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.

所有的爪蟾实验在里尔第一大学的动物实验室根据欧盟委员会的准则（86/609 / EEC），实验室动物实验的规定执行。动物方案经当地机构审查委员会（科米特德Ethique连接实验安尼米尔北部 - 加来海峡，CEEA 07/2010）。 1.卵母细胞处理<ol><li>准备麻醉剂溶液:溶于1升的无菌水1克三卡因甲基磺酸粉末。 </li><li>切入女性爪蟾到这个解决方案，覆盖了烧杯，以避免逃逸，并等待大约45分钟的动物完全镇静剂（没有任何反应，一条腿捏）。 </li><li>用肥皂清洗青蛙和用自来水冲洗然后将其在清洁的铝箔回动物。 </li><li>夹紧腹部的侧部和在卵巢水平与钳皮肤。使大约1厘米长的切口，用剪刀用70％乙醇，使用前清洗。确保部分足够深，以达到潜在的腹壁，使卵巢组织中的几个卵母细胞发育的不同阶段被发现的切除。 </li><li>解剖卵巢裂片，在培养皿中（50毫米）与ND96介质（96毫摩尔NaCl，2mM的氯化钾冲洗4次，1毫氯化镁 ，1.8毫氯化钙 ，5mM的HEPES调节至pH 7.4，用NaOH为辅用50微克/ ml链霉素/青霉素，225微克/毫升丙酮酸钠，30微克/毫升大豆胰蛋白酶抑制剂，1微升/毫升四环素），除去血液和碎屑所有痕迹。 注:四环素允许最佳的保护和显微注射治疗后恢复良好。 </li><li>其存储在覆盖的培养皿在14℃下浸没在介质中，从而允许它们的保存一周。 </li><li>缝合腹壁并与兽医可吸收吨皮肤hread和缝合针（3或4针将是必要的）。 </li><li>放置在一个烧杯中的动物没有水和潮湿的皮肤用自来水直到蛙被再次移动。盖烧杯，以防止逃逸。 </li><li>通过使用下，用10倍的倍率在立体显微镜2对钳子的仔细分离中在5至10的卵母细胞群在介质卵巢瓣。在第六阶段选择的卵母细胞。他们可以通过我承认）的形状和颜色，用棕色色素动物极和黄色植物极有明确的皮带二）其规模优于1.2毫米直径10分开。 </li><li> 45分钟期间孵育（ 溶组织梭菌，溶解在ND96无四环素1毫克/毫升胶原酶A）中在缓慢搅拌下培养皿胶原酶溶液所选的卵母细胞，以促进卵母细胞defolliculation（胶原酶消化卵母细胞/滤泡细胞连接）。冲洗卵母细胞3或4次，ND96介质保持在14C。 注意:不要孵化的卵母细胞少于或多于45分钟，由于破坏卵母细胞表面的蛋白质，造成生存能力差的风险。胶原酶治疗可引起自发性GVBD。 </li><li>在3-4小时的中等孵化的卵母细胞。在双目放大镜删除滤泡细胞用细镊子，并保持它们在19℃的ND96网上平台。 </li></ol> 2.准备mRNA合成<ol><li>通过使用PME我酶酶切线性化5微克以下质粒。 注:pcDNA6.2 /含1599个氨基酸eIF4G1基因野生型（eIF4G1-WT; NM_198241）V5-DEST，包含eIF4G1显性基因（eIF4G1-DN）pcDNA6.2 / V5-DEST有突变c.2105T "G c.2106A&gt; C，c.2120-&gt; G，c.2122T&gt; A，2125T&gt; -和c.2126T"G在eIF4G1和eIF4E的相互作用在位置612到相应的蛋白质618的区域干扰互动betwe恩eIF4G1和eIF4E的8，pcDNA6.2 / C-EmGFP含氯霉素乙酰转移酶（GFP）。准备2的混合物为每个3质粒:第一包括PME I酶和第二不含酶作为对照。 </li><li>使用经典乙醇沉淀过程质粒DNA重悬它于20μl不含核酸酶的H 2 O的</li><li>使用分光光度计测定其浓度。浓度要优于150毫微克/微升转录的cRNA。 </li><li>运行1微升样本以及它们与5微升加样缓冲液在0.8％琼脂糖凝胶对照来验证质粒线性化。 </li><li>使用体外转录，并根据制造商的说明纯化的cRNA的试剂盒。转录的cRNA再悬浮于20μl不含核酸酶的H 2 O的样品可以贮存于-80℃下，如果需要的。 </li><li>准备含有0.2M MOPS（pH值7.0），20米MOPS 10X迁移缓冲液3M乙酸钠和10mM EDTA（pH8.0）中。消毒用0.45μm过滤溶液。库存保护免受光在RT以避免溶液氧化该溶液中。 </li><li>先后清洗电泳槽用NaOH，HCl和双过滤水的O彻底冲洗/ N，以避免RNA酶，防止RNA降解。 </li><li>投含有MOPS和甲醛1.5％的琼脂糖凝胶。温馨直至完全溶解琼脂糖，让它冷却至55℃。下通风橱中，添加0.1体积的MOPS 10X的，甲醛的6.6％和4微克溴化乙锭（10毫克/毫升）。倾通风柜下凝胶并等待凝胶变硬大约1小时。 注意:甲醛和溴化乙锭是有毒的。 </li><li>制备样品迁移在一个0.2毫升的试管用1微升的cRNA，甲醛的8.8％，甲酰胺的60％和0.1体积的MOPS 10X的。孵育3分钟，在70℃，把管冰浴10分钟。离心机样品几秒钟5000×g的。加入2微升凝​​胶上样缓冲液中。 注意:甲酰胺是有毒的。 </li><li>运行样品20分钟，在90V。浸泡在无核酸酶H 2 OO / N凝胶分析它来检查质量的cRNA之前脱色凝胶。 </li><li>确定的cRNA浓度使用分光光度计，并将它们存储在-80℃。 </li></ol> 3.显微注射合成的RNA和卵母细胞成熟刺激<ol><li>准备必要的设备，卵母细胞显微注射。使用微量拉马拉小玻璃毛细管。体视显微镜下，断绝毛细管的末端与镊子以创建一个平端。使用1ml注射器微孔0.45μm的过滤在相对的末端填充毛细管微量矿物油。安装微量到显微注射吸管。选择一个精确的微量校正由制造商提供良好的精度，适当的玻璃毛细管中使用时。 </li><li>执行注射defolliculation后1-2小​​时，需要延迟对卵母细胞的卵母细胞的恢复保持在14℃。使用培养皿的底部刮下用镊子创建用于卵母细胞的粘合表面和ND96介质填充它们。 </li><li>安排沿着在培养皿允许逐次注射的卵母细胞与毛细管微量的在45℃的角度的刮车道的卵母细胞。 </li><li>注入在卵母细胞赤道区域，着色动物区域下方样品在细胞质一个最佳扩散。插入毛细管尖端的上大约150-200微米深度只有最薄的部分。 </li><li>注入的cRNA 30纳克来自不同质粒在一个容积为60 NL在第一卵母细胞分别获得。注射后，等待5-10秒钟取出毛细管尖端，以避免样品逃生（ 图2A）之前。 注意:不要超过120 NL。作为注入慢慢地，你可以。建议用蒸馏水的控制。 </li><li>手动将培养皿到下一个卵母细胞注入。 注:确保尖端未通过产生一个小的脉冲出槽液后2〜3显微注射堵塞。 </li><li>在24孔培养板转移注入的卵母细胞（10卵母细胞/孔）装有3毫升新鲜ND96介质和离开他们在19℃。 </li><li>孵育在ND96的卵母细胞用孕酮（PG）（2微克/毫升）在19℃以触发细胞成熟（GVBD），15小时或分别从pcDNA6.2 / V5-DESTeIF4G1和pcDNA6所得聚腺苷酸化的cRNA的显微注射后4小时0.2 / C-EmGFP用作对照（图2A）。 注意:共注射荧光标记物与的cRNA是一个很好的工具，以确保的cRNA注入并保持在卵母细胞。执行使用了GFP标记感兴趣的cRNA这样的初步试验。 </li><li> Microinject如在＃3.5不同比率（1:3; 2:2; 3:1）的多聚腺苷酸化eIF4G1-WT和eIF4G1-DN的cRNA以忒ST的eIF4G1-WT的cRNA的突变表型（图2D）的翻译作用。 </li></ol> 4.胚泡破裂测定<ol><li>通过观察在黑色动物极用立体显微镜一个白点的存在下计数的PG刺激后的成熟卵母细胞的数目。重复计数每隔一小时，直至第二十四小时（图2B，2C）。 </li></ol> 5.免疫印迹（WB）分析<ol><li>均质化组的10卵母细胞通过来回运动用微量尖，在4℃在200μl在下列缓冲液:50mM HEPES，pH值7.4，500 mM氯化钠，0.05％SDS，5mM的MgCl 2的，1毫克/ ml牛血清白蛋白，10毫克/毫升亮肽素，10毫克/ ml抑肽酶，10mg / ml的大豆胰蛋白酶抑制剂，10毫克/毫升苄脒，1mM的PMSF，1mM的钒酸钠。 </li><li>离心样品15分钟以10,000×g离心以除去脂质（上层相）和膜（下层相）Fractions。收集细胞质部分，存储一个等分试样，以确定蛋白质浓度，并完成与的Laemmli或双Tris样品缓冲液，其余（1:1）。 