We thank Anne Wochele for her assistance in the laboratory and Vera Augsburger for help with the automated gel sequencer. We thank the Deutsche Forschungsgemeinschaft for funding by grants BE4038/2 and BE4038/5 within the &#8220;priority programmes&#8221; SPP1316 and SPP1617.

<ol>
	<li>Simpson, C. G., Brown, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer+extension+assay.">Primer extension assay.</a> Methods Mol. Biol. 49, 249-256 (1995).</li><li>Sanger, F., Nicklen, S., Coulson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+sequencing+with+chain-terminating+inhibitors.">DNA sequencing with chain-terminating inhibitors.</a> Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 (1977).</li><li>Fekete, R. A., Miller, M. J., Chattoraj, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescently+labeled+oligonucleotide+extension:+a+rapid+and+quantitative+protocol+for+primer+extension.">Fluorescently labeled oligonucleotide extension: a rapid and quantitative protocol for primer extension.</a> Biotechniques. 35, 90-94 (2003).</li><li>Schuster, C. F., 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=Characterization+of+a+mazEF+Toxin-Antitoxin+Homologue+from+Staphylococcus+equorum.">Characterization of a mazEF Toxin-Antitoxin Homologue from Staphylococcus equorum.</a> J. Bacteriol. 195, 115-125 (2013).</li><li>Nolle, N., Schuster, C. F., Bertram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+paralogous+yefM-yoeB+loci+from+Staphylococcus+equorum+encode+functional+toxin-antitoxin+systems.">Two paralogous yefM-yoeB loci from Staphylococcus equorum encode functional toxin-antitoxin systems.</a> Microbiology. 159, 1575-1585 (2013).</li><li>Yamaguchi, Y., Park, J. H., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+in+bacteria+and+archaea.">Toxin-antitoxin systems in bacteria and archaea.</a> Annu. Rev. Genet. 45, 61-79 (2011).</li><li>Schuster, C. F., Bertram, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+are+ubiquitous+and+versatile+modulators+of+prokaryotic+cell+fate.">Toxin-antitoxin systems are ubiquitous and versatile modulators of prokaryotic cell fate.</a> FEMS Microbiol. Lett. 340, 73-85 (2013).</li><li>Goeders, N., Van Melderen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxin-antitoxin+systems+as+multilevel+interaction+systems.">Toxin-antitoxin systems as multilevel interaction systems.</a> Toxins. 6, 304-324 (2014).</li><li>Yamaguchi, Y., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+interferases,+sequence-specific+endoribonucleases+from+the+toxin-antitoxin+systems.">mRNA interferases, sequence-specific endoribonucleases from the toxin-antitoxin systems.</a> Progress in molecular biology and translational science. 85, 467-500 (2009).</li><li>Park, J. H., Yamaguchi, Y., Inouye, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacillus+subtilis+MazF-bs+(EndoA)+is+a+UACAU-specific+mRNA+interferase.">Bacillus subtilis MazF-bs (EndoA) is a UACAU-specific mRNA interferase.</a> FEBS Lett. 585, 2526-2532 (2011).</li><li>Zhu, L., 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=et+al.Staphylococcus+aureus+MazF+specifically+cleaves+a+pentad+sequence,+UACAU,+which+is+unusually+abundant+in+the+mRNA+for+pathogenic+adhesive+factor+SraP.">et al.Staphylococcus aureus MazF specifically cleaves a pentad sequence, UACAU, which is unusually abundant in the mRNA for pathogenic adhesive factor SraP.</a> J. Bacteriol. 191, 3248-3255 (2009).</li><li>Zhu, L., 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+mRNA+interferases,+MazF-mt3+and+MazF-mt7+from+Mycobacterium+tuberculosis+target+unique+pentad+sequences+in+single-stranded+RNA.">The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA.</a> Mol. Microbiol. 69, 559-569 (2008).</li><li>Fu, Z., Donegan, N. P., Memmi, G., Cheung, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+MazFSa,+an+endoribonuclease+from+Staphylococcus+aureus.">Characterization of MazFSa, an endoribonuclease from Staphylococcus aureus.</a> J. Bacteriol. 189, 8871-8879 (2007).</li><li>Marmur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+the+isolation+of+deoxyribonucleic+acid+from+micro-organisms.">A procedure for the isolation of deoxyribonucleic acid from micro-organisms.</a> J. Mol. Biol. 3, 208-218 (1961).</li><li>Emory, S. A., Belasco, 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=The+ompA+5'+untranslated+RNA+segment+functions+in+Escherichia+coli+as+a+growth-rate-regulated+mRNA+stabilizer+whose+activity+is+unrelated+to+translational+efficiency.">The ompA 5' untranslated RNA segment functions in Escherichia coli as a growth-rate-regulated mRNA stabilizer whose activity is unrelated to translational efficiency.</a> J. Bacteriol. 172, 4472-4481 (1990).</li><li>Cole, S. T., Bremer, E., Hindennach, I., Henning, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+the+promoters+for+the+ompA+gene+which+encodes+a+major+outer+membrane+protein+of+Escherichia+coli.">Characterisation of the promoters for the ompA gene which encodes a major outer membrane protein of Escherichia coli.</a> Mol. Gen. Genet. 188, 472-479 (1982).</li><li>Schleifer, K. H., Kilpper-B&#228;lz, R., Devriese, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staphylococcus+arlettae+sp.+nov.,+S.+equorum+sp.+nov.+and+S.+kloosii+sp.+nov.:+Three+New+Coagulase-Negative,+Novobiocin-Resistant+Species+from+Animals.">Staphylococcus arlettae sp. nov., S. equorum sp. nov. and S. kloosii sp. nov.: Three New Coagulase-Negative, Novobiocin-Resistant Species from Animals.</a> Syst. Appl. Microbiol. 5, 501-509 .</li><li>Yu, H., Goodman, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+HIV-1+and+avian+myeloblastosis+virus+reverse+transcriptase+fidelity+on+RNA+and+DNA+templates.">Comparison of HIV-1 and avian myeloblastosis virus reverse transcriptase fidelity on RNA and DNA templates.</a> J. Biol. Chem. 267, 10888-10896 (1992).</li><li>Ying, B. W., Fourmy, D., Yoshizawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substitution+of+the+use+of+radioactivity+by+fluorescence+for+biochemical+studies+of+RNA.">Substitution of the use of radioactivity by fluorescence for biochemical studies of RNA.</a> RNA. 13, 2042-2050 (2007).</li></ol>

