Project Details
Projekt
Formation, maturation and nuclear export of pre-ribosomal subunits
Subject Area
Biochemistry
Term
from 2004 to 2017
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 5425172
During eukaryotic ribosome biogenesis, nascent 60S and 40S subunits are formed in the nucleus followed by nuclear export and final maturation in the cytoplasm. We study the mechanisms of ribosome biogenesis in the yeast Saccharomyces cerevisiae, employing cell biological, biochemical and structural analyses. In the past we have investigated late nuclear maturation steps of both the pre-60S and pre-40S subunit and the coupling to nuclear export and cytoplasmic maturation, with a focus on selected and essential biogenesis factors. Important findings and conclusions achieved during the previous funding period were: (i) the dynein-related AAA ATPase Rea1 functions in two successive steps removing the biogenesis factors Ytm1 and Rsa4, respectively, from the evolving pre-60S subunit; (ii) a molecular understanding of how Rsa4 itself is recruited to the pre-60S surface involving another essential biogenesis factor, Nsa2, that occupies a hallmark structure of the ribosome, the peptidyl transferase center; (iii) the first cryo-EM structure of an immature 60S subunit, revealing how the nuclear export factor Arx1 is bound to the nascent chain exit tunnel, probably to prevent premature recruitment of ribosome-associated factors active in co-translational targeting, processing and folding; (iv) the first X-ray crystal structure of the eukaryotic Rio2 kinase, enabling us to perform a detailed structure/function relationship analysis, which showed quite unexpectedly that Rio2 performs a role as ATPase to drive late steps in pre-40S biogenesis. For the next funding period, we will extend our studies on these and several other ribosome biogenesis factors to increase our mechanistic insight into this most complicated process of ribosome synthesis. Specifically, we aim to gain (i) cryo-EM structures of different pre-60S particles, which will enable us to understand the spatio-temporal structural rearrangements, including rRNA processing events, and correlate them with distinct biogenesis factors that are associated with these dynamic substructures, (ii) to intensify our efforts to gain x-ray crystal structures of biogenesis factors and complexes, especially the Rea1-Rix1-Rsa4-Nsa2 relay, which can generate mechanical force to remove factors and shape the pre-60S particle, (iii) and study the role of GTPases Nug1, Nug2 and Lsg1, and the unconventional kinases Rio1 and Rio2, to find out how these energy-consuming enzymes trigger ribosome biogenesis and perform quality control. From all these investigations we expect profound knowledge of how eukaryotic ribosomes are generated, which may have also an impact on other linked cellular pathways dependent on de novo ribosome synthesis, both under healthy und disease conditions.
DFG Programme
Research Grants
