This research was supported by grant from INSERM, University of Lille 1, University of Lille 2, Regional Hospital Center of Lille (CHR de Lille). MCCH acknowledges supports from the Fondation de France and wishes to thank IRCL, Pr. Sonenberg for the gift of the V5-plasmids, Dr. Dissous (Pasteur Institute, Lille) for the gift of anti-GFP antibodies, UMS 3387 (University of Rennes) where Xenopus oocytes are purchased and Dr Taymans (JPArc, Lille) for critical reading of the manuscript text.

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The authors have nothing to disclose.

תרגום הוא מנגנון המעורב בphysiopathology של הפרעות אנושיות רבות, כולל כמה מחלות ניווניות. למשל במחלת פרקינסון כמה דיווחים הציעו ירידת הערך בתרגום הקשורים מוטציות תורשתיות 8,12,13. כמה דגמים סלולריים זמינים ללמוד תרגום. כאן, על מנת לל?...

חלבונים הם מרכיבים חיוניים של חיים סלולריים ובכך בקנה מידה גדולה יותר של האורגניזם. הם להבטיח רוב הפונקציות סלולריות כוללים מבנה, תחבורה, קטליזה תגובה, רגולציה, ביטוי גנים וכו &#39;הביטוי שלהם הוא התוצאה של מנגנון מורכב של תרגום המאפשר המרה של mRNA לחלבון. התרגום הוא נתון לבקרה שונות להסתגל ולהסדיר ביטוי גנים על פי צרכי התא, במהלך התפתחות והתמיינות, הזדקנות, לחצים פיסיולוגיים או פתולוגי גילויים. תרגום מחולק ל 3 שלבים (ייזום, התארכות וסיום) ומתנות 3 מערכות תרגום ייזום כדי לענות על צרכי אלה: כובע תלוי, כובע-עצמאי באמצעות מגזר הפנימי הריבוזום כניסה (IRES) מבנים וכובע-עצמאי משפרי תרגום ( CITE). רוב mRNA אוקריוטים מתורגמים בכובע-depeאופן ndent באמצעות כובע 5&#39;-טריפוספט 7-methylguanosine המשמש כתכונת זיהוי במהלך סינתזת חלבון. כובע זה נקשר לeIF4E, רכיב של מורכב eIF4F עם eIF4G1 וeIF4A. קשור עם שותפים אחרים כמו פולי () חלבון מחייב (PABP), eIF2-GTP-Met-tRNA Met, גורמי חניכת תרגום אלה מאפשרים לcircularize mRNA ולשפר את הנגישות שלה לצורות מורכבות 43s עד ייזום אוגוסט קודון הכרת 1. אירוע זה מתאים לסוף כלומר ייזום תרגום, הצעד הראשון של תרגום. תרגום Cap-עצמאי משמש קידוד mRNA לחלבונים חיוניים בתנאים הדגישו כי לגרום להתפשטות תאים למשל ואפופטוזיס. מנגנון זה כולל מבנים משניים בmRNA 5&#39;- אזור מתורגם (UTR) נקרא IRES, סוף carboxy מסוף של eIF4G1 הקשורים eIF4A והמורכב 43s. כריכה של זה 43s מראש הייזום גomplex לIRES יוזם את התרגום העצמאי הכובע ללא הצורך בגורם eIF4E 2,3. לבסוף, מנגנון תרגום אחר עדיין לא הבין היטב תומך בפעילות תרגום כובע-העצמאי הזה, לפי תנאים הדגישו באמצעות CITE מבנים הנמצאים בתוך ה- mRNA 4 UTR. דרך אלה מצבים השונים של התרגום השונה על ידי צעדי החניכה שלהם, תרגום משחק תפקיד קריטי בהומאוסטזיס סלולארי וכל שינוי באחד מהתהליכים אלה ובכך ישפיעו האורגניזם עם קטן לתופעות בקנה מידה גדולות. ואכן, החניכה היא צעד שיעור הגבלת שלטון תהליכי התרגום הנכון של mRNA לחלבונים ולכן היעד של מספר רב של בקרות ונקודות תקנה 5. בין אם מדובר באפשרות השנייה או לרכיבים של תהליכים אלה, אם אחד הופך להיות פגומים, זה יהיה לטרוד את האיזון שנקבע בתא ובכך עלול להוביל לקונדי פתולוגייםמשא. בהקשר זה, מוטציות בגורמי תרגום היו מעורבות במספר הפרעות, כולל הפרעות ניווניות כגון `leukoencephalopathy עם ההיעלמות לבנה matter&#39; (מקטע eIF2B1-5) 6, בתסמונת וולקוט-Rallison (קידוד EIF2AK3 גן ל&quot;הערכה&quot;) 7, שעלול להיות ב המחלה (p.R1205H eIF4G1) פרקינסון 