The authors have nothing to disclose that would present a conflict of interest.

형광 프라이머 신장은 TSP- 또는 RNA 이차 가공 식별 어느 쪽의 RNA의 5 &#39;말단을 결정하기위한 간단하고 신속한 방법이다. 때문에 형광 프라이머의 사용, 반응이 설정 될 수 있으며, (방사성 표지 된 프라이머 경우와 달리) 추가 보안 조치없이 실행합니다. 샘플은 형광으로 검출되는 바와 같이 전기 영동 X 선 필름은 일반적으로 사용되는 방사성 방법에 비해 신속한 분석을 허용 진행되는 동안, 이들...

프라이머 신장도 1은 하나의 기본 해상도까지 특정 RNA 분자의 5 &#39;말단을 결정하기 위해 분자 방법이다. 이러한 5&#39;-RACE (cDNA의 신속한 증폭이 종료 됨) 같은 다른 방법의 장점은 빠른 처리 시간을 용이 RNA 분자의 서로 다른 길이의 혼합물을 분석 할 수있는 능력이다. 이 방법은 RNA 분자가 특정 길이의 cDNA 단편을 생성하는 특정 형광 프라이머를 사용하여 전사 반응을 반대로 실시함으로써 작동한다. 이들의 cDNA 분자 변성 폴리 아크릴 아마이드 겔에 전통적인 생거 시퀀싱 반응이 함께 실행 의한 형광 표지 된 프라이머를 사용하여 그 형광을 검출 할 수있다. 된 cDNA 단편의 길이는 5 &#39;말단 RNA의 맵핑을 허용 시퀀싱 사다리 비교에 의해 평가된다. 전통적으로, 프라이머 신장 반응을 함께 사용방사성 동위 원소와 X 선 필름에의 cDNA 분자들을 검출한다. 그들의 감도가 약간 낮다이라도 인해 건강 위험, 폐기물 처리 문제 및 취급의 용이성, 새로운 프로토콜은 자동화 된 시퀀서와 프라이머 신장의 검출을위한 형광을 이용한다. 형광 프라이머 (로모 손 년 이상) 장기간 안정적으로 형광 표지 된 프라이머를 사용하여 무선 표지 반복 절차가 생략 될 수있다. 우리는 여기에서 설명하는 방법은 자동화 겔 시퀀서를 이용하지만, 약간의 수정과, 모세관 시퀀서는 cDNA의 분리 및 검출 3에 사용될 수있다. 겔 분석 병렬 특성은 가능한 RNA 절단 또는 프로세싱 심지어 적은 양을 검출 할 수있다. 또 다른 이점은 검출 될 수 단말 절단 또는 심지어 하나의베이스 처리로서,이 방법의 고해상도이다. RNA 절단 또는 가공, t의 검출에 관련하여프라이머 확장 ypically 두 가지 종류가 구별된다. 다른 경우에, 처리가 생체 내에서 수행하고, 생성 된 RNA 정제 반면 한 경우, 효소 처리, 정제 된 RNA 정제 효소를 사용하여 시험 관내에서 수행된다. 프라이머 신장 시험 관내에서 수행에 두 경우 RNA가 RNA의 소스에 따라, 그러나, 실시되고, 상기 방법은 시험 관내 또는 생체 내 프라이머 신장이라고 하나. 여기 제시 프로토콜에서는 때문에 사용 용이성 (정제없이 필요한 단백질)과 동시에 전사 시작점 및 처리를 결정하는 가능성 단독 생체 프라이머 신장에 초점. 