Najmeh Nikpour and Fiona Alum provided suggestions and edited this manuscript. Department of Biochemistry at McGill University supported HM and VM with the CIHR Training Initiative in Chemical Biology. This work was supported by Canadian Institute of Health Research (CIHR) grant 119464 (to R. S.).

הפרוטוקול שלהלן מתאר את ההליך לביצוע assay עריכת RNA המבוסס על הקרינה. Assay יכול להתבצע בצינור PCR יחיד, 96 היטב, או צלחות תלוי בהיקף של הניסוי 384 גם. בהמשך לכך אות הקרינה ניתן לקרוא במערכת איתור PCR בזמן אמת מתאימה. Assay כאן מתואר בהקשר של צלחות 384 גם. 1. Culturing ט תאי brucei <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את מדיום גידול לט brucei procyclic תאי טופס. עבור 1 ליטר של מדיום: <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפזר אבקת SDM-79 25.4 גרם במי 800 מיליליטר miliQ. </li><li style=";text-align:right;direction:rtl"> הוסף 2 גרם של NaHCO 3 וה-pH ל 7.3 עם 10 M NaOH. </li><li style=";text-align:right;direction:rtl"> מוסיף מים nanopure לנפח סופי של 900 מיליליטר, לסנן לעקר. </li><li style=";text-align:right;direction:rtl"> להוסיף סרום שור עוברי (FBS), פתרון וhemin (2.5 מ&quot;ג / מיליליטר) פניצילין, סטרפטומיצין לריכוזים סופיים של 10% (V / V), 100 U / ml ו7.5 מ&quot;ג / ליטר בהתאמה. </li></ol></li><li style=";text-align:right;direction:rtl"> לגדול 300 מיליליטר של ט &lt;em&gt; סוג 1.7A brucei פראי (טופס procyclic) תאים ב28 ° C, רועד בסל&quot;ד 70 לצפיפות של 1.5 x 10 7 תאים / מיליליטר. הערה: זה אמור לייצר 3 מיליליטר של editosome הפעיל עם ~ 0.5 מ&quot;ג של חלבון סך הכל, מספיק ל600 תגובות עריכה. </li><li style=";text-align:right;direction:rtl"> קציר התאים על ידי צנטריפוגה ב6,000 XG 10 דקות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> שטוף את הכדור עם 50 מיליליטר של חיץ PBSG המקורר (10 מ&quot;מ Na 2 HPO 4, 10 מ&quot;מ אא 2 PO 4, 145 mM NaCl, ו6 גלוקוז מ&quot;מ), וספין למטה התאים שוב על ידי צנטריפוגה ב 10,000 XG 10 דקות ב 4 ° C. </li></ol> 2. בידוד של גולמי המיטוכונדריה הערה: יש לבצע את כל הצעדים על קרח או על 4 מעלות צלזיוס לשמר editosome פעילות. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Resuspend התאים שנקטפו ב30 מיליליטר של חיץ DTE (1 מ&quot;מ טריס-HCl pH 8.0 ו1 mM EDTA). השתמש homogenizer 40 מיליליטר סטרילי Dounce (טרום צונן) לשבש את קרום התא על ידי מלטף את מעלה ומטה לפחות 10 פעמים על קרח. </li&gt; <li style=";text-align:right;direction:rtl"> מייד להוסיף סוכרוז 4.3 מיליליטר של 60% (w / v, כלומר 1.75 ז) לhomogenate לריכוז סופי של 0.25 מ צנטריפוגה ב XG 15,800 עבור 10 דקות ב 4 ° C, כדי להפיל מעדיף מיטוכונדריה. </li><li style=";text-align:right;direction:rtl"> Resuspend את כדור המיטוכונדריה ב4.6 מיליליטר של חיץ STM (20 מ&quot;מ טריס-HCl pH 8.0, 250 סוכרוז מ&quot;מ ו 2 מ&quot;מ MgCl 2). הוספת 13.8 μl של 0.1 M CaCl 2 ו -4 μl של DNase RNase