8. לכן חשוב לבצע מחקרים תאיים ומולקולריים של חלבוני מוטציה אלה כדי להגדיל את הידע שלנו על התפתחות מחלה ועל התהליך הכללי של חניכת תרגום. כדי לבצע את המחקרים הללו, זה חיוני כדי לבחור את הדגמים המתאימים ביותר לקיים את ההשלכות של מוטציות אלה Xenopus laevis ביציות מותאמות במיוחד גם בשל המאפיינים הפיסיולוגיים וביוכימיים שלהם:. סינכרוניות הפיזיולוגית (חסום בG2 שלב של מחזור התא) , קיבולת גבוהה של סינתזת חלבון (200-400 ng / יום / ביצית), מספר גבוה של OOC חולץytes מאותה חיה (800-1,000 ביציות / נקבה) וגודל תא (1.2-1.4 מ&quot;מ קוטר) המאפשר המניפולציה שלהם. Microinjection של ביציות Xenopus עם mRNA מסונתז בקלות ניתן לבצע כדי לנתח את הפעולות תרגום. בתצוגה זו מציג יתרונות אחרים. בהתחשב במהירות של התקדמות מיוזה ותרגום לאחר microinjection mRNA (~ שעה 24), ביצית Xenopus מייצגת מערכת מהירה בהשוואה למערכות מחדש סלולריות (שחולצו מE.coli, חיידקי חיטה או reticulocyte ארנב ...) שבו mRNA הוא מתורגם עם שיעור מופחת ותרגום במהירות נמוכה יותר. אז, את ההשפעות של מוטציה הציגה בmRNA תהיה במהירות לצפייה ולמד בקלות בכמה ביציות. יתרון נוסף של ביציות Xenopus הוא שmRNAs האימהי הוא סמוי ותרגום חלבון חסום לפני גירוי פרוגסטרון. תוספת של פרוגסטרון היא אפוא אמצעי טוב של השליטה האינדוקציה התרגום. p cytoplasmicolyadenylation אינו מתרחש במהלך oogenesis. זה מתחיל בהבשלת ביצית בביציות מגורה פרוגסטרון בצו זמני וממשיך לאורך התפתחות מוקדמת ויכול לשמש כדי לחקור את השלבים השונים של תרגום. Polyadenylation של Mos mRNA הוא בין הראשונים שמתרחשים והוא שייך עם אורורה / Eg2, היסטון-כמו B4 mRNA לכיתה של גנים &quot;התבגרות המוקדמת&quot; כהגדרתו בCharlesworth et al. (2004) 9. האינדוקציה translational של mRNA &quot;מאוחר&quot; כגון Cyclin A1 וCyclin B1 מתרחשת בערך בזמן של התמוטטות נבטי שלפוחית ​​(GVBD). Mos mRNA מקודד קינאז סרין / תראונין חלבון. התרגום שלה הוא קריטי שכן הוא גורם למפל קינאז מפה שבעקיפין מפעיל את הבשלת הביצית. ואכן, בתגובה לפרוגסטרון, polyadenylation של Mos mRNA מוגבר באמצעות תהליך הכרוך אורורה / Eg2 חלבונים רגולטוריים וחלבוני RNA מחייב אחרים עם ההדואר 3&#39;UTR של Mos mRNA. polyadenylation המוגבר של Mos mRNA מוביל לעלייה של רמת חלבון mos, אשר בתורו מפעילה MEK1. תהליך זה מתווך את ההפעלה של קינאז מוסדר האיתות תאי 2 (ERK2) (איור 1). מפל איתות זה יכול לגרום לאז M-שלב התבגרות קידום גורמים, מורכב נוצר על ידי קינאז Cyclin B וCdc2, וסופו של דבר גורם לחידוש meiotic. לכן בXenopus laevis ביציות, יכול לשמש בקלות המחקר של mRNA האימהי כגון Mos לבדוק translatability עם כמה נקודות קצה מpolyadenylation היעילה שלהם לתרגום של כמה Mos איתות רכיבים, הכולל גם את קביעת שיעור GVBD. מערכת זו היא מעניינת ולכן כדי להעריך את ההשלכות הראשונות של מוטציות בגורמי חניכת תרגום ללא התערבות של mRNA עיבד חדש או של יעילות transfection, בעיות לעתים קרובות מתרחשות עם eukaryמחקרי תא otic. כאן, פרוטוקול הוקם בי mRNAs eIF4G1 מוטצית microinjected בXenopus laevis ביציות ובתרגום של mRNA האימהי נבדק. בנוכחות של פגם בהתקדמות GVBD, Mos polyadenylation mRNA שהוא חיוני להתקדמות דרך מחזור תא ביצית meiotic ולתרגום הבא של mRNAs כיתה המוקדם והמאוחר הוא הוברר. זירחון אורורה / Eg2 וERK הוא גם למד כדי לאשר את התוצאה של deregulation.Thus mos, ביציות Xenopus מייצגות דרך פשוטה כדי לנתח את הפעולות שונות של תרגום mRNA.