그러나, 시험 관내 프라이머 확장은 동일한 방법으로 설정 원칙적으로,이 프로토콜은 시작점을 제공 할 수있다. 여기에 예시 된 방법은 그것들이 높은 의무만큼 많은 세균 종에 적용될 수있다핵산의 순도 높은 수율 준비. 우리 실험실 연구 독소 항독소 (맞대기) 시스템 4,5, 프라이머 신장 방법은 광범위하게 사용되는 필드의 규정 범위에 초점을 맞춘다. TA-시스템은 안정적이고 내생 활성 독성 단백질과 독성 6,7 중화 대부분 불안정한 단백질 또는 RNA 항독소로 구성 원핵 생물의 게놈에 존재하는 작은 유전 적 요소입니다. 독소 활성은 때때로하는 RNase 활성 8,9 의해 대부분 복제, 세포벽 합성 또는 다른 메커니즘의 억제에 의해 발휘되지만. 일반적으로, RNA 분해 효소 특이성 프라이머 신장 방법 중 하나는 다른 테스트를 수행하여 결정된다. 벽개 및 전체 길이 단편의 혼합물을 동시에 그들의 5 &#39;끝나는 결정하기 위해 분석 될 수있는 프라이머 신장 반응,이 애플리케이션에 매우 적합하다. 시험 관내 및 생체 프라이머 확장에 믹스를 사용하여,특정 독소의 RNase 절단은, 예를 들어, 서열 특이성 10-13 결정될 수있다. <img alt="그림 1" fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> . 프라이머 확장 절차 1. 개요 그림 세균 배양 실험의 필요에 따라 배양 및 처리됩니다. 총 RNA는 세포의 cDNA를 수득 대상 특정 형광 DNA 프라이머를 사용하여 역전사 반응에 DNA의 흔적을 제거하기의 DNase I로 처리하고 실시로부터 추출된다. 게놈 DNA 또는 플라스미드를 추출하여 이후의 cDNA 조각과 크기 비교를 위해 형광 생거 시퀀싱 반응에 사용된다. 프라이머 신장 산물은 변성 우레아 폴리 아크릴 아미드 겔상에서 생거 시퀀싱 제품을 함께 실행하고 자동화 레이저 현미경으로 분석한다. 라 cDNA를 밴드와 함께 선까지입니다 시퀀싱베이스세인트 5 &#39;의 cDNA 끝 (파란색 화살표)의 기본. Fekete 추가 정보, 등. (3) <a href="https://www.jove.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 전체 프라이머 신장 절차의 개요는도 1에서 발견 될 수있다. 요약하면, 박테리아 세포 수확, 배양, 세포 펠렛을 용해 및 RNA 추출. 정제 된 RNA이어서 역전사 템플릿으로 동작 할 수있다 DNA 분자의 흔적을 제거하기의 DNase I로 처리된다. 특정 형광 프라이머는 관심 영역에 교배와 이후 단일 가닥 보완 DNA (cDNA를) 결과, 전사 역, RNA에 추가됩니다. 시퀀싱 사다리 형광 프라이머를 이용하는 전통적인 생거 시퀀싱에 의해 생성 및 프라이머 신장 된 cDNA 단편의 나란히 변성 폴리 아크릴 아미드 겔상에서 분리된다. 결과겔은 관심의 5 &#39;말단의 식별을 허용 형광 밴드를 비교하여 분석된다. 전사 시작 지점 및 처리 사이트는 다음 서열 비교​​에 의해 개별적으로 평가된다.

