Name: genome-wide quantification of translation in budding yeast by ribosome profiling
Uploaded: 2017-12-21T14:00:00
Description: Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

Dette arbejde blev støttet af de nationale kontorer i sundhed tilskud AG040191 og AG054566 til VML. Denne forskning blev udført mens VML var en AFAR forskning tilskud modtager fra den amerikanske sammenslutning for aldring forskning.

1. Uddrag forberedelse
<ol>
	<li>Streak gærstammer fra frosne bestande for enkelt kolonier på YPD plader (1% gærekstrakt, 2% pepton, 2% glukose og 2% agar). Inkuber plader ved 30 ° C i 2 dage.</li>
	<li>Podes gær fra en YPD tallerken (brug en enkelt koloni) til 15 mL YPD medium (1% gærekstrakt, 2% pepton, 2% glukose) i en 50 mL konisk centrifugeglas og vokse natten under omrystning (200-250 rpm) ved 30 ° C.</li>
	<li>Fortynd kultur i 500 mL af YPD medium i en 2 L sterile kolbe, således at OD600 &#60;...

<ol>
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Y., 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+use+of+duplex-specific+nuclease+in+ribosome+profiling+and+a+user-friendly+software+package+for+Ribo-seq+data+analysis.">The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.</a> RNA. 21 (10), 1731-1745 (2015).</li><li>Popa, A., 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=RiboProfiling:+a+Bioconductor+package+for+standard+Ribo-seq+pipeline+processing.">RiboProfiling: a Bioconductor package for standard Ribo-seq pipeline processing.</a> F1000Res. 5, 1309 (2016).</li><li>Dunn, J. G., Weissman, J. 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R., Green, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dom34+rescues+ribosomes+in+3'+untranslated+regions.">Dom34 rescues ribosomes in 3' untranslated regions.</a> Cell. 156 (5), 950-962 (2014).</li><li>Ingolia, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribosome+profiling:+new+views+of+translation,+from+single+codons+to+genome+scale.">Ribosome profiling: new views of translation, from single codons to genome scale.</a> Nat Rev Genet. 15 (3), 205-213 (2014).</li><li>Gerashchenko, M. V., Lobanov, A. V., Gladyshev, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+ribosome+profiling+reveals+complex+translational+regulation+in+response+to+oxidative+stress.">Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress.</a> Proc Natl Acad Sci U S A. 109 (43), 17394-17399 (2012).</li><li>Weinberg, D. E., 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=Improved+ribosome-footprint+and+mRNA+measurements+provide+insights+into+dynamics+and+regulation+of+yeast+translation.">Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation.</a> Cell Rep. 14 (7), 1787-1799 (2016).</li><li>Park, J. E., Yi, H., Kim, Y., Chang, H., Kim, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+poly(A)+tail+and+translation+during+the+somatic+cell+cycle.">Regulation of poly(A) tail and translation during the somatic cell cycle.</a> Mol Cell. 62 (3), 462-471 (2016).</li><li>Howard, M. T., Carlson, B. A., Anderson, C. B., Hatfield, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+redefinition+of+UGA+codons+is+regulated+by+selenium+availability.">Translational redefinition of UGA codons is regulated by selenium availability.</a> J Biol Chem. 288 (27), 19401-19413 (2013).</li><li>Gerashchenko, M. V., Gladyshev, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonuclease+selection+for+ribosome+profiling.">Ribonuclease selection for ribosome profiling.</a> Nucleic Acids Res. 45 (2), e6 (2017).</li><li>Gerashchenko, M. V., Gladyshev, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+inhibitors+cause+abnormalities+in+ribosome+profiling+experiments.">Translation inhibitors cause abnormalities in ribosome profiling experiments.</a> Nucleic Acids Res. 42 (17), e134 (2014).</li><li>O'Connor, P. B., Andreev, D. E., Baranov, P. 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Ribo-Seq tilgang har vist sig som en kraftfuld teknologi til analyse af mRNA oversættelse i vivo på genome-wide niveau 3. Undersøgelser ved hjælp af denne fremgangsmåde, der tillader overvågning oversættelse med single-codon opløsning, har bidraget til forståelsen af translationel forordning. Trods sine fordele har Ribo-Seq flere begrænsninger. Ribosomale RNA (rRNA) fragmenter er altid Co renset under isolation af ribosomet-beskyttet fodspor faldende udbytte af nyttige sekventer...