<table><tbody><tr><td>Tricaine methane sulphonate</td><td>Sandoz</td><td>MS-222</td><td>oocytes handling</td></tr><tr><td>Forceps</td><td>Moria</td><td>Dumont MC40</td><td></td></tr><tr><td>Streptomycin/penicillin</td><td>Sigma</td><td>781</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P2256</td><td></td></tr><tr><td>Soybean trypsin inhibitor</td><td>Sigma</td><td>T9128</td><td></td></tr><tr><td>Tetracyclin</td><td>Sigma</td><td>T7660</td><td></td></tr><tr><td>Veterinarian absorbable Vicryl thread</td><td>Johnson&amp;amp;Johson Intl</td><td>JV1205</td><td></td></tr><tr><td>Collagenase A</td><td>Roche diagnostics</td><td>10103586001</td><td></td></tr><tr><td>PmeI</td><td>New England Biolabs</td><td>R0560S</td><td>Preparation of synthetic mRNA</td></tr><tr><td>DNAse/RNAse free H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Life Technologies</td><td>10977</td><td></td></tr><tr><td>Sodium Acetate</td><td>Merck</td><td>6268</td><td></td></tr><tr><td>Absolute ethanol</td><td>Sigma</td><td>02854</td><td></td></tr><tr><td>Nano Drop</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>TBE 10X</td><td>Eppendorf</td><td>0032006.507</td><td></td></tr><tr><td>Ethidium bromide 10 mg/ml</td><td>Life Technologies</td><td>15585-011</td><td></td></tr><tr><td>mMESSAGE mMACHINE Kit</td><td>Ambion by Life Technologies</td><td>AM1344</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td></td></tr><tr><td>EDTA</td><td>Fluka</td><td>03609</td><td></td></tr><tr><td>Agarose</td><td>Life Technologies</td><td>16500-500</td><td></td></tr><tr><td>Formaldehyde 37%</td><td>Merck</td><td>1.04003.1000</td><td></td></tr><tr><td>Formamide</td><td>Fluka</td><td>47671</td><td></td></tr><tr><td>Gel Doc Imager</td><td>Biorad</td><td></td><td></td></tr><tr><td>Oocyte Pipet with 100 8&quot; Glass Capillaries</td><td>Drummond Scientific Company</td><td>3-000-510-X</td><td>microinjection</td></tr><tr><td>Replacement Glass 8&quot;</td><td>Drummond Scientific Company</td><td>3-000-210-G8</td><td></td></tr><tr><td>0.45 &amp;micro;m filter</td><td>Millipore</td><td>SLHA025NB</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P0130</td><td></td></tr><tr><td>Hepes</td><td>Sigma</td><td>H3375</td><td>Western Blot</td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>SDS</td><td>Biorad</td><td>161-0301</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>A3294</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma</td><td>A4612</td><td></td></tr><tr><td>leupeptin</td><td>Sigma</td><td>L8511</td><td></td></tr><tr><td>aprotinin</td><td>Sigma</td><td>A1153</td><td></td></tr><tr><td>benzamidine</td><td>Sigma</td><td>B6506</td><td></td></tr><tr><td>PMSF</td><td>Sigma</td><td>P7626</td><td></td></tr><tr><td>sodium vanadate</td><td>Sigma</td><td>S6508</td><td></td></tr><tr><td>Laemmli buffer</td><td>Biorad</td><td>161-0737</td><td></td></tr><tr><td>NuPAGE