Investigating the role of microRNA in translational control in Xenopus oocytes

一个让生物学家着迷的关键问题是一个复杂的有机体是如何从一个单细胞——卵子中产生的。发育是通过控制特定基因的使用时间和地点来驱动的。虽然过去人们认为基因表达的大部分控制发生在DNA复制产生RNA时，但现在越来越明显的是，在发育过程中，控制更多的是通过调节先前合成的RNA基因拷贝用于制造蛋白质的时间和地点来实现的。我们的实验室一直在研究早期发育中蛋白质合成的关键调节因子CPEB（代表细胞质聚腺苷化元件结合蛋白），它在各种生物体的结构和功能上都是保守的。我们研究了非洲爪蟾（Xenopus laevis）蛙卵中的CPEB。非洲爪蟾是研究脊椎动物发育的重要生物，非常适合用于生化实验。这种蛋白质通过与其他蛋白质的mrna结合来调节其他蛋白质的水平，这些蛋白质对早期胚胎发育至关重要。我们想知道CPEB如何调节蛋白质合成。最近，我们发现CPEB与另一种保守蛋白Xp54共纯化，Xp54是一种RNA解旋酶，可能因此影响结合的mRNA或蛋白质的构象。该复合物的其他成员包括X4E-T和P100蛋白，已知它们在调节酵母、苍蝇和人类细胞中的蛋白质水平方面很重要；保护意味着它们的重要性。令人惊讶的是，在酵母和人类中，CPEB复合物成分存在于细胞质中不同的颗粒中（所谓的P体），这些颗粒是未翻译mRNA存在的地方，可能会被降解，并且还包含一类新的小rna，即调节蛋白质合成的microRNA。我们尚未鉴定所有与cpeb相关的mrna和mirna。我们将使用microRNA阵列来检测这些小rna中的哪些存在于卵母细胞和CPEB复合体中，并研究miRNA在调节卵母细胞蛋白质合成中的作用。了解卵母细胞中的因子和机制不仅对我们理解酵母和人类体细胞中的基因表达具有重要意义，而且对神经元（CPEB调节翻译）和癌细胞（一些mirna大量发生显著改变）中的基因表达也具有重要意义。

One of the key questions which fascinates biologists is how a complex organism arises from a single cell, the egg. Development is driven by controlling when and where particular genes are used. While it used to be thought that most control of gene expression occurred when DNA is copied to give RNA, it is now becoming apparent that, during development, control more often occurs by regulating when and where previously-synthesised RNA gene copies are used to make protein. Our lab has been studying a critical regulator of protein synthesis in early development called CPEB (standing for cytoplasmic polyadenylation element binding protein ), which is conserved in structure and function in a wide variety of organisms. We investigate CPEB in the eggs of a frog, Xenopus laevis, an important organism for studying vertebrate development, and highly suitable for biochemical experiments. This protein regulates the levels of other proteins that are crucial for early embryonic development by binding to their mRNAs. We want to know how CPEB regulates protein synthesis. Recently we showed that CPEB co-purifies with another conserved protein, Xp54, which is an RNA helicase and may thus influence the conformation of bound mRNA or proteins. Additional members of the complex include X4E-T, and P100 proteins, which are known to be important in regulating protein levels in yeast, fly and human cells; the conservation implying their importance. Surprisingly, in yeast and man, CPEB complex components are present in distinct granules in the cytoplasm (so called P Bodies), places where untranslated mRNA resides, and where it may be degraded, and which also contain a new class of small RNAs, microRNA, which regulate protein synthesis. We have yet to identify all CPEB-associated mRNAs and miRNAs. We will use microRNA arrays to examine which of these small RNAs are present in oocytes, and in the CPEB complex, and study the role of miRNA in regulating protein synthesis in oocytes. Knowledge of the factors and mechanisms in oocytes will have critical implications not only for our understanding of gene expression in yeast and human somatic cells, but also in neurons, where CPEB regulates translation and in cancer cells, where some miRNAs are significantly altered in abundance.