Structure And Function Analysis Of Yeast Ribosome Assembly Factors

Ribosomes are large conserved molecular machines that catalyze translation of mRNA to synthesize proteins.Ribosome assembly is one of the most energetically demanding of cellular processes.In Saccharomyces cerevisiae,ribosome formation requires a large number of transiently associated assembly factors that coordinate processing and folding of pre-rRNA and binding of ribosomal proteins.It begins in the nucleous where the 35S pre-rRNA is transcribed.Many assembly factors are cotrancriptionally assembled with the nacent pre-rRNA to form the earlist precursor of small subunit ribosome.Understanding the structure and assembly of the 90S pre-ribosome is a major task in the ribosome biogenesis filed.In this thesis,we first provide a structural and functional perspective of the Krrl-Fafl complex within the pre-90S ribosome.In the second part we will investigate the role of the deubiquinase Otu2 in the regulation of ribosome biogenesis.Krrl and Fafl are two interacting proteins present in early 90S precursor particles of the small ribosomal subunit.Here,we determined a co-crystal structure of the core domain of Krrl bound to a 19-residue fragment of Fafl at 2.8 A resolution.The structure reveals that Krrl consists of two packed K homology(KH)domains,KH1 and KH2,and resembles archaeal Dim2-like proteins.We show that KH1 is a divergent KH domain that lacks the RNA-binding GXXG motif and is involved in binding another assembly factor,Kril.KH2 contains a canonical RNA-binding surface and additionally associates with an a-helix of Fafl.Specific disruption of the Krrl-Fafl interaction impaired early 18S rRNA processing at sites A0,A1,and A2 and caused cell lethality,but it did not prevent incorporation of the two proteins into pre-ribosomes.The Krrl-Fafl interaction likely maintains a critical conformation of 90S pre-ribosomes required for pre-rRNA processing.Our results illustrate the versatility of KH domains in protein interaction and provide insight into the role of Krrl-Fafl interaction in ribosome biogenesis.It is generally believed that most ribosome assembly factors have been found in yeast.Here we identified Otu2,a member of the OUT deubiquinase,as a putative novel ribosome assembly factor.The otu2 deletion strain of yeast is liable,yet shows hypersensitivity to some ribosome inhibitors.Deletion of Otu2 results in the reduction of 40S and 60S subunits as well as translating ribosomes.This suggests that Otu2 is involved in ribosome biogenesis or ribosome degradation.We show that Otu2 is localized in the cytoplasm.We show that Otu2 is associated with ribosome fractions in sucrose density gradients.Through proteomic analyses and immunoprecipitation,we found that Otu2 interacts with pre-40S assembly factors,suggesting that Otu2 can incorporate into the pre-40S particle and regulate ribosome synthesis.We have determined the crystal structure of Otu2 catalytic domain at 3.0 A.The Otu2 structure resembles many human deuibiquitinase structures and is made up of two globular clamps separated by a large,relatively shallow cleft.This cleft is highly conserved and forms the binding site for ubiquitin.Based on the OTUD5-ubiquitin complex structure,ubiquitin can be modeled onto the Otu2 structure.Otu2 has two homologs in human,OTUD6A and OTUD6B.OTUD6A has a linkage specificity for the Lys27,Lys29,Lys33 types of ubiquitin chain,while OTUD6B has no activity against all types of ubiquitin chain.The substrate of Otu2 is unknown.The ribosomal proteins Rps31 and Rp130 are expressed as ubiquitin fusion proteins in yeast and other eukaryotes.The presence of Otu2 in pre-40S led us to hypothesize that Otu2 may cleave the ubiquitin-Rps31 fusion protein.We found that Otu2 shows a weak deubiquinase activity on ubiquitin-Rps31 in vitro.However,the in vivo processing of Rps31 is not affected in the otu2 deletion stain,suggesting that Otu2 may not act on ubiquitin-Rps31 or is not the only deubiquinase for ubiquitin-Rps31.Our results provide the initial clue to Otu2 function in ribosome biogenesis,but the exact substrate and function of Otu2 need to be further studied.