Novex 4-12% Bis-Tris Protein Gels, 1.0 mm, 15 well</td><td>Life Technologies</td><td>NP0323BOX</td><td></td></tr><tr><td>SDS-PAGE electrophoresis, mini-Protean TGX</td><td>Biorad</td><td>456-1036 and -1096</td><td></td></tr><tr><td>horizontal semi-dry blotting system</td><td>W.E.P. Compagny</td><td></td><td></td></tr><tr><td>Glycine</td><td>Biorad</td><td>161-0718</td><td></td></tr><tr><td>Tris-HCl</td><td>Biorad</td><td>161-0719</td><td></td></tr><tr><td>Hybond ECL Membrane</td><td>Amersham Life Science</td><td>10401180</td><td></td></tr><tr><td>Methanol</td><td>VWR</td><td>20846-292</td><td></td></tr><tr><td>Ponceau Red (0.5%)</td><td>Sigma</td><td>P3504</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P2287</td><td></td></tr><tr><td>anti-GFP</td><td>Life Technologies</td><td>A11122</td><td></td></tr><tr><td>anti-V5</td><td>Santa Cruz Biotechnology</td><td>sc-58052</td><td></td></tr><tr><td>anti-Eg2</td><td>Santa Cruz Biotechnology</td><td>sc-27884</td><td></td></tr><tr><td>anti-Eg2-P</td><td>Cell Signaling</td><td>C39D8</td><td></td></tr><tr><td>anti-ERK2</td><td>Santa Cruz Biotechnology</td><td>sc-1647</td><td></td></tr><tr><td>anti-ERK2-P (Tyr204)</td><td>Santa Cruz Biotechnology</td><td>sc-7976</td><td></td></tr><tr><td>anti-Rsk</td><td>Santa Cruz Biotechnology</td><td>sc-231</td><td></td></tr><tr><td>anti-mos</td><td>Santa Cruz Biotechnology</td><td>sc-86</td><td></td></tr><tr><td>anti-mouse horseradish peroxidase labeled secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc-2005</td><td></td></tr><tr><td>anti-rabbit horseradish peroxidase labeled secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc-2812</td><td></td></tr><tr><td>anti-goat horseradish peroxidase labeled secondary antibody</td><td>Santa Cruz Biotechnology</td><td>sc-2378</td><td></td></tr><tr><td>Advanced ECL Detection System</td><td>Amershan Life Science</td><td>RPN2135</td><td></td></tr><tr><td>PBS 1x</td><td>Sigma</td><td>P4417</td><td>polyadenylation assay</td></tr><tr><td>TRIZOL (Qiazol Lysis Reagent)</td><td>Qiagen</td><td>79306</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>31998-8</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>278475-1L</td><td></td></tr><tr><td>RNeasy mini kit</td><td>Qiagen</td><td>74106</td><td></td></tr><tr><td>RTL buffer&amp;nbsp;</td><td>Qiagen</td><td>79216</td><td></td></tr><tr><td>RNAse free DNAse set</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Primers</td><td>Eurogentec</td><td></td><td></td></tr><tr><td>T4 RNA ligase 1&amp;nbsp;</td><td>New England Biolabs</td><td>M0204S</td><td></td></tr><tr><td>High capacity c-DNA Reverse Transcription kit</td><td>Applied Biosystems, Life Technologies</td><td>4368813</td><td></td></tr><tr><td>Taq polymerase</td><td>Life Technologies</td><td>10342020</td><td></td></tr></tbody></table>