注意:Bis-Tris凝胶和加载缓冲液具有更中性的pH，保护蛋白质水解</li><li>加热20微克的样品在95℃下进行10分钟。每个样品加载到丙烯酰胺凝胶的孔中。将凝胶在一个适当的缓冲罐。 </li><li> 1小时，在200五执行电泳</li><li>实现一个WB在TBS pH 8.0的液（Tris盐酸15mM的，氯化钠150mM的，吐温0.1％，含10％牛血清白蛋白）与山羊抗 - 极光激酶A /为Eg2（1:3000，2小时）或用兔抗-Aurora A /为Eg2-P（1:1000，O / N在4°C），鼠抗ERK2（1:3000，2小时），羊抗ERK2-P（Tyr204上）（1:3000，2小时），兔抗金属氧化物半导体（1:5000，4小时），兔抗GFP（1:3000，2小时）antibodies.Use抗体小鼠抗V5（1:10,000，2小时）和兔抗替代Rsk（1 :1000，2小时）（作为上样对照）。 </li><li>洗刷该膜在TBS-Tween中孵育1小时，用3次，每次10分钟，或者抗小鼠或抗兔或抗山羊（IgM抗体）辣根过氧化物酶标记的二抗以1稀释:5000，1:7500和1 :分别为5000。 </li><li>执行10分钟3次洗涤在TBS-Tween中并检测抗原 - 抗体复合物与高级的ECL检测系统。 </li></ol> 6.多聚腺苷酸含量<ol><li>每样条件5卵母细胞在1.5ml。用1毫升无核酸酶PBS 1X（pH值7.4）清洗。以下步骤将根据通风柜制成。 </li><li> RNA提取使用传统的有机过程（参见制造商的说明）。 </li><li>回收通过离心（10,000×g离心）5分钟的RNA在4℃。除去上清，空气干燥RNA的10分钟。 </li><li>于30μl不含核酸酶的H 2 O的重悬的RNA沉淀</li><li>取在一个新的管15微升样品，并添加85微升不含核酸酶的H 2 O的清理本身样品根据生产商的说明改善其在硅胶上的柱质量。洗脱使用30微升无核酸酶H 2 O的的RNA </li><li>运行1微升的样品，用5微升加样缓冲液在0.8％琼脂糖凝胶上以检查RNA的完整性。 </li><li>确定使用分光光度计RNA浓度。 </li><li>制备每种条件2连接混合物（即，eIF4G1-WT，eIF4G1-DN和H 2 O的控制）与以下试剂:10单位RNA连接酶，0.1体积的10X缓冲液，1mM ATP中，PEG8000 50％10％ 0.1底漆P1（5&#39;-P-GGTCACCTTGATCTGAAGC-NH2-3&#39;）11和使终体积到10微升用无核酸酶的 H 2 O的微克加在从卵母细胞无PG刺激获得的第一混合物的RNA和从在PG中获得的第二混合物的RNA刺激卵母细胞。 </li><li>孵育1小时，在37℃，然后20分钟在65℃下使酶失活。 </li><li>美国EA的cDNA反转录（RT）套件。准备RT混合物中，每管:0.1倍体积的10X RT缓冲液，0.1引物P2（5&#39;-GCTTCAGATCAAGGTGACCTTTTT）10，4毫米的dNTP混合，逆转录酶50单位和微克完全无核酸酶H 2 O的最终体积的10微升。加10微升先前获得的连接反应。执行对RT由生产所述的条件下。 </li><li>制备聚合酶链反应（PCR）混合物，每管:0.1体积缓冲器10X，1.5毫米氯化镁2，133μMdNTP混合液，0.2μM的特异性引物，0.2μM的引物P2，0.025 单位 Taq聚合酶，1μl的cDNA的和完整的无核酸酶H 2 O操作的50微升最终体积。执行的PCR在以下条件下:50℃，2分钟，95℃，10分钟，[95℃1分钟，56℃，30秒，72℃30秒] * 40个循环，72℃ 10分钟9。的特异性引物如下:金属氧化物半导体，GTTGCATTGCTGTTTAAGTGGTAA组蛋白样B4，AGTGACAAACTAGGCTGATATACT;细胞周期蛋白A1，CATTGAACTGCTTCATTTTCCCAG;细胞周期素B1:GTGGCATTCCAATTGTGTATTGTT 9。 </li><li>制备3％的琼脂糖凝胶。添加在每个PCR产物10微升样缓冲液。运行与10微升在110伏的样品的凝胶为最佳的迁移。后10和20分钟分析凝胶来观察的大小反映了尾多聚（A），因此核糖核酸成熟是一个先决条件，其翻译的长度的变化。 </li></ol>

