Strukturelle Basis der Polyadenylierung zytoplasmatischer Boten-RNA durch den GLD-2/GLD-3 Proteinkomplex
Zusammenfassung der Projektergebnisse
The cytoplasmic non-canonical poly(A) polymerase GLD-2 is a highly conserved enzyme in higher eukaryotes, which is involved in key regulatory post-transcriptional processes in the germline, embryo and brain. To understand the mechanism underlying its polyadenylation acitivty, we structurally and functionally characterized the C.elegans GLD-2/GLD-3 complex. We solved two structures of the GLD-2 polymerase in complex with its C. elegans specific, non homologous co-factors GLD-3 and RNP-8. GLD-2, which is inactive on its own, is activated by its co-factors by stabilization and additional contribution to RNA binding. In addition, we solved the structure of the 4 KH domains of GLD-3, which fold into a globular structural unit, most likely serving as a platform for protein-protein interaction. Although we have crystallizable GLD-2/GLD-3 and GLD-2/RNP-8 complexes and although we could detect reasonable RNA binding affinity in Fluorescence Anisotropy measurements, we were not able to obtain crystals of an RNA bound polymerase complex. In addition, we were not able to obtain crystals of GLD-2 with a longer GLD-3 (KH domains 1-5) to locate the position of the 4 KH domains relative to GLD-2. However, our structural and biochemical data are of relevance for and may guide future in vitro and in vivo studies of GLD-2 not only in C. elegans, but also in other organisms.
Projektbezogene Publikationen (Auswahl)
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(2010), Four KH domains of the C. elegans Bicaudal-C ortholog GLD-3 form a globular structural platform, RNA 2010: Nov 16 (11): 2058-67
Nakel K., Hartung SA., Bonneau F., Eckmann CR., and Conti E.
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(2015), Structural Basis for the activation of the C. elegans non-canonical poly(A)-polymerase GLD-2 by GLD-3, PNAS 2015: Jul 14; 112(28): 8614-9
Nakel K., Bonneau F., Eckmann CR., and Conti E.
