Investigating the role of microRNA in translational control in Xenopus oocytes

Lead Research Organisation: University of Cambridge
Department Name: Biochemistry

Abstract

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.

Technical Summary

Early development is regulated by temporal and spatial changes in translation of pre-existing mRNAs. The cytoplasmic polyadenylation element binding protein (CPEB) is a critical regulator of translation in all metazoa. CPEB binds CPE elements in the 3' UTR of specific mRNAs, and represses their translation in oocytes, and activates their translation by polyadenylation in eggs. Towards understanding the mechanism of CPEB action, we have identifed 4 polypeptides that interact with CPEB in Xenopus oocytes: the RNA helicase Xp54, P100/Pat1, 4E-T and eIF4E, the cap-binding protein. These proteins have been implicated in translational regulation and/or early development and, intriguigingly, are found in distinct cytoplasmic bodies, in other model systems. In mammalian cells, untranslated RNA (which may be degraded), including mRNAs translationally repressed by microRNAs, collect within or nearby to P bodies. The results from several labs thus attest to a highly unusual convergence of conserved factors and mechanisms in yeast, oocytes and mammalian cells / unifying RNA decay, translational repression and microRNA-mediated regulation of gene expression. We propose to combine the expertise of the Standart lab in the biochemistry of maternal RNA regulation with the microRNA expertise of the Miska group to dissect the roles of microRNA in the Xenopus oocyte. In this cell, specific RNAs are tightly regulated at the level of translation to control the cell cycle. Our interrelated aims are to i) identify CPEB-associated mRNAs, maternal miRNA and CPEB-associated miRNA, ii) examine whether miRNAs repress translation in oocytes, iii) investigate the role of maternal miRNP components in early development, and iv) localise CPEB complex proteins, targets and miRNP components in oocytes. Knowledge of the factors and mechanisms in oocytes will have critical implications not only for our understanding of gene expression in the soma, but also in neurons, and in cancer cells.
 
Description 1. The first identification of miRNAs, piRNAs and endo-siRNAs in Xenopus tropicalis germline and soma (Armisen et al., 2009)

2. The first identification of maternal piRNA-interacting proteins, Xiwi and Xilli (Wilczynska et al., 2009). The demonstration that tethered Argonaute 2 and GW182, components of miRNP, but not the piRNP proteins Xiwi and Xili, repress translation in Xenopus oocytes (Minshall et al. 2010; Wilczynska et al., in preparation). These studies indicate that the miRNP pathway is active in oocytes while the piRNA pathway does not regulate translation in these cells.

3. The identification of maternal miR-16, which co-immunoprecipitates with CPEB (cytoplasmic polyadenylation element-binding protein), as a co-regulator of cyclin E1 mRNA translation in Xenopus oocytes (Wilczynska et al., in preparation). We propose a model in which the 3' untranslated region miR-16 sites act in collaboration with the CPE elements to tightly regulate the expression of this important cell cycle mRNA during meiotic maturation.

I provided full details in my final report to the BBSRC. The publications from this research have been highly cited, and we presented this work at several national and international meetings, with poster presentations and selected talks.
Exploitation Route Many investigators have built on our work, particularly in the field of small non-coding RNAs in vertebrates
Sectors Education,Healthcare,Other

 
Description Our research findings have been used by us and other scientists in the years to subsequent to grant, and together they have formed the basis of the current view of how small non-coding RNAs including microRNA and piRNA regulate gene expression
Sector Education
 
Description PhD Studentship
Amount £90,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2007 
End 09/2011
 
Description Royal Society International Collaboration
Amount £8,000 (GBP)
Organisation The Royal Society 
Sector Academic/University
Country United Kingdom
Start 02/2010 
End 01/2012
 
Description P-bodies and RNA-binding proteins 
Organisation Pierre and Marie Curie University - Paris 6
Country France 
Sector Academic/University 
PI Contribution Exchange of reagents and training in experimental methods. Continued active collaborations in projects related to BBSRC funding
Collaborator Contribution Exchange of reagents and training in experimental methods. Continued active collaborations in projects related to BBSRC funding
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