<table><tbody><tr><td>AMV Reverse Transcriptase (20-25 U/&amp;micro;l)</td><td>NEB / Roche</td><td>NEB: M0277-T / Roche: 10109118001</td><td></td></tr><tr><td>DNase I (RNase free)</td><td>Ambion (life technologies)</td><td>AM2222</td><td></td></tr><tr><td>FastPrep-24 Instrument&amp;nbsp;</td><td>MPBio</td><td>116004500</td><td></td></tr><tr><td>Fluorescently labeled primers</td><td>Biomers</td><td>n/a</td><td>5&amp;rsquo; DY-681 modification of &amp;ldquo;ordinary&amp;rdquo; DNA oligonucleotides. Compatible dyes such as the LICOR IRDye 700/800 are also available from other suppliers such as IDTdna.</td></tr><tr><td>Li-Cor 4200 Sequencer incl. ImagIR Data collection software</td><td>Li-Cor</td><td></td><td>Product discontinued</td></tr><tr><td>NanoDrop 2000</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Nuclease free water</td><td>Ambion (life technologies)</td><td>AM9915G</td><td></td></tr><tr><td>Plasmid mini preparation kit</td><td>QIAGEN</td><td>12125</td><td></td></tr><tr><td>RapidGel-XL-40% Concentrate</td><td>USB</td><td>US75863</td><td></td></tr><tr><td>RNA STORAGE BUFFER</td><td>Ambion (life technologies)</td><td>AM7000</td><td></td></tr><tr><td>Roti-Aqua-P/C/I</td><td>Carl Roth</td><td>X985.3</td><td>Alternative: &amp;ldquo;Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)&amp;rdquo; from Ambion (AM9720)</td></tr><tr><td>SUPERase&amp;bull;In RNase Inhibitor</td><td>Ambion (life technologies)</td><td>AM2696</td><td></td></tr><tr><td>Thermo Sequenase fluorescently labelled primer cycle sequencing kit with 7-deaza-dGTP</td><td>GE Healthcare</td><td>RPN2538</td><td></td></tr><tr><td>TRIzol reagent</td><td>life technologies</td><td>15596-026</td><td></td></tr><tr><td>Zirconia/Silica Beads 0.1 mm</td><td>BioSpec</td><td>11079101z</td><td></td></tr></tbody></table>

fluorescence based primer extension technique to determine transcriptional starting points and cleavage sites of rnases in vivo

형광 계 프라이머 신장 (FPE)는 전사 시작점 또는 RNA 분자의 프로세싱 사이트를 결정하기 위해 분자 방법이다. 이것은 특정 형광 표지 된 프라이머 및 폴리 아크릴 아미드 겔 전기 영동을 변성하여 수득 된 cDNA 단편의 후속 분석을 이용하여 관심있는 RNA의 역전사에 의해 달성된다. 동시에, 전통적인 생거 시퀀싱 반응은 자신의 정확한 해당 기지의 cDNA 단편의 단부를 매핑 겔상에서 실행된다. 제품은 복제되어야 복수 후보 서열 5&#39;-RACE (cDNA를 엔드 신속한 증폭), 대조적으로, 프라이머 신장에 의해 생성 된 cDNA 단편의 대부분은 동시에 하나 겔 실행에서 검출 할 수있다. 또한, (결과를 최종 분석에 역전사에서) 모든 절차는 하나의 작업 일에 완료 할 수 있습니다. 형광 표지 된 프라이머를 사용함으로써, 유해 방사성 동위 원소의 사용은 표지 된 시약을회피 할 수 있고, 제품은 전기 영동 과정에서 검출 될 수 있기 때문에 처리 시간이 감소된다. 다음의 프로토콜에서는 확실하고 신속 S.에서 (예를 들어, 독소 항독소 시스템 구성 요소) 시작점 및 RNA 프로세싱 사이트 전사을 추론의 RNA의 5 &#39;말단을 검출하는 생체 내 형광 프라이머 신장 방법을 서술 구균, E. 대장균 및 기타 세균.