ללא לריכוזים סופיים של 0.3 מ&quot;מ ו -9 U / ml, בהתאמה. דגירה את התערובת במשך שעה 1 על קרח. </li><li style=";text-align:right;direction:rtl"> הוספת 4.6 מיליליטר של חיץ STE (20 מ&quot;מ טריס-HCl pH 8.0, סוכרוז 250 מ&quot;מ ו2 mM EDTA) כדי להשבית DNase I. צנטריפוגה ב XG 15,800 עבור 10 דקות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> Resuspend את הכדור ב400 μl של חיץ תמוגה (10 מ&quot;מ טריס-HCl pH 7.2, 10 מ&quot;מ MgCl 2, 100 מ&quot;מ KCl, 1 מיקרוגרם / מיליליטר pepstatin, 1 מ&quot;מ DTT, ומעכבי פרוטאז 1x שלמים ללא EDTA) והעברה ל צינור microfuge. </li><li style=";text-align:right;direction:rtl"> הוסף 10% טריטון X-100 לריכוז סופי של 1%<html> nd דגירה lysate ל15 דקות ב 4 ° C על הכתף צינור. </li><li style=";text-align:right;direction:rtl"> נקה את lysate המיטוכונדריה על ידי צנטריפוגה פעמיים ב17,000 XG במשך 15 דקות ב 4 ° C; שמירה על supernatant פינה בכל פעם. </li></ol> 3. Editosome טיהור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> יוצקים 10 מיליליטר 10% -30% (v / v) גליצרול שיפוע ליניארי (טבלה 1) בצינור ultracentrifuge באמצעות חיץ שיפוע 2x hhe (40 HEPES מ&quot;מ pH 7.9, 20 מ&quot;מ Mg (OAC) 2, 100 מ&quot;מ KCl, ו 2 mM EDTA) ומכונת שיפוע על ידי ביצוע הוראות במדריך. </li><li style=";text-align:right;direction:rtl"> מוציא בזהירות 500 μl של פתרון מהחלק העליון של שיפוע גליצרול ועדינות לטעון 500 μl של lysate המיטוכונדריה פינה. ספין על 178,000 XG במשך 6 שעות על 4 מעלות צלזיוס באמצעות ultracentrifuge. </li><li style=";text-align:right;direction:rtl"> לאסוף 500 שברים μl ברצף מהחלק העליון לחלק התחתון של השיפוע ב 4 ° C. לאחר מכן הצמדתי להקפיא את השברים באמצעות חנקן נוזל חנות ב -80 ° C עד שימוש. </li></ol>s = &quot;jove_title&quot;&gt; 4. RNA הכנה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לחשל את תבנית ה-DNA המתאימה המכילה רצף משלים רצף אמרגן T7 (טבלה 2) עם oligonucleotide אמרגן T7 (5&#39;-TAATACGACTCACTATAGGG-3 &quot;) ביחס טוחנת 1:01 על ידי חימום ב90 מעלות צלזיוס במשך 3 דקות וקירור ב RT לפחות 10 דקות. </li><li style=";text-align:right;direction:rtl"> לתמלל RNA באמצעות ערכת שעתוק במבחנה על ידי ביצוע הוראות במדריך. </li><li style=";text-align:right;direction:rtl"> עצור את תגובת השעתוק על ידי הוספת נפח שווה של 7 M צבע אוריאה (שתנן 7 M, 0.05% Cynol קסילן, ו0.05% bromophenol כחולים). לרוץ על מסנן מעוקר 9% denaturing ג&#39;ל polyacrylamide (acrylamide 9%, 7 M אוריאה, 1x TBE). </li><li style=";text-align:right;direction:rtl"> השתמש בהצללת אולטרה סגול (UV) עם מנורת UV בגלים קצרים כדי לאתר וRNA בהתאמה בלו. מניחים את חתיכת ג&#39;ל נכרתה בצינור microfuge ולהוסיף 400 μl של חיץ elution ג&#39;ל (20 מ&quot;מ טריס-HCl pH 7.5, 250 מ&quot;מ NaOAc, 1 mM EDTA ו0.25% SDS). Elute הלילה בשעה RT על הכתף צינור. </li><li style=";text-align:right;direction:rtl"> Precipitate RNA eluted