<ol>
	<li>Jackson, R. J., Hellen, C. U., Pestova, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+eukaryotic+translation+initiation+and+principles+of+its+regulation.">The mechanism of eukaryotic translation initiation and principles of its regulation.</a> Nat Rev Mol Cell Biol. 11 (2), 113-127 (2010).</li><li>Holcik, M., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+in+stress+and+apoptosis.">Translational control in stress and apoptosis.</a> Nat Rev Mol Cell Bio. 6 (4), 318-327 (2005).</li><li>Lopez-Lastra, M., Rivas, A., Barria, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+in+eukaryotes:+the+growing+biological+relevance+of+cap-independent+translation+initiation.">Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation.</a> Biol Res. 38 (2-3), 121-146 (2005).</li><li>Terenin, I. M., Andreev, D. E., Dmitriev, S. E., Shatsky, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mechanism+of+eukaryotic+translation+initiation+that+is+neither+m7G-cap-,+nor+IRES-dependent.">A novel mechanism of eukaryotic translation initiation that is neither m7G-cap-, nor IRES-dependent.</a> Nucleic Acids Res. 41 (3), 1807-1816 (2013).</li><li>Sonenberg, N., Hinnebusch, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+translation+initiation+in+eukaryotes:+mechanisms+and+biological+targets.">Regulation of translation initiation in eukaryotes: mechanisms and biological targets.</a> Cell. 136 (4), 731-745 (2009).</li><li>Van der Knaap, M. S., Leegwater, P. A., K&#246;nst, A. A., Visser, A., Naidu, S., Oudejans, C. B., Schutgens, R. B., Pronk, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+each+of+the+five+subunits+of+translation+initiation+factor+eIF2B+can+cause+leukoencephalopathy+with+vanishing+white+matter.">Mutations in each of the five subunits of translation initiation factor eIF2B can cause leukoencephalopathy with vanishing white matter.</a> Ann Neurol. 51 (2), 264-270 (2002).</li><li>Sen&#233;e, V., Vattem, K. M., Del&#233;pine, M., Rainbow, L. A., Haton, C., Lecoq, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wolcott-Rallison+Syndrome:+clinical,+genetic,+and+functional+study+of+EIF2AK3+mutations+and+suggestion+of+genetic+heterogeneity.">Wolcott-Rallison Syndrome: clinical, genetic, and functional study of EIF2AK3 mutations and suggestion of genetic heterogeneity.</a> Diabetes. 53 (7), 1876-1883 (2004).</li><li>Chartier-Harlin, M. C., Dachsel, J. C., Vilari&#241;o-G&#252;ell, C., Lincoln, S. J., Lepr&#234;tre, F., Hulihan, M. M., 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=Translation+initiator+EIF4G1+mutations+in+familial+Parkinson+disease.">Translation initiator EIF4G1 mutations in familial Parkinson disease.</a> Am J Hum Genet. 89 (3), 398-406 (2011).</li><li>Charlesworth, A., Cox, L. L., MacNicol, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+polyadenylation+Element+(CPE)-+and+CPE-binding+Protein+(CPEB)-independent+mechanisms+regulate+early+class+maternal+mRNA+translational+activation+in+Xenopus+oocytes.">Cytoplasmic polyadenylation Element (CPE)- and CPE-binding Protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes.</a> J Biol Chem. 279 (17), 17650-17659 (2004).</li><li>Dumont, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oogenesis+in+Xenopus+laevis.">Oogenesis in Xenopus laevis.</a> J Morphol. 136, 153-180 (1972).</li><li>Rassa, J. C., Wilson, G. M., Brewer, G. A., Parks, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spacing+constraints+on+reinitiation+of+paramyxovirus+transcription:+the+gene+end+U+tract+acts+as+a+spacer+to+separate+gene+end+from+gene+start+sites.">Spacing constraints on reinitiation of paramyxovirus transcription: the gene end U tract acts as a spacer to separate gene end from gene start sites.</a> Virology. 274, 438-449 (2000).</li><li>Lin, W., Wadlington, N. L., Chen, L., Zhuang, X., Brorson, J. R., Kang, U. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+PINK1+attenuates+HIF-1&#945;+induction+by+preventing+4E-BP1-dependent+switch+in+protein+translation+under+hypoxia.">Loss of PINK1 attenuates HIF-1&#945; induction by preventing 4E-BP1-dependent switch in protein translation under hypoxia.