도 6에 도시 된 바와 같이, 프라이머 신장 반응 관심 사체의 전사 시작 포인트를 결정하는데 사용될 수 있고 (통상적으로 -10 -35 원소로 식별 됨) 프로모터 영역을 추론 할 수 있습니다. 최상위 (가장 긴)의 cDNA 단편의 mRNA의 5 &#39;말단을 나타내며, 따라서 시퀀싱 사다리에 비해 용이하게 매핑 될 수있다. <img alt="그림 6" src="/files/ftp_upload/52134/52134fig6highres.jpg" /> <st...

Watch this Scientific Journal Video about Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo at JoVE.com

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

형광 기반 프라이머 확장 기술은 전사 시작 포인트 및 RNases의 쪼개짐 사이트를 확인하는<em&gt; 생체</em

Fluorescence based primer extension (FPE) is a molecular method to determine transcriptional starting points or processing sites of RNA molecules. This is ...

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Biology

Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

26.9K 조회수.  University of Tübingen. We here describe a fluorescence based primer extension method to determine transcriptional starting points from bacterial transcripts and RNA processing in vivo using an automated gel sequencer.

비디오: Fluorescence Based Primer Extension Technique to Determine Transcriptional Starting Points and Cleavage Sites of RNases In Vivo

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

바인딩, 전사, 번역, 및 회전율 세포 활성화 중 글로벌 이벤트를 표시 하는 녹음 방송 요인의 실시간 분석

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

연구

유전학

Prediction and Validation of Gene Regulatory Elements Activated During Retinoic Acid Induced Embryonic Stem Cell Differentiation

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to tissue and cell-type specific regulation of gene expression during the cellular differentiation. Thus, their identification and further investigation is important in order to understand how cell fate is determined. Integration of gene expression data (e.g., microarray or RNA-seq) and results of chromatin immunoprecipitation (ChIP)-based genome-wide studies (ChIP-seq) allows large-scale identification of these regulatory regions. However, functional validation of cell-type specific enhancers requires further in vitro and in vivo experimental procedures. Here we describe how active enhancers can be identified and validated experimentally. This protocol provides a step-by-step workflow that includes: 1) identification of regulatory regions by ChIP-seq data analysis, 2) cloning and experimental validation of putative regulatory potential of the identified genomic sequences in a reporter assay, and 3) determination of enhancer activity in vivo by measuring enhancer RNA transcript level. The presented protocol is detailed enough to help anyone to set up this workflow in the lab. Importantly, the protocol can be easily adapted to and used in any cellular model system.

In this work we provide an experimental workflow of how active enhancers can be identified and experimentally validated.

예측 및 유전자 규제 요소의 검증은 레티노 산 유도 배아 동안 활성화 된 세포 분화 줄기

Embryonic development is a multistep process involving activation and repression of many genes. Enhancer elements in the genome are known to contribute to ...

발생학

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

번역 개시 포유류 mRNAs에 복잡 한 대형의 Toeprinting 분석

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

생화학

Artificial RNA Polymerase II Elongation Complexes for Dissecting Co-transcriptional RNA Processing Events

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA polymerase II and co-transcriptional capping and splicing of the precursor RNA to form the mature mRNA. During mRNA synthesis, the RNA polymerase II elongation complex is a target for regulation by a large collection of transcription factors that control its catalytic activity, as well as the capping, splicing, and 3&#8217;-processing enzymes that create the mature mRNA. Because of the inherent complexity of mRNA synthesis, simpler experimental systems enabling isolation and investigation of its various co-transcriptional stages have great utility.
In this article, we describe one such simple experimental system suitable for investigating co-transcriptional RNA capping. This system relies on defined RNA polymerase II elongation complexes assembled from purified polymerase and artificial transcription bubbles. When immobilized via biotinylated DNA, these RNA polymerase II elongation complexes provide an easily manipulable tool for dissecting co-transcriptional RNA capping and mechanisms by which the elongation complex recruits and regulates capping enzyme during co-transcriptional RNA capping. We anticipate this system could be adapted for studying recruitment and/or assembly of proteins or protein complexes with roles in other stages of mRNA maturation coupled to the RNA polymerase II elongation complex.