mRNA oversættelse er en af de grundlæggende processer i cellen, som spiller en vigtig rolle i reguleringen af protein udtryk. Derfor, mRNA oversættelse er stramt kontrolleret som svar på forskellige interne og eksterne fysiologiske stimuli 1,2. Mekanismer for Translationel forordning er dog stadig forsømt. Her beskriver vi protokol for genome-wide kvantificering af oversættelse i spirende gær af ribosomet profilering. Det overordnede mål med ribosomet profilering teknik er at studere og kvantificere oversættelse af specifikke mRNAs forskellige cellulære betingelser. Denne teknik bruger next generation sequencing kvantitativt analysere ribosomet belægning i hele genomet og giver mulighed for overvågning sats af protein syntese i vivo på enkelt codon opløsning 3,4. I øjeblikket, denne metode giver den mest avancerede metode til at måle niveauet af protein oversættelse, og har vist sig for at være en nyttig opdagelse værktøj giver oplysninger, der ikke kan blive afsløret af andre i øjeblikket tilgængelige teknik, fx microarrays eller oversættelse tilstand array analyse (TSAA) 5. Som ribosomet profilering rapporter om de samlede ændringer i udskrift niveauer og translationel output, giver det også meget større følsomhed i forhold til andre metoder.
Denne tilgang er baseret på dyb sekventering af ribosomet-beskyttet mRNA fragmenter 3. Under protein oversættelse, ribosomer beskytte ~ 28 nt dele af mRNA (kaldet fodspor) 6. Ved at bestemme rækkefølgen af ribosomet-beskyttet fragmenter, kan Ribo-Seq kortlægge placeringen af ribosomer på den oversatte mRNA og identificere hvilke regioner af mRNA er tilbøjelige til at være aktivt oversat til protein 3,7. Desuden måler vi kvantitativt oversættelse af mRNA ved at tælle antallet af fodspor, der justeres i forhold til en given mRNA udskrift.
For at isolere de ribosom-beskyttet fragmenter, behandles cellelysater i første omgang med en oversættelse hæmmer til stall ribosomer efterfulgt af ribonuklease fordøjelsen. Der henviser til, at frie mRNA og dele af oversatte mRNAs ikke beskyttet af ribosomes er nedbrudt af ribonuklease, kan ribosomet-beskyttet mRNA fragmenter inddrives ved rensende intakt ribosomet-fodaftryk komplekser. Disse mRNA fodspor er derefter omdannes til cDNA bibliotek og analyseret af dyb sekventering (figur 1). Parallelt til ribosomet profilering er intakt mRNA udvundet fra den samme prøve og sekventeret. Ved at sammenligne niveauet for oversættelse identificeret ved Ribo-Seq med mRNA overflod målinger, kan vi identificere gener, der er specifikt op - eller nedreguleret på niveau med oversættelse og beregne oversættelse effektivitet af mRNA på genom-plan. Mens den protokol, der er beskrevet i denne artikel er specifikke for gær, bør det også være nyttig for forskere, der vil forsøge at etablere Ribo-Seq-protokollen i andre systemer.