על ידי הוספת 1 מיליליטר של אתנול 100% קרים ודוגר גם ב-80 מעלות צלזיוס למשך 30 דקות או -20 מעלות צלזיוס למשך הלילה. </li><li style=";text-align:right;direction:rtl"> צנטריפוגה ב XG 16,000 ל30 דקות ב 4 ° C, כדי גלולה למטה RNA. </li><li style=";text-align:right;direction:rtl"> שטוף את הכדור עם 1 מיליליטר של 75% אתנול. צנטריפוגה ב XG 16,000 עבור 20 דקות ב 4 ° C. </li><li style=";text-align:right;direction:rtl"> Resuspend את כדור RNA כראוי במי RNase ללא כדי להשיג את הריכוזים הרצויים, כפי שמוצג בטבלה 2. </li></ol> 5. Assay RNA עריכת הקרינה מבוססת <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לתגובה אחת, לשלב pmol 1 (μl 1) של preA6Rbz ו2.5 pmol (μl 1) של gA6Rbz (יחס טוחנת 1:2.5) בצינור microfuge, לדגור על 70 מעלות צלזיוס במשך 3 דקות ולתת לו לשבת על RT במשך שעה לפחות 10 דקות. </li><li style=";text-align:right;direction:rtl"> בינתיים מכין תערובת הורים באמצעות טבלה 3, ללא preA6Rbz וgA6Rbz, לתגובת העריכה המכילה חיץ 1x hhe (25 HEPES מ&quot;מ pH 7.9, 10 מ&quot;מ Mg (OAC) 2, 50 מ&quot;מ KCl ו10 mM EDTA), 1 מ&quot;מה-ATP, 5 מ&quot;מ CaCl 2, 16 ng / μl של RNA Torula שמרים, 0.1% טריטון X-100, וeditosome המטוהרים. </li><li style=";text-align:right;direction:rtl"> הוספת preA6Rbz annealed וgA6Rbz כדי להשלים את מיקס מאסטר. </li><li style=";text-align:right;direction:rtl"> לוותר 18 μl של מיקס מאסטר (לוח 3) לתוך בארות המכילות גם 2 μl מים RNase חינם (בארות ללא תרכובות) או 2 μl של 200 תרכובות כימיות מיקרומטר וכולל דגימות שליטה בצלחת על פי איור 5. </li><li style=";text-align:right;direction:rtl"> חותם את הצלחת עם אוטם צלחת ומסובב את הצלחת, כדי להסיר כל בועות אוויר. לדגור על C ° 28 במשך 4 שעות. </li><li style=";text-align:right;direction:rtl"> הוסף 25 pmol (2 μl) של המתחרה gA6Rbz היטב כל אחד. הנח אוטם טרי, לסובב את הצלחת ומניח אותו על מכונה ה-PCR בזמן אמת. לתכנת את הניסוי הבא: שלב 1: 85 מעלות צלזיוס למשך 5 דקות; שלב 2: 24 מעלות צלזיוס למשך 10 דקות; שלב 3: להפסיק. </li><li style=";text-align:right;direction:rtl"> הוסף 15 pmol (μl 1) של מצע סריג היטב כל אחד לנפח סופי של μl 23. חותם את הצלחת עם אוטם טרי.מהירות לסובב את הצלחת ומניח אותה בחזרה במכונה ה-PCR בזמן אמת. </li><li style=";text-align:right;direction:rtl"> תכנית ניסוי חדש בשלבים הבאים: שלב 1: 37 מעלות צלזיוס במשך 1 דקות; שלב 2: קראו; שלב 3: עבור לשלב 1, 40 פעמים; שלב 4: להפסיק. </li><li style=";text-align:right;direction:rtl"> התקנת הצלחת על ידי בחירת כל הבארות הדורשות קריאה ולבחור את מסנן FAM. קלט נפח כמו 23 μl ולהתחיל לרוץ. </li><li style=";text-align:right;direction:rtl"> לחשב את השיפוע של הערכים המתקבלים מכל טוב / מדגם להשיג מדידה קינטית על ידי התוויית המדרונות על גרף עמודות לצורך הניתוח. הערה: קריאה הקינטית משפרת את יחס אות לרעש בין המדגם והרקע; כמדגם הרקע יהיה מדרון קרוב לאפס. יש קריאת נקודת סיום רעש רקע גבוה יותר. </li></ol>