Here, we describe the assembly of RNA polymerase II (Pol II) elongation complexes requiring only short synthetic DNA and RNA oligonucleotides and purified Pol II. These complexes are useful for studying mechanisms underlying co-transcriptional processing of transcripts associated with the Pol II elongation complex.

인공 RNA 폴리머라제 II 연신복합체 해부 공동 전사 RNA 처리 이벤트

Eukaryotic mRNA synthesis is a complex biochemical process requiring transcription of a DNA template into a precursor RNA by the multi-subunit enzyme RNA ...

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be bound by many different RBPs that will regulate its production, stability, activity, and degradation. As such, much has been done to understand the dynamics that exist between these two types of molecules. A particularly important breakthrough came with the emergence of &#8216;cross-linking and immunoprecipitation&#8217; (CLIP). This technique allowed stringent investigation into which RNAs are bound by a particular RBP. In short, the protein of interest is UV cross-linked to its RNA substrates in vivo, purified under highly stringent conditions, and then the RNAs covalently cross-linked to the protein are converted into cDNA libraries and sequenced. Since its conception, many derivative techniques have been developed in order to make CLIP amenable to particular fields of study. However, cross-linking using ultraviolet light is notoriously inefficient. This results in extended exposure times that make the temporal study of RBP-RNA interactions impossible. To overcome this issue, we recently designed and built much-improved UV irradiation and cell harvesting devices. Using these new tools, we developed a protocol for time-resolved analyses of RBP-RNA interactions in living cells at high temporal resolution: Kinetic CRoss-linking and Analysis of cDNAs (&#967;CRAC). We recently used this technique to study the role of yeast RBPs in nutrient stress adaptation. This manuscript provides a detailed overview of the &#967;CRAC method and presents recent results obtained with the Nrd1 RBP.

Kinetic cross-linking and analysis of cDNA is a method that allows investigation of the dynamics of protein-RNA interactions in living cells at high temporal resolution. Here the protocol is described in detail, including the growth of yeast cells, UV cross-linking, harvesting, protein purification, and next generation sequencing library preparation steps.

χCRAC를 사용하여 높은 시간 분해능에서 생체 내 단백질-RNA 상호 작용 역학 모니터링

The interaction between RNA-binding proteins (RBPs) and their RNA substrates exhibits fluidity and complexity. Within its lifespan, a single RNA can be ...

Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers and modulators. While these dynamic states remain hidden to most structural methods, R1&#961; relaxation dispersion (RD) spectroscopy allows the study of conformational dynamics in the micro- to millisecond regime at atomic resolution. The use of 1H as the observed nucleus further expands the time regime covered and gives direct access to hydrogen bonds and base pairing.
The challenging steps in such a study are high-purity and high-yield sample preparation, potentially 13C- and 15N-labeled, as well as setup of experiments and fitting of data to extract population, exchange rate, and secondary structure of the previously invisible state. This protocol provides crucial hands-on steps in sample preparation to ensure the preparation of a suitable RNA sample and setup of 1H R1&#961; experiments with both isotopically labeled and unlabeled RNA samples.

We present a protocol to measure micro- to millisecond dynamics on 13C/15N-labeled and unlabeled RNA with 1H R1&#961; relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy. The focus of this protocol lies in high-purity sample preparation and setup of NMR experiments.

RNA의 1 H R 1θ 이완 분산 실험의 샘플 준비 및 설치의 실제적인 양상

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA 메틸트랜스퍼라제 억제제를 스크리닝하기 위한 연속 형광 기반 엔도뉴클레아제 결합 DNA 메틸화 분석

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

접합 효율 모니터링을 위한 기자 기반 셀룰러 분석

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

iCLIP - Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation1. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs2.
Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP)3-5. These approaches were prone to identifying indirect or non-physiological interactions6. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced7,8. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq)9,10. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking11,12.
Despite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide13. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1)14. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation14.

The spatial arrangement of RNA-binding proteins on a transcript is a key determinant of post-transcriptional regulation. Therefore, we developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) that allows precise genome-wide mapping of the binding sites of an RNA-binding protein.

iCLIP - 개인 염기 해상도와 단백질 RNA 상호 작용의 Transcriptome 전체 매핑

The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional ...

생물학

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

싱글 생활 세포에서 mRNA의 전사의 속도론을 측정

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

형광 기반 프라이머 확장 기술은 전사 시작 포인트 및 RNases의 쪼개짐 사이트를 확인하는

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