<table>
	<tbody>
		<tr>
			<td>0.45 &#956;M membrane filters</td>
			<td>Millipore</td>
			<td>HVLP04700</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9261</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL centrifugal filters (100 kDa MWCO)</td>
			<td>Millipore</td>
			<td>UFC510024</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.0</td>
			<td>Invitrogen</td>
			<td>AM9850G</td>
			<td></td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.5 (pH 8.0 at 4&#176;C)</td>
			<td>Invitrogen</td>
			<td>15567-027</td>
			<td></td>
		</tr>
		<tr>
			<td>10X TBE buffer</td>
			<td>Invitrogen</td>
			<td>AM9863</td>
			<td></td>
		</tr>
		<tr>
			<td>10% TBE-urea gel</td>
			<td>Invitrogen</td>
			<td>EC6875BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>15% TBE-urea gel</td>
			<td>Invitrogen</td>
			<td>EC6885BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>2 M MgCl2</td>
			<td>RPI</td>
			<td>M24500-10.0</td>
			<td></td>
		</tr>
		<tr>
			<td>2X TBE-urea sample buffer</td>
			<td>Invitrogen</td>
			<td>LC6876</td>
			<td></td>
		</tr>
		<tr>
			<td>3M NaOAc, pH 5.5</td>
			<td>Invitrogen</td>
			<td>AM9740</td>
			<td></td>
		</tr>
		<tr>
			<td>5&#39; Deadenylase (10 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>DA11101K</td>
			<td></td>
		</tr>
		<tr>
			<td>5X Nucleic acid sample loading buffer</td>
			<td>Bio-Rad</td>
			<td>161-0767</td>
			<td></td>
		</tr>
		<tr>
			<td>8% TBE gel</td>
			<td>Invitrogen</td>
			<td>EC6215BOX</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)</td>
			<td>Invitrogen</td>
			<td>AM9722</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue light transilluminator</td>
			<td>Clare Chemical Research</td>
			<td>DR-46B</td>
			<td></td>
		</tr>
		<tr>
			<td>Chrome-steel beads, 3.2 mm</td>
			<td>BioSpec Products</td>
			<td>11079132c</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryogrinder</td>
			<td>Biospec product</td>
			<td>3110BX</td>
			<td></td>
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		<tr>
			<td>Cycloheximide</td>
			<td>RPI</td>
			<td>C81040-5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Data Acquisition System</td>
			<td>DATAQ Instruments</td>
			<td>DI-245</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) solution mix (10 mM)</td>
			<td>NEB</td>
			<td>N0447L</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Invitrogen</td>
			<td>AM9510</td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient fractionation system</td>
			<td>Brandel</td>
			<td>BR-184X</td>
			<td></td>
		</tr>
		<tr>
			<td>High-fidelity DNA polymerase (2,000 U/mL)</td>
			<td>NEB</td>
			<td>M0530S</td>
			<td>Supplied with 5X Phusion HF Buffer</td>
		</tr>
		<tr>
			<td>Next-generation sequencing library quantification kit</td>
			<td>Kapa Biosystems</td>
			<td>KK4824</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic acid gel stain</td>
			<td>Invitrogen</td>
			<td>S11494</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima XE-90 ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>A94471</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) mRNA isolation kit</td>
			<td>Invitrogen</td>
			<td>61011</td>
			<td></td>
		</tr>
		<tr>
			<td>Rec J exonuclease (10 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>RJ411250</td>
			<td></td>
		</tr>
		<tr>
			<td>Reverse transcriptase (200 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>18080093</td>
			<td>Supplied with 5X first-strand buffer and 0.1 M DTT</td>
		</tr>
		<tr>
			<td>RNA fragmentation buffer</td>
			<td>NEB</td>
			<td>E6186A</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase I (100 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>AM2295</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase inhibitor (20 U/&#956;L)</td>
			<td>Invitrogen</td>
			<td>AM2696</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone rubber caps</td>
			<td>BioSpec Products</td>
			<td>2008</td>
			<td></td>
		</tr>
		<tr>
			<td>ssDNA ligase (100 U/&#956;L)</td>
			<td>Epicentre</td>
			<td>CL9021K</td>
			<td>Supplied with 10X CircLigase II buffer and 50 mM MnCl2</td>
		</tr>
		<tr>
			<td>Stainless steel microvials, 1.8 mL</td>
			<td>BioSpec Products</td>
			<td>2007</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>RPI</td>
			<td>S24060-5000.0</td>
			<td></td>
		</tr>
		<tr>
			<td>SW-41 Ti rotor</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 polynucleotide kinase (10,000 U/mL)</td>
			<td>NEB</td>
			<td>M0201S</td>
			<td>Supplied with 10X T4 polynucleotide kinase buffer</td>
		</tr>
		<tr>
			<td>T4 RNA ligase 2 truncated KQ (200,000 U/mL)</td>
			<td>NEB</td>
			<td>M0373S</td>
			<td>Supplied with 10X T4 RNA ligase buffer and 50% PEG8000</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Bio-Rad</td>
			<td>1851148</td>
			<td></td>
		</tr>
		<tr>
			<td>Thinwall polyallomer tubes, 13.2 mL</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>X100-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>UV monitor</td>
			<td>Bio-Rad</td>
			<td>7318160</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae strain BY4741</td>
			<td>Open Biosystems</td>
			<td>YSC1048</td>
			<td></td>
		</tr>
	</tbody>
</table>

