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.

סיומת פריימר ניאון היא שיטה פשוטה ומהירה לקביעת קצוות &quot;5 של RNA, או לTSP- או הזדהות עיבוד RNA משנית. בשל השימוש בצבעי יסוד ניאון, ניתן להגדיר את התגובות ולרוץ ללא אמצעי אבטחה נוסף (שלא כמו במקרה של פריימרים שכותרתו רדיואקטיבית). כדגימות זוהו על ידי הקרינה, הם יכולים להיות ?...

סיומת פריימר 1 היא שיטה מולקולרית כדי לקבוע קצוות &#39;5 של מולקולות RNA ספציפיות עד רזולוציה בסיסית אחד. היתרון לשיטות אחרות כגון 5&#39;-RACE (הגברה מהירה של cDNA מסתיימת) הוא זמן אספקה ​​המהיר והיכולת לנתח תערובת של אורכים שונים של מולקולות RNA בקלות. שיטה זו פועלת על ידי חשיפת מולקולות RNA להפוך תגובות שעתוק באמצעות פריימרים ספציפיים ניאון, יצירה שברי cDNA של אורכים מסוימים. מולקולות cDNA אלה לרוץ לצד תגובות סנגר רצף מסורתי 2 על ג&#39;לים polyacrylamide denaturing ויכולות להיות מזוהות על ידי הקרינה שלהם בשל השימוש בפריימרים שכותרתו fluorescently. האורכים של ברי cDNA מוערכים על ידי השוואה לסולם הרצף, המאפשרים המיפוי של 5 קצות RNA &quot;. באופן מסורתי, תגובות סיומת פריימר משמשות בשילובעם איזוטופים רדיואקטיביים לזיהוי תרכובות cDNA על צילומי רנטגן. בשל סכנות בריאותיות, בעיות סילוק פסולת ולהקל על טיפול, פרוטוקולים חדשים יותר לנצל הקרינה לגילוי סיומת פריימר עם sequencers האוטומטי, אם כי הרגישות שלהם נמוכה במקצת. באמצעות פריימרים שכותרתו fluorescently, ניתן להשמיט את הליך רדיו תיוג החוזר, כצבעי יסוד ניאון הם יציבים במשך זמן רב (יותר משנה בידיים שלנו). השיטה שאנו מתארים כאן מנצלת רצף ג&#39;ל אוטומטי, אך עם שינויים קלים, sequencers נימים יכול לשמש גם להפרדת cDNA וגילוי 3. הטבע המקביל של ניתוח ג&#39;ל מאפשר לזהות אפילו כמות קטנה של מחשוף RNA או עיבוד. יתרון נוסף הוא ברזולוציה הגבוהה של שיטה זו, כמחשוף או עיבוד של אף אחד בסיס מסוף ניתן לאתרם. בהקשר לזיהוי של מחשוף RNA או עיבוד, typically שני סוגים שונים של הרחבות פריימר הם מכובדים. במקרה אחד, הטיפול אנזימטי נעשה במבחנה באמצעות RNA מטוהר ואנזים מטוהר, ואילו במקרה האחר, העיבוד נעשה in vivo ו- RNA וכתוצאה מכך הוא מטוהרים. בשני המקרים RNA הוא נתון לרחבת פריימר בוצעה במבחנה, עם זאת, בהתאם למקור של RNA, השיטה היא גם נקראת סיומת פריימר vivo במבחנה או ב. בפרוטוקול אנו מציגים כאן, אנו מתמקדים אך ורק על in vivo סיומת פריימר, בגלל קלות שימוש (אין חלבונים מטוהרים צורך) ואת האפשרות לקבוע נקודות התחלת תעתיק ועיבוד באותו הזמן. עם זאת, במבחנה הרחבות פריימר באופן עקרוני להגדיר באותה הצורה ופרוטוקול זה יכול לשמש כנקודת התחלה. השיטה