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S., 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=Protein-dependent+ribozymes+report+molecular+interactions+in+real+time.">Protein-dependent ribozymes report molecular interactions in real time.</a> Nature. 20, 717-722 (2002).</li><li>Famulok, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allosteric+aptamers+and+aptazymes+as+probes+for+screening+approaches.">Allosteric aptamers and aptazymes as probes for screening approaches.</a> Current opinion in molecular therapeutics. 7, 137-143 (2005).</li><li>Teng, B., Burant, C. F., Davidson, N. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+an+apolipoprotein+B+messenger+RNA+editing+protein.">Molecular cloning of an apolipoprotein B messenger RNA editing protein.</a> Science. 260, 1816-1819 (1993).</li><li>Jurica, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Searching+for+a+wrench+to+throw+into+the+splicing+machine.">Searching for a wrench to throw into the splicing machine.</a> Nature chemical biology. 4, 3-6 (2008).</li><li>Spahn, C., Prescott, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Throwing+a+spanner+in+the+works:+antibiotics+and+the+translation+apparatus.">Throwing a spanner in the works: antibiotics and the translation apparatus.</a> Journal of molecular medicine. 74, 423-439 (1996).</li></ol>

The authors have nothing to disclose.

שיטת הקרנת תפוקה גבוהה רומן לזהות מעכבים נגד מורכב עריכת RNA של trypanosomes הוצגה, מתן כלי חדש לגילוי תרופות נגד מחלות הנגרמות על ידי trypanosomatids. assay ribozyme מבוסס סריג כבר נעשה שימוש נרחב למטרות שונות 20-22; עם זאת, יש לנו מנוצלים הקיבולת של assay ribozyme מבוסס סריג לניטור במבחנה<...

