Investigating the role of microRNA in translational control in Xenopus oocytes

让生物学家着迷的一个关键问题是，一个复杂的有机体是如何从一个单一的细胞--卵--产生的。发展是通过控制何时何地使用特定基因来推动的。虽然过去人们认为，大多数基因表达的控制发生在DNA复制给RNA时，但现在变得明显的是，在发育过程中，控制更多地发生在调节以前合成的RNA基因复制用于制造蛋白质的时间和地点。我们的实验室一直在研究一种早期蛋白质合成的关键调节因子，称为CPEB(代表细胞质多腺苷化元件结合蛋白)，它在多种生物体中在结构和功能上都是保守的。我们研究了非洲爪蛙卵中的CPEB，这是研究脊椎动物发育的重要生物，非常适合进行生化实验。这种蛋白质通过与其mRNAs结合来调节对早期胚胎发育至关重要的其他蛋白质的水平。我们想知道CPEB是如何调节蛋白质合成的。最近，我们发现CPEB与另一种保守的蛋白质Xp54共同纯化，Xp54是一种RNA解旋酶，因此可能会影响结合的mRNA或蛋白质的构象。该复合体的其他成员包括X4E-T和P100蛋白，它们被认为在调节酵母、苍蝇和人类细胞中的蛋白质水平方面具有重要作用；这种保守意味着它们的重要性。令人惊讶的是，在酵母和人类中，CPEB复杂成分存在于细胞质(所谓的P小体)中的不同颗粒中，这些颗粒是未翻译的mRNA驻留的地方，也是它可能被降解的地方，其中还包含一类新的小RNA，即微RNA，它调节蛋白质的合成。我们还没有确定所有与CPEB相关的mRNAs和miRNAs。我们将使用microRNA阵列来检查这些小RNA中的哪些存在于卵母细胞中，以及在CPEB复合体中，并研究miRNA在调控卵母细胞蛋白质合成中的作用。了解卵母细胞中的这些因素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，而且对神经元和癌细胞中的基因表达也具有重要意义。在神经元中，CPEB调控翻译，在癌细胞中，一些miRNAs发生显著变化。

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.