</a> J Neurosci. 34 (8), 3079-3089 (2014).</li><li>Martin, I., Kim, J. W., Lee, B. D., Kang, H. C., Xu, J. C., Jia, H. 2., Stankowski, J., 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=Ribosomal+protein+s15+phosphorylation+mediates+LRRK2+neurodegeneration+in+Parkinson's+disease.">Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson's disease.</a> Cell. 57 (2), 472-485 (2014).</li><li>Cohen, S., Au, S., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis+oocytes.">Microinjection of Xenopus laevis oocytes.</a> J Vis Exp. (24), (2009).</li><li>Willis, J., DeStephanis, D., Patel, Y., Gowda, V., Yan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+DNA+damage+checkpoint+using+Xenopus+egg+extracts.">Study of the DNA damage checkpoint using Xenopus egg extracts.</a> J Vis Exp. (69), e4449 (2012).</li><li>Keiper, B. D., Rhoads, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+recruitment+of+Xenopus+maternal+mRNAs+in+response+to+poly(A)+elongation+requires+initiation+factor+eIF4G-1.">Translational recruitment of Xenopus maternal mRNAs in response to poly(A) elongation requires initiation factor eIF4G-1.</a> Dev Biol. 206 (1), 1-14 (1999).</li><li>Wakiyama, M., Imataka, H., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+eIF4G+with+poly(A)-binding+protein+stimulates+translation+and+is+critical+for+Xenopus.oocyte+maturation.">Interaction of eIF4G with poly(A)-binding protein stimulates translation and is critical for Xenopus.oocyte maturation.</a> Curr Biol. 10 (18), 1147-1150 (2000).</li><li>Sheets, M. D., Wu, M., Wickens, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyadenylation+of+c-mos+mRNA+as+a+control+point+in+Xenopus+meiotic+maturation.">Polyadenylation of c-mos mRNA as a control point in Xenopus meiotic maturation.</a> Nature. 374 (6522), 511-516 (1995).</li><li>Gebauer, F., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+function+of+Mos:+the+control+switch+of+vertebrate+oocyte+meiosis.">Synthesis and function of Mos: the control switch of vertebrate oocyte meiosis.</a> Bioessays. 19 (1), 23-28 (1997).</li><li>Le Sommer, C., Lerivray, H., Lesimple, M., Hardy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+as+a+model+to+study+alternative+splicing+in+vivo.">Xenopus as a model to study alternative splicing in vivo.</a> Biol Cell. 99 (1), 55-65 (2007).</li><li>Au, S., Cohen, S., Pant&#233;, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Xenopus+laevis.oocytes+as+a+system+for+studying+nuclear+transport+of+viruses.">Microinjection of Xenopus laevis.oocytes as a system for studying nuclear transport of viruses.</a> Methods. 51 (1), 114-120 (2010).</li><li>Gotoh, T., Villa, L. M., Capelluto, D. G., Finkielstein, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulatory+pathways+coordinating+cell+cycle+progression+in+early+Xenopus+development.">Regulatory pathways coordinating cell cycle progression in early Xenopus development.</a> Results Probl Cell Differ. 53, 171-199 (2011).</li><li>Schmidt, E. K., Clavarino, G., Ceppi, M., Pierre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUnSET,+a+nonradioactive+method+to+monitor+protein+synthesis.">SUnSET, a nonradioactive method to monitor protein synthesis.</a> Nat Methods. 6 (4), 275-277 (2009).</li><li>Moreno, J. A., Halliday, M., Molloy, C., Radford, H., Verity, N., Axten, J. M., 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=Oral+treatment+targeting+the+unfolded+protein+response+prevents+neurodegeneration+and+clinical+disease+in+prion-infected+mice.">Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.</a> Sci Transl Med. 5 (206), 206ra138 (2013).</li><li>Doyle, K. M., Kennedy, D., Gorman, A. M., Gupta, 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=Unfolded+proteins+and+endoplasmic+reticulum+stress+in+neurodegenerative+disorders.">Unfolded proteins and endoplasmic reticulum stress in neurodegenerative disorders.</a> J Cell Mol Med. 15 (10), 2025-2039 (2011).</li><li>Gandin, V., Sikstr&#246;m, K., Alain, T., Morita, M., McLaughlan, S., Larsson, O., Topisirovic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J Vis Exp. (87), e51455 (2014).</li></ol>