xenopus laevis as a model to identify translation impairment

סינתזת חלבון היא תהליך בסיסי לביטוי גנים המשפיעים על תהליכים ביולוגיים מגוונים בעיקר הסתגלות לתנאי סביבה. צעד הייזום, הכולל הרכבה של תת-יחידות ריבוזומלי בmRNA קודון החניכה, הגורם מעורב ייזום כולל eIF4G1. פגמים בשלב זה שיעור הגבלה של תרגום צמודים להפרעות שונות. כדי לחקור את ההשלכות הפוטנציאליות של deregulations כזה, Xenopus laevis ביציות מהוות מודל אטרקטיבי עם מעלות גבוהות של שימור של מנגנונים תאיים ומולקולריים חיוניים עם אדם. בנוסף, במהלך התבגרות meiotic, ביציות תעתיק מודחקות וכל החלבונים הדרושים מתורגמים מקיימים מראש mRNAs, נגזר אימהי. מודל זה מאפשר זול mRNA אקסוגניים להשתלב בצורה מושלמת עם תרגום יעיל. כאן מתואר פרוטוקול להערכת תרגום עם גורם עניין (כאן eIF4G1) באמצעות storאד mRNA האימהי שהם ראשון להיות polyadenylated ותורגם במהלך הבשלת ביצית כקריאת נתונים פיזיולוגית. בתחילה, ה- mRNA synthetized ידי שעתוק במבחנה של פלסמידים של עניין (כאן eIF4G1) מוזרקים בביציות וקינטיקה של הבשלת ביצית על ידי זיהוי התפלגות רמינל שלפוחיות נקבע. יעד mRNA האימהי למד הוא סרין / Mos תראונין חלבון-קינאז. polyadenylation והתרגום הבא שלה נחקרים יחד עם הביטוי וזירחון של חלבונים של Mos מפל איתות מעורב בהבשלת ביצית. וריאציות של הפרוטוקול הנוכחי לשים קדימה פגמי translational גם הציעו להדגיש תחולתה הכללית. לאור ראיות שמתעוררות סינתזת חלבון חריגה עשויה להיות מעורבת בפתוגנזה של הפרעות נוירולוגיות, מודל כזה מספק את ההזדמנות כדי להעריך בקלות ירידת ערך זו ולזהות מטרות חדשות.

התבגרות הקינטית של ביציות Xenopus ונחישות של GVBD ביצית אחוז לאחר 24 שעות של גירוי PG (2B דמויות, 2C): על מנת ללמוד את ההשלכות של התרגום של המוטציה eIF4G1-DN, התגובה לPG ב Xenopus laevis ביציות microinjected עם קרנה eIF4G1-DN הוא בהשוואה לeIF4G1-WT ול...

Watch this Scientific Journal Video about Xenopus laevis as a Model to Identify Translation Impairment at JoVE.com

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis כמודל לזיהוי תרגום ירידת ערך

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

xenopus-laevis-as-a-model-to-identify-translation-impairment

Research

JoVE Journal

Biology

Xenopus laevis as a Model to Identify Translation Impairment

10.7K Views.  INSERM UMR-S1172, CHRU Lille, University of Lille 1 and Lille 2. Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Video: Xenopus laevis as a Model to Identify Translation Impairment

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Immunofluorescence כמותי מודדים הגלובלי לשפות אחרות תרגום

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemistry

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Polysome פרופיל לישמניה, תאים אנושיים, Testis העכבר

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

הגדרת התוכנית של תרגום mRNA אימהי במהלך התבגרות במבחנה באמצעות בדיקה של כתב ביצית יחיד

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

Developmental Biology

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

מדידה של שיעורי הטעויות הספציפיים של פטרת מערכות כתבת

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

Immunology and Infection

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

הערכה תפקודית של חוש הריח מסלולים ב ראשנים של צפרדע רפואית חי

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as ...

Neuroscience

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

בדוגמנות Vivo של הגנום האנושי החולנית באמצעות Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

ייצור של<em&gt; Xenopus tropicalis</em&gt; ביצה מחלץ לזהות RNAs microtubule הקשורים

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Analysis of Translation in the Developing Mouse Brain using Polysome Profiling

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell types. This balance is tightly controlled by gene expression that is fine-tuned at multiple levels, including transcription, post-transcription and translation. In this regard, a growing body of evidence highlights a critical role of translational regulation in coordinating neural stem cell fate decisions. Polysome fractionation is a powerful tool for the assessment of mRNA translational status at both global and individual gene levels. Here, we present an in-house polysome profiling pipeline to assess translational efficiency in cells from the developing mouse cerebral cortex. We describe the protocols for sucrose gradient preparation, tissue lysis, ultracentrifugation and fractionation-based analysis of mRNA translational status.

The development of the mammalian brain requires proper control of gene expression at the level of translation. Here, we describe a polysome profiling system with an easy-to-assemble sucrose gradient-making and fractionation platform to assess the translational status of mRNAs in the developing brain.

ניתוח תרגום במוח העכבר המתפתח באמצעות פרופיל פוליזום

The proper development of the mammalian brain relies on a fine balance of neural stem cell proliferation and differentiation into different neural cell ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

הערכת סלקטיבי mRNA התרגום בתאי יונקים ידי Polysome פרופיל

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...