslinking. É importante que o complexo resultante seja estável para suportar a ultracentrifugação no gradiente de glicerol. Além disso, as condições do buffer devem permitir a interação, mas ser rigorosas o suficiente para evitar ligações não específicas. Nos experimentos aqui mostrados, utilizamos uma solução tampão já estabelecida para reações in vitro de emenda15.<...

O splicing é um processo altamente dinâmico que requer a vinculação e liberação de uma infinidade de fatores de forma coordenada. Esses fatores de emenda incluem proteínas de ligação de RNA, ATPases, helicases, quinases de proteínas e fosfattases, ligaduras de ubiquitina, entre outros1,2,3; e para permitir que os rearranjos moleculares ocorram, alguns desses fatores se ligam muito transitoriamente aos subcoplexos de emenda, tornando muito desafiador o isolamento e identificação desses complexos intermediários RNP.
Aqui, utilizamos o método Grafix4,5 para estabilizar a interação do fator de emenda da levedura Cwc24 com o complexo deato B6 para permitir a identificação de outros fatores ligados concomitantemente a esse subcoplexo e determinar se a ligase ubiquitina Prp19 desempenha algum papel na vinculação ou liberação do complexo Cwc24 para o Complexo de Dezenove (NTC) e até o final de 5' do intron antes da ativação e a primeira reação de transesterificação leva lugar. A vantagem de expor as macromoléculas a uma concentração crescente do crosslinker ao longo do gradiente de glicerol é que ele evita inter-complexos crosslinks4,5, e, portanto, a formação de agregados.
Este método foi utilizado como complementação para a co-doença da proteína e ensaios puxados para baixo, que, apesar de permitir o isolamento de grandes complexos, pode não ser confiável para manter interações transitórias dentro de grandes complexos dinâmicos7,8. O uso de reagentes de fixação no gradiente de glicerol estabiliza a vinculação de tais fatores, permitindo a confirmação de interações de proteínas específicas com subcomplexos de emenda. Como o crosslinker escolhido era quimicamente irreversível, as proteínas presentes nas frações recuperadas foram analisadas por mancha de ponto após a centrifugação gradiente.

<table><tbody><tr><td>Anti-Calmodulin Binding Protein Epitope</td><td>Millipore</td><td>07-482</td><td></td></tr><tr><td>ECL anti-Rabbit IgG</td><td>GE Healthcare</td><td>NA934</td><td></td></tr><tr><td>EconoSystem</td><td>Bio-Rad</td><td>1-800-424-6723</td><td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td></tr><tr><td>EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Fraction Recovery System</td><td>Beckman Coulter</td><td>270-331580</td><td>Tube-perforating device that was connected to the parts of the EconoSystem</td></tr><tr><td>Gradient Master Model 107ip</td><td>Biocomp</td><td>107-201M</td><td></td></tr><tr><td>Mixer Mill MM 200</td><td>Retsch</td><td>207460001</td><td>Ball Mill device</td></tr><tr><td>Rotor F12-6x500Lex</td><td>Thermo Scientific</td><td>096-062375</td><td></td></tr><tr><td>Sorvall RC 6 Plus Centrifuge</td><td>Thermo Scientific</td><td>36-101-0816</td><td></td></tr><tr><td>Swinging Bucket Rotor P40ST</td><td>Hitachi</td><td></td><td></td></tr><tr><td>Ultracentrifuge CP 80 NX</td><td>Hitachi</td><td>901069</td><td></td></tr><tr><td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td><td>Beckman Coulter</td><td>344059</td><td></td></tr></tbody></table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

O emendamento pré-mRNA é um processo muito dinâmico que envolve muitos rearranjos moleculares dos subcoplexos de emenda durante a montagem, processamento de RNA e liberação dos componentes complexos. A centrifugação de gradiente de glicerol tem sido usada para a separação de complexos de proteínas ou RNP (RiboNucleoProtein) para estudos funcionais e estruturais. Aqui, descrevemos a utilização do Grafix (Gradient Fixation), que foi desenvolvido pela primeira vez para purificar e estabilizar complexos macromoleculares para microscopia crio-elétron de partículas únicas, para identificar interações entre fatores de emenda que se ligam transitoriamente ao complexo de emendas. Este método baseia-se na centrifugação das amostras em uma concentração crescente de um reagente de fixação para estabilizar complexos. Após a centrifugação de extratos totais de levedura carregados em gradientes de glicerol, as frações recuperadas são analisadas por mancha de ponto para a identificação dos sub complexidades e determinação da presença de fatores de emenda individuais.

Para analisar o perfil de sedimentação do Cwc24-TAP e determinar se o método Grafix foi eficaz para estabilizar sua vinculação a subcomplexos de emenda, separamos extratos totais de leveduras de células expressando Cwc24-TAP através da centrifugação em gradientes de glicerol, na presença ou ausência de glutaraldeído como agente de ligação cruzada. Amostras de 24 frações de 500 μL foram então analisadas por mancha de slot com anticorpo contra a porção CBP da tag TAP. Os resultados mostram que, na ausê...

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Aqui, descrevemos a utilização do Grafix (Gradient Fixation), uma centrifugação de gradiente de glicerol na presença de um crosslinker, para identificar interações entre fatores de emenda que se ligam transitoriamente ao complexo de emendas.

Utilização de Grafix para a detecção de Interagentes Transintes de Saccharomyces cerevisiae Subcomplexos Spliceosome

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

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Universidad San Sebastian

Research

JoVE Journal

Biochemistry

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

3.5K visualizações.  University of Sao Paulo. Aqui, descrevemos a utilização do Grafix (Gradient Fixation), uma centrifugação de gradiente de glicerol na presença de um crosslinker, para identificar interações entre fatores de emenda que se ligam transitoriamente ao complexo de emendas.

Vídeo: Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Única molécula fluorescência In Situ Hybridization (smFISH) análise em Budding fermento crescimento vegetativo e meiose

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Genética

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

Uma tela de supressor de Deep-sequenciamento-assistida, espontânea na fissão levedura Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Identificação de complexos de proteínas com proteômica quantitativa em S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biologia

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

Ensaios de ACT1-CUP1 determinam as sensibilidades específicas do substrato de mutantes spliceossomais em leveduras em brotamento

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

Detecção de Intermediários de Recombinação Homóloga via Ligadura de Proximidade e PCR Quantitativa em Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

Complementação da Atividade de Emenda por um Complexo Galectin-3 - U1 snRNP on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

Bioquímica

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

Identificação rápida de químicos Genéticos Interações em<em&gt; Saccharomyces cerevisiae</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

Correção rápida in Vivo e isolamento de complexos translacionais de células eucarióticas

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

Identificação Global de Redes de Interação Co-Translacional por Perfil Seletivo Ribosome

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

Utilização de Grafix para a detecção de Interagentes Transintes de Saccharomyces cerevisiae Subcomplexos Spliceosome

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