The authors have nothing to disclose.

翻译是一个涉及许多人类疾病，包括几种神经退行性疾病的病理生理机制。例如，在帕金森氏病的几份报告建议与遗传性基因突变8,12,13相关的翻译的损害。 一些细胞模型可用来研究翻译。这里，为了研究在eIF4G1的突变，作为显性负突变减少eIF4G1和eIF4E的伙伴8之间的相互作用的平移后果，在非洲爪蟾卵母细胞中。该模型具有的优点是简单，因为它仅由一...

蛋白是细胞生命的较大规模的生物体的基本要素，因此。它们确保大部分的细胞功能，包括结构，运输，反应催化，调节，基因表达等。它们的表达是翻译的允许的mRNA转化成蛋白质的复杂机制的结果。翻译进行各种控制，以适应并根据该小区需要调节基因表达，发育和分化，衰老，生理应力或病理表现期间。 翻译分为3个阶段（起始，延伸和终止），并提出3开始翻译系统，以满足这些需求: 通过内部核糖体进入段（IRES）的结构和总量控制的独立翻译增强（帽依赖性，依赖帽CITE）。 大多数真核生物基因的翻译在帽DEPE经由 7-甲基-5&#39;-三磷酸帽在作为在蛋白质合成的识别特征ndent方式。此帽结合eIF4E的，的eIF4F复杂与eIF4G1和eIF4A的成分。合并有其他合作伙伴，如聚（一）结合蛋白（PABP）的eIF2-GTP-MET-tRNA的满足，这些翻译起始因子允许环化表达，提高其辅助形式43S复杂，直到AUG起始密码子识别 1。这个事件对应于翻译起始即翻译的第一步骤的结束。 章独立翻译为紧张的条件下诱导，例如细胞增殖和凋亡下必需的蛋白质使用的基因编码。此机制涉及的二级结构在mRNA的5&#39;非翻译区（UTR）称为IRES，eIF4G1的羧基末端与eIF4A和43S复合物有关。该43S启动前C中的结合omplex到IRES启动而不需要eIF4E的因素2,3的帽依赖性翻译。 最后，另一种转换机制仍然没有得到很好的理解支持这种依赖帽的翻译活动强调的条件下，通过 CITE位于mRNA的UTR 4内的结构。 通过翻译通过起始步骤不同的这些各种模式，翻译在细胞内环境稳定的重要作用，并在这些过程中的一个的任何改变从而将影响生物体有小到大规模的效果。事实上，起始是限速步骤理事mRNA的正确翻译流程成蛋白质，因此许多控制和调节点5的目标。无论是对于后者，或这些过程的组成部分，如果一个结果是有缺陷的，它会扰乱在细胞内建立平衡，从而有可能导致病理康迪系统蒸发散。在此背景下，翻译因子的突变已经参与了一些障碍，包括神经退行性疾病，如`白质脑病与消失的白色matter&#39;（eIF2B1-5亚基）6，沃尔科特，Rallison综合征（用于PERK EIF2AK3基因编码）7，潜在帕金森氏病（eIF4G1 p.R1205H）8。因此重要的是要进行的细胞和分子这些突变蛋白的研究，以增加对疾病的发展和对翻译起始的一般过程我们的知识。 