genome-wide quantification of translation in budding yeast by ribosome profiling

Oversættelse af mRNA i proteiner er en kompleks proces, der involverer flere lag af forordningen. Det antages ofte, at ændringer i mRNA transskription afspejler ændringer i proteinsyntesen, men mange undtagelser er blevet observeret. For nylig, en teknik kaldet ribosomet profilering (eller Ribo-Seq) fremstod som en kraftfuld metode, der giver mulighed for identifikation, med høj nøjagtighed, hvilke regioner af mRNA oversættes til proteiner og kvantificering af oversættelse på genom-plan. Vi præsenterer her, en generaliseret protokol for genome-wide kvantificering af oversættelse ved hjælp af Ribo-Seq i spirende gær. Desuden giver kombinere Ribo-Seq data med mRNA overflod målinger os samtidig kvantificere oversættelse effektivitet af tusindvis af mRNA udskrifter i samme prøve og sammenligne ændringer i disse parametre i svar til eksperimentelle manipulationer eller i forskellige fysiologiske stater. Vi beskriver en detaljeret protokol for generation af ribosomet fodspor ved hjælp af nukleasen fordøjelse, isolation af intakt ribosomet-fodaftryk komplekser via saccharose gradient fraktionering og forberedelse af DNA biblioteker til dyb sekvensering sammen med passende kvalitetskontrol er nødvendige for at sikre præcis analyse af i vivo oversættelse.

Detaljerede rørledninger til bioinformatic analyse af ribosomet profilering data er blevet beskrevet tidligere 8,9. Derudover har flere forskergrupper udviklet bioinformatik værktøjer til differentieret gen expression analyse og behandling af sequencing data, som er specifikke for ribosomet profilering metode 10,11,12 ,<sup...

Watch this Scientific Journal Video about Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling at JoVE.com

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translationel forordning spiller en vigtig rolle i kontrollen af protein overflod. Her, beskriver vi en høj overførselshastighed metode til kvantitativ analyse af oversættelse i spirende gær Saccharomyces cerevisiae.

Genome-wide kvantificering af oversættelse i spirende gær af ribosomet profilering

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Research

JoVE Journal

Biochemistry

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Løsning affinitetsrensede proteinkomplekser ved Blå Native PAGE og protein Korrelation Profilering

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Dyb proteomet profilering af Isobar mærkning, omfattende væskekromatografi, massespektrometri og Software-assisteret kvantificering

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Quantification of Hypopigmentation Activity In Vitro

Kvantificering af hypopigmentering aktivitet In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Nitropeptid profilering og identifikation illustreret af angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

Oversættelse af Ribosome affinitets rensning (TRAP) for RNA-isolation fra endotelceller in vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA-sekvens genkendelse af DNA Primase ved hjælp af Primase profilering med høj gennemløb

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution

Dual DNA-linealer til at studere mekanismen af Ribosome translokation med enkelt-nukleotid opløsning

The ribosome translocation refers to the ribosomal movement on the mRNA by exactly three nucleotides (nt), which is the central step in protein synthesis. ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Complexome-profilering med høj opløsning ved Cryoslicing BN-MS-analyse

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...