תהליך עריכת רנ&quot;א, שינוי mRNA לאחר תעתיק, התגלה לראשונה בtrypanosomatids 1. מאז, עבודה משמעותית כבר נערכה בלומדת את המנגנון עומד מאחורי עריכת רנ&quot;א בTrypanosoma brucei 2,3. בסדרה של תגובות אנזימטיות, editosome, מורכב ליבה של כ 20 חלבונים, יוצר mRNAs המיטוכונדריה הבוגר לרכיבים מרובים של מערכת זרחון חמצוני להפקת אנרגיה. סדר אירועי קטליטי הוא מחשוף endonucleolytic, uridylate בנוסף (U) או מחיקה, וקשירה, כפי שהוכתב על ידי RNAs מדריך (gRNAs) 4. בנוסף לחלבוני editosome המורכבים הליבה, מספר גורמי אבזר יש גם זוהה 5-7. חלבונים אלה נראים בעיקר מקובצים במתחמים עצמאיים. עם זאת, את סדר ההרכבה חלבון במתחם editosome הליבה ודפוסי האינטראקציה של מתחם הליבה עם האבזרמתחמים הם עדיין לא נקבעו. מיקוד תהליך עריכת רנ&quot;א בtrypanosomatids עשויה לספק dissectors הכימי המסייעים בלימוד ההרכבה והתפקוד של מתחם editosome. יתר על כן, מחקרים תפקודיים בכמה חלבוני editosome הראו חיוניות פני שלבי חיים שונים, המצביע על הפוטנציאל שלהם כתרופת מטרות 8-12. לכן, מעכבים מצאו של editosome יכולים גם לפעול כתרכובות עופרת נגד trypanosomatids. זה זמן, כמו תרופות הקיימות כיום נגד מחלות הנגרמות על ידי trypanosomatid הן רעילות, לא יעילות ויקרות 13,14. Assay יעיל ונוח במבחנה יש צורך לחקור את היקום הכימי למעכבים ספציפיים שחוסמים עריכת רנ&quot;א. שלושה מבחני פותחו ומשמשים לניטור פעילות editosome: (א) עגול מלאה בassay עריכת רנ&quot;א המבחנה 15, (ב) לפני שהבקיע בassay עריכת רנ&quot;א המבחנה 16,17,nd (ג) ribozyme פטיש (HHR) מבוסס assay 18. שני מבחני הראשונים מסתמכים על הדמיה ישירה של המוצר בעריכה (ATPase 6 mRNA) עם העזרה של קרינה רדיואקטיבית. Assay מבוסס HHR משתמש בגרסה שונה של mRNA ATPase 6 שהוא מודל להתנהג כribozyme על עריכה. Ribozyme הפונקציונלי אז דווקא דבק מצע RNA רדיואקטיבי, המשרת ככתב. לאחרונה, Moshiri et al. פיתח &#39;לערבב ולמדוד &quot;מבוסס HHR בassay כתב מבחנה כדי לפקח על עריכת RNA שבו מצע RNA רדיואקטיבי מוחלף בהעברת תהודה הקרינה אנרגיה (סריג) מצע 19. יתרונות העיקרון של assay זה הם: (א) זה הוא שילוב מהיר ונוח וסוג המידה של assay, כמו הייצור של ribozyme הפעיל ומחשוף מצע מתרחש בו זמנית באותו הצינור בנפח נמוך (כלומר 20 μl), (ב רגישות) זה ימנע את השימוש בחומרים שכותרתו רדיואקטיבית, (ג) שהואfforded ידי מכשור הקרינה בפורמט צלחת מייקר כייל, ואות גבוהה יחס רעש (ד). שימוש assay זה, ההשפעה של מעכבים ידועים RNA עריכת האנזים נגד editosome מטוהר אושרה 19. ניסוי זה תוקף את assay לזיהוי מהיר של מעכבי עריכת רנ&quot;א, בעיקר נגד editosomes השלם מט brucei. איור 1 הוא סכמטי צעד אחר צעד מפורט של בassay עריכת רנ&quot;א מבחנה הקרינה מבוסס. פרוטוקול זה יכול לשמש לניטור עריכת RNA במבחנה או בקלות להיות מותאם להקרנת ספריות מתחם של קני מידה שונות.

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rna catalyst as a reporter for screening drugs against rna editing in trypanosomes

התקדמות משמעותית נעשתה בקביעת המנגנון של עריכת רנ&quot;א המיטוכונדריה בtrypanosomes. בדומה לכך, התקדמות ניכרת נעשתה בזיהוי הרכיבים של מתחם editosome כי לזרז עריכת רנ&quot;א. עם זאת, זה עדיין לא ברור כיצד חלבונים אלו לעבוד יחד. תרכובות כימיות המתקבלות ממסך תפוקה גבוהה נגד editosome עלולות לחסום או להשפיע שלב אחד או יותר במחזור העריכה. לכן, זיהוי של תרכובות כימיות חדשות יפיק בדיקות מולקולריות רבות ערך עבור לנתח את פונקצית editosome והרכבה. במחקרים קודמים, במבחני חוץ גופית עריכה בוצעו באמצעות RNA שכותרתו רדיו. מבחני אלה הם זמן רב, לא יעיל ואינם מתאים למטרות תפוקה גבוהה. כאן, המבוסס על הקרינה הומוגני &quot;לערבב ולמדוד&quot; פטיש ribozyme במבחנה assay כתב לפקח על עריכת רנ&quot;א, מוצג. רק כתוצאה של עריכת RNA שלribozyme פטיש מצע oligoribonucleotide תהודה העברת אנרגיית הקרינה (סריג) עובר מחשוף. זה בתורו גורם להפרדה של fluorophore מהמרווה ובכך לייצר אות. לעומת זאת, כאשר פונקצית editosome היא עצורה, אות הקרינה תהיה הרווה. זה assay רגיש והפשוט ביותר שצריכה להיות באופן כללי החלים על לפקח בעריכת RNA חוץ גופית או הקרנת תפוקה גבוהה של כימיקלים שיכולים לעכב את פונקצית editosome.

