Project Details
Projekt
Formation of the repressed nanos mRNP in the early Drosophila embryo
Applicant
Professor Dr. Elmar Wahle
Subject Area
General Genetics and Functional Genome Biology
Biochemistry
Biochemistry
Term
from 2019 to 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 427447367
Maternal mRNAs are RNAs that are provided from the mother to a developing oocyte. Such RNAs govern early development and are regulated by post-transcriptional mechanisms. One such RNA is the nanos mRNA, which encodes the determinant for posterior development of early Drosophila embryos. The Nanos protein is synthesized exclusively at the posterior pole of the embryo. Most of the nanos mRNA is homogeneously distributed throughout the embryo and is translationally repressed. Its poly(A) tail is removed (deadenylation), and the RNA is slowly degraded. Both translational repression and degradation of the RNA depend on Smaug Recognition Elements (SREs) in the 3‘ UTR, which are binding sites for the regulatory protein Smaug. SRE-dependent translational repression and deadenylation can be reproduced in a cell-free extract from early Drosophila embryos. Deadenylation is known to be catalyzed by the Ccr4-Not complex. We have found that translational repression involves the slow formation of a stable protein complex on the SREs. This complex contains seven proteins: Smaug, Cup, eIF4E, Me31B, Tral, PABPC and Belle. Cup is known to bind Smaug. Cup also binds the cap-binding translation initiation factor eIF4E and is thought to competitively displace the initiation factor eIF4G. While this contributes to the repression of translation, repression can also function in the absence of a cap structure, suggesting that a second mechanism is also used.We have purified the seven constituents of the repressor complex and are able to assemble a repressor complex on SRE-containing reporter RNAs that will then repress translation in a subsequent reaction. When a certain cell extract is used to assay translation, Smaug and Cup are sufficient for the formation of a stable repressor complex on the RNA; the additional components of the repressor complex are presumably recruited from the translation extract. This experiment and others identify the Smaug-Cup-RNA complex as the core of the repressed mRNP. Our proposal aims to analyze the mechanism of repression. We hypothesize that a two-pronged mechanism is operating, employing eIF4E binding as explained above and, in addition, the binding of Me31B and Tral, two known translation repressors. We will test the idea that a complex of these two proteins sequesters the RNA in a form that is inaccessible to ribosomes. We will also examine how components of the repressor complex recruit the CCR4-NOT complex.
DFG Programme
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