מאוירת כאן יכולה להיות מיושמת על מינים רבים של חיידקים, כל עוד הם ניתנים לגבוהיםהכנת טוהר ותשואה גבוהה של חומצות גרעין. המחקר במעבדה שלנו מתמקד בהיקף הרגולציה של מערכות רעלן-נסיוב (ת א) 4,5, תחום שבו נעשתה שימוש נרחב בשיטת הרחבה פריימר. מדד ת&quot;א מערכות אלמנטים גנטיים קטנים הנמצאים בגנומים פרוקריוטים שמורכבים מחלבון רעיל יציב ואופן אנדוגני פעיל וחיסון אנטי-חלבון או RNA בעיקר לא יציב שמנטרל רעילות 6,7. לפעמים פעילות רעלן מופעלת על ידי עיכוב של שכפול, סינתזת דופן תא או מנגנונים אחרים, אבל לרוב על ידי פעילות RNase 8,9. בדרך כלל, סגוליות RNase נקבע על ידי ביצוע בדיקות שונות, שאחד מהם היא שיטת סיומת פריימר. תגובות סיומת פריימר הם גם מתאימות ליישום זה, כתערובת של שברי אורך בקעו ומלאים ניתן לנתח בו זמנית כדי לקבוע את &#39;5 מסתיים. באמצעות שילוב של במבחנה וברחבות פריימר vivo,מחשוף RNase רעלן ספציפי, לדוגמא, ניתן לקבוע סגוליות רצף 10-13. <img alt="איור 1" fo:content-width="6in" src="/files/ftp_upload/52134/52134fig1highres.jpg" width="600" /> איור 1. סקירה כללית של הליך סיומת פריימר. תרבויות חיידקים מודגרת וטופלו בהתאם לצרכי הניסוי. RNA סה&quot;כ מופק מהתאים, שטופל בDNase אני כדי להסיר עקבות DNA ונתונים לתגובת שעתוק לאחור באמצעות פריימרים יעד ספציפיים ניאון DNA מניב cDNA. DNA או פלסמידים הגנומי מופקים ולאחר מכן משמש לתגובות סנגר רצף ניאון להשוואת גודל עם שברי cDNA. מוצרי סיומת פריימר מנוהלים על צד מוצרים סנגר רצף על ג&#39;ל polyacrylamide האוריאה denaturing וניתחו עם לייזר ומיקרוסקופ אוטומטיים. בסיס הרצף ששורות עם להקת cDNA הוא להרח בסיס של 5 סוף cDNA &#39;(החץ הכחול). מידע נוסף ב Fekete, et al. 3 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52134/52134fig1highres.jpg" target="_blank">אנא לחץ כאן כדי לצפות בגרסה גדולה יותר של דמות זו.</a> ניתן למצוא סקירה של ההליך כולו סיומת פריימר באיור 1. בקצרה, תאי חיידקים בתרבית, שנקטפו, התא גלולה lysed וRNA חילוץ. RNA המטוהר לאחר מכן טופל בDNase אני כדי להסיר עקבות של מולקולות DNA אשר יכול לפעול כתבניות לטרנסקריפטאז ההפוך. פריימרים ניאון ספציפיים מתווספים ל- RNA, כלאה אל האזור של עניין, ולאחר מכן להפוך את תמלילי עדויות, וכתוצאה מכך דנ&quot;א יחיד שנתקע משלים (cDNA). סולם רצף נוצר על ידי סנגר רצף מסורתי העסקת פריימרים ניאון ומופרד על ג&#39;ל polyacrylamide denaturing צד של שברי cDNA סיומת פריימר. וכתוצאה מכךג&#39;ל מנותח על ידי השוואת להקות ניאון, המאפשרת זיהוי של קצוות &#39;5 של עניין. נקודות התחלת תעתיק ואתרי עיבוד מוערכים בנפרד על-ידי השוואות רצף.