כדי להדגים הנדרשים להקמת מסך בקנה מידה גדולה בכל הצעדים הדרושים, איורים 2-5 הם ניסויי שליטת נציג הקשורים לאיכות של assay. אלה הם ניסויי שליטה חיוניים לassay עקבי על פני כמה ימים של הקרנה או להשוואה בין מסכים שונה. הערכת יחס ...

Watch this Scientific Journal Video about RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes at JoVE.com

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

RNA Catalyst ככתב להקרנת סמים נגד RNA עריכה בtrypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

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8.4K Views.  McGill University. A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

Video: RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

הדמיה ב-RNA לוקליזציה Xenopus ביציות

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

בתעתיק במבחנה תקרת של mRNA בלוציפראז Gaussia ואחריו transfection Cell הלה

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

בידוד הכרומטין על ידי טיהור RNA (ציוץ)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

דור מצע לEndonucleases של מערכות CRISPR / קאש

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

דלדול של רנ&quot;א ריבוזומלי לMetagenomic גוט יתוש RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

רנ&quot;א<em&gt; באתר</em&gt; הכלאה בעוברי הר שלמים והיסטולוגיה הסלולרית מותאמת לElasmobranchs הימי

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

הליך מיון כימי לGlucocorticoid איתות עם מערכת כתב דג הזברה הזחל בלוציפראז

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

זיהוי של חלבונים קושרי RNA על ידי<em&gt; במבחנה</em&gt; RNA הנפתח ב adipocyte תרבות

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

שימוש מעשי של RNA הפרעה: משלוח אוראלי של זוגיות גדילי RNA ב ליפוזום ספקים עבור ג ' וקים

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

Imaging FITC-dextran as a Reporter for Regulated Exocytosis

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated exocytosis is fundamental for intercellular communication and is a key mechanism for the secretion of neurotransmitters, hormones, inflammatory mediators, and other compounds, by a variety of cells. At least three distinct mechanisms are known for regulated exocytosis: full exocytosis, where a single SG fully fuses with the plasma membrane, kiss-and-run exocytosis, where a single SG transiently fuses with the plasma membrane, and compound exocytosis, where several SGs fuse with each other, prior to or after SG fusion with the plasma membrane. The type of regulated exocytosis undertaken by a cell is often dictated by the type of secretory trigger. However, in many cells, a single secretory trigger can activate multiple modes of regulated exocytosis simultaneously. Despite their abundance and importance across cell types and species, the mechanisms that determine the different modes of secretion are largely unresolved. One of the main challenges in investigating the different modes of regulated exocytosis, is the difficulty in distinguishing between them as well as exploring them separately. Here we describe the use of fluorescein isothiocyanate (FITC)-dextran as an exocytosis reporter, and live cell imaging, to differentiate between the different pathways of regulated exocytosis, focusing on compound exocytosis, based on the robustness and duration of the exocytic events.

Here we detail a method for live cell imaging of regulated exocytosis. This method utilizes FITC-dextran, which accumulates in lysosome-related organelles, as a reporter. This simple method also allows distinguishing between different modes of regulated exocytosis in cells that are difficult to manipulate genetically.

הדמיה FITC-לתוספי כעיתונאית אקסוציטוזה מוסדר

Regulated exocytosis is a process by which cargo, which is stored in secretory granules (SGs), is released in response to a secretory trigger. Regulated ...