Study Of Late Nuclear Pre-60S Ribosomes In Yeast

Ribosomes are ribonucleoprotein complexes in all living cells responsible for the protein synthesis.Ribosome biogenesis in eukaryotes is an extremely sophisticated and highly dynamic process involving transcription,modification,folding and processing of rRNAs and binding of ribosomal proteins that is regulated by 76 different small nucleolar RNAs and more than 200 assembly factors including structural proteins that may serve as chaperones or scaffolds and enzymatic proteins such as GTPases,AAA-ATPases,ATP-dependent RNA helicases and kinases.Loss-of-function mutations of certain assembly factors in human result in dyregulation of ribosome biogenesis and may led to several human diseases referred to as ribosomopathies.However,the structure and function of most assembly factors are largely unknown.Therefore,understanding the mechanism of ribosome biogenesis in yeast will provide a framework to insight into how human ribosomes are synthesized and help us find an effective treatment for those ribosomopathies.Nog2 is a regulatory GTPase that participates in the processing of 27SB pre-rRNA.The recruitment of Nog2 requiring the prior binding of all the other B factors to pre-60S particles is one of the last structural remodeling events preceding C₂ cleavage.Nog2associates with pre-60S particles in the nucleolus and releases from pre-60S particles before nuclear export.Thus,we can obtain native nuceloplasmic pre-60S ribosomes from Saccharomyces cerevisiae via affinity purification using the epitope-tagged Nog2 as a bait protein.The structures of multiple pre-60S intermediates are solved by the aid of single-particle cryo-EM.And one of these structures named state 1 was solved at a nominal resolution of 3.08?.According to the high quality density map of state 1,we identified the location of 19 assembly factors as well as a partial sequence of internal transcribed spacer 2?ITS2?and built their atomic models.In particular,two essential assembly GTPase Nog1 and Nog2 may act as hub proteins to interact with various assembly factors and functional rRNA elements,revealing their significant roles in structural rearrangement checkpoints and nuclear export.Moreover,we also located 5 additional assembly factors in the density map of state 2.Besides,our compositional and structural different Nog2 lifetime pre-60S intermediates present a detailed view of three major remodeling events before nuclear export:removal of the ITS2 RNA,rotation of the 5S ribonucleoprotein?RNP?and construction of the functional sites including peptidyl transferase center?PTC?and polypeptide exit tunnel?PET?.In addition,we found the depletion of Sda1 hindered rotation of the 5S RNP in Gal-Sda1 Nog2-TAP samples.In summary,the abundant structural information in our data not only interprets and validates massive previous genetic and biochemical results of eukaryotic ribosome maturation,but also provides a valuable resource for the formation of new hypotheses and models.