<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

הארכת הקרינה מבוססת פריימר (FPE) היא שיטה מולקולרית כדי לקבוע נקודת התחלת שעתוק או אתרי עיבוד של מולקולות RNA. זו מושגת על ידי שעתוק לאחור של ה- RNA של עניין באמצעות פריימרים שכותרתו fluorescently ספציפיים וניתוח בדיעבד של שברי cDNA וכתוצאה מכך על ידי denaturing ג&#39;ל אלקטרופורזה polyacrylamide. במקביל, תגובה סנגר רצף מסורתי מנוהלת על הג&#39;ל למפות את הקצוות של שברי cDNA לבסיסים המקביל המדויקים שלהם. בניגוד ל5&#39;-RACE (Rapid ההגברה של קצות cDNA), שבו המוצר חייב להיות משובט ומספר מועמדים רבים רצף, התפזורת של שברי cDNA נוצרו על ידי סיומת פריימר יכולה להתגלות בו זמנית בטווח ג&#39;ל אחד. בנוסף, ההליך כולו (משעתוק לאחור לניתוח סופי של התוצאות) ניתן להשלים ביום עבודה אחד. על ידי שימוש בפריימרים שכותרתו fluorescently, השימוש באיזוטופ רדיואקטיבי מסוכן שכותרתו חומרים כימייםניתן להימנע וזמני עיבוד צומצמו ניתן לאתר מוצרים במהלך הליך אלקטרופורזה. בפרוטוקול הבא, אנו מתארים שיטת סיומת פריימר ניאון in vivo באופן מהיר כדי לזהות קצוות &#39;5 של RNAs להסיק תעתיק נקודות התחלה ואתרי עיבוד RNA (למשל, על ידי רכיבי מערכת רעלן-אַנְטִיטוֹקסִין) בס aureus, E. coli וחיידקים אחרים.

כפי שמתואר באיור 6, תגובת סיומת פריימר ניתן להשתמש כדי לקבוע את נקודות התחלת תעתיק של תמלילים של עניין ויכולה לעזור להסיק אזורי אמרגן (מזוהה בדרך כלל על ידי -10 ו-35 אלמנטים). בר cDNA העליון (הארוך ביותר) מייצג את הקצה 5 &#39;של ה- mRNA, ולכן יכול להיות ממופה בקלות בהשוואה...

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.

טכניקת הארכת פריימר בהתבסס הקרינה לקביעת נקודות התחלת תעתיק ומחשוף אתרים של RNases<em&gt; In vivo</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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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

26.9K Views.  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.

Video: 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.

ניתוח בזמן אמת של גורם שעתוק כריכה, תמלול, תרגום, מחזור ולהציג אירועים גלובליים במהלך ההפעלה הסלולרית

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

Genetics

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.

חיזוי ותיקוף של גן בקרה אלמנטים הופעל במהלך החומצה הרטינואית מושרה גזע עובריים התמיינות תאים

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

Developmental Biology

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.

ניתוח Toeprinting של תרגום חניכה היווצרות מורכבות על יונקים mRNAs

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

Biochemistry

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.

RNA מלאכותי פולימראז השני מכלולי התארכות לניתוח אירועים בעיבוד RNA

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.

ניטור דינמיקת אינטראקציה חלבון-רנ"א ב-Vivo ברזולוציה טמפורלית גבוהה באמצעות χCRAC

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.

היבטים מעשיים של הכנת מדגם והגדרת 1H R1ρ הרפיה ניסויים של RNA

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;

בדיקת מתילציה רציפה של דנ"א מצומד אנדונוקלאז מבוססת פלואורסצנציה לסינון מעכבי מתיל-טרנספראז של DNA

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.

בדיקה סלולרית מבוססת כתב לניטור יעילות חיבור

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 - Transcriptome רחב מיפוי של חלבון-RNA אינטראקציות עם רזולוציה נוקלאוטיד יחיד

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

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.

מדידת קינטיקה של שעתוק mRNA בתאים חיים יחיד

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

טכניקת הארכת פריימר בהתבסס הקרינה לקביעת נקודות התחלת תעתיק ומחשוף אתרים של RNases

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