为了进行这些研究，有必要选择最适当的模式来观察这些突变的后果非洲爪蟾卵母细胞特别适合由于他们的生理生化特征:生理同步（阻挡在细胞周期的阶段G2）蛋白质合成的高容量（200-400毫微克/天/卵母细胞），高数提取OOC的从同一动物ytes（800-1000的卵母细胞/孔）和细胞尺寸（1.2-1.4毫米直径），这便于它们的操作。 爪蟾卵母与合成mRNA的显微注射能够容易地进行剖析翻译步骤。这种观点认为它提出的其他优点。由于减数分裂进展和翻译后核显微注射（〜24小时）的速度， 非洲爪蟾卵母细胞代表一个快速系统相比，重组蜂窝系统（从大肠杆菌中提取，小麦胚芽或兔网织红细胞...），其中的mRNA是翻译具有减小翻译速率和以较低的速度。所以，在与mRNA引入一个突变的影响将是迅速地观测和容易地研究了几个卵母细胞。 爪蟾卵母的另一个优点是，产妇的mRNA是潜在的，蛋白质翻译孕酮刺激前阻止。因此，加用孕激素是控制翻译归纳的很好的手段。细胞质polyadenylation不会发生卵子发生过程中。它开始时在一时间顺序孕酮刺激的卵母细胞的卵母细胞成熟和持续贯穿早期发展，并且可以用于研究不同步骤的翻译。 MOS mRNA的多聚腺苷酸化是最先出现，并与极光A /为Eg2，组蛋白样B4基因属于类如查尔斯沃思等人定义的"早熟" 的基因。（2004）9。平移感应"迟到"表达，如细胞周期蛋白A1和细胞周期素B1左右出现的生发泡破裂（GVBD）的时间。金属氧化物半导体的mRNA编码的丝氨酸/苏氨酸 - 蛋白激酶。它的翻译是至关重要的，因为它导致了MAP激酶级联反应，间接地激活卵母细胞成熟。事实上，响应于孕酮，MOS mRNA的聚腺苷酸化是通过涉及极光A /为Eg2调节蛋白和其它RNA结合蛋白与第一个过程增强MOS mRNA的3&#39;UTRË。这种增加的MOS mRNA的聚腺苷酸化导致增加MOS蛋白质水平，从而激活MEK1。此过程介导的胞外信令调节的激酶2（ERK2）（图1）的活化。然后，该信号级联可以触发成熟M相促进因子，通过细胞周期蛋白B和的Cdc2激酶形成的复杂，并最终导致在减数分裂恢复。 因此，在非洲爪蟾卵母细胞 ，产妇的mRNA如马鞍山的研究可以方便地使用他们的高效聚腺苷酸化有几个端点，以测试他们译的几个MOS信号元件，其中也包括GVBD率的测定翻译。这个系统是有趣的，因此要评估翻译起始因子突变的第一个后果，而没有转染效率新的mRNA转录或干扰，问题往往与eukary发生耳细胞研究。 这里，协议被建立，其中突变eIF4G1的mRNA显微注射在爪蟾卵母细胞和母体mRNA翻译进行测试。在在GVBD进展的缺陷，金属氧化物半导体的mRNA聚腺苷酸化是用于进程必需通过卵母细胞减数分裂细胞周期和为早期和晚期的mRNA类随后翻译的存在下确定。磷酸极光A /为Eg2和ERK还研究确认的MOS deregulation.Thus的结果， 爪蟾卵母代表来分析不同步骤mRNA翻译的一个简单的方法。

<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

蛋白质的合成是一个基本过程的基因表达影响不同的生物学过程特别是适应环境条件。引发步骤，其中涉及核糖体亚基在mRNA起始密码子，参与起始因子包括eIF4G1的组装。在翻译这个限速步骤的缺陷都与不同的疾病。为了研究这种放松管制的潜在后果， 非洲爪蟾卵母细胞构成具有高程度的保护与人类基本的细胞和分子机制的一个有吸引力的模式。此外，在母细胞成熟，卵母细胞转录抑制和所有必需的蛋白从先前存在的，母体衍生的mRNA翻译。这种廉价的模型使外源基因，成为一种有效的翻译完美地融为一体。这里描述了一种协议，用于使用保存位置评估翻译用感兴趣的因子（此处eIF4G1）编产妇的mRNA是第一个被聚腺苷酸化，并在卵母细胞成熟作为生理读数翻译。起初，合成出的mRNA 通过体外转录感兴趣的质粒（此处eIF4G1）的卵母细胞和卵母细胞成熟的动力学通过胚泡破裂检测注入被确定。所研究的母体mRNA靶是丝氨酸/苏氨酸 - 蛋白激酶MOS。其多聚腺苷酸化和随后的翻译与所述MOS信号级联涉及卵母细胞成熟的蛋白质的表达和磷酸化研究在一起。当前协议的变化，提出翻译缺陷也提出来强调其普遍适用性。在光新兴的证据表明，异常蛋白合成可能参与神经障碍的发病机制，这样的模式提供了机会，轻松地评估这个障碍，并确定新的目标。

爪蟾卵母细胞和确定所述卵母细胞百分比GVBD的24小时的PG刺激（ 图2B，2C）后动力学成熟: 为了研究所述eIF4G1-DN突变的平移后果， 在非洲爪蟾给PG响应蟾卵母细胞显微注射的cRNA eIF4G1-DN进行比较eIF4G1-WT和其它控制条件（H 2 O的，GFP）。该控制使评估卵母细胞显微注射的发生不论eIF4G1过表达的注射的cRNA或性质。 10卵母...

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.

非洲爪蟾作为一种模型来确定翻译减值

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 次观看.  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.

视频: 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.

定量荧光测量全球本地化翻译

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

科研

生物化学

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.

在利什曼原虫、人体细胞和小鼠睾丸中的 Polysome 分析

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.

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

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

发育生物学

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.

通过实时 FRET 显微镜和免疫荧光评估早期斑马鱼 Noonan 综合征模型中 ERK 活性调节的有效方案

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.

功能金报告系统测量特定分枝杆菌误调速率

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 ...

免疫与感染

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.

活的嗅觉途径的功能评价

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

神经科学

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.

在体内病态的人类基因组建模使用斑马鱼

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

生物学

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.

生产<em&gt;热带爪蟾</em&gt;卵提取物识别微管相关的RNA

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.

使用多体分析在发育中的鼠标大脑中进行翻译分析

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.

选择性的mRNA翻译在哺乳动物细胞中的核糖体性能分析评估

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