Structural And Functional Study Of Ribosome Assembly Factors Utp24 And Mtr4

Correct assembly of ribosome is essential for protein synthesis and cell survival.Eukaryotic ribosome assembly starts with the transcription of a precursor ribosomal RNA(pre-rRNA)in the nucleolus.The pre-rRNA is processed by multiple steps of nucleolytic cleavage to generate mature rRNAs.In the budding yeast Saccharomyces cerevisiae,more than 200 protein assembly factors and 76 snoRNAs are involved in ribosome biogenesis.They temporarily associate with pre-rRNAs,forming various pre-ribosomal particles.The 90S pre-ribosome is the earliest assembly intermediate of small ribosomal subunit and is formed co-transcriptionally on the 5' part of nascent pre-rRNA with stepwise association of-70 assembly factors,U3,U14,snR30 and snR10 snoRNAs and a subset of ribosomal proteins.After the pre-rRNA is cleaved at A0,A1 and A2 sites,the 90S is transformed into the pre-40S ribosome that matures into the small subunit in the cytoplasm.This research study the structure and function of ribosome assembly factors Utp24 and Mtr4 with structural biology,yeast genetics and biochemical approaches.Utp24 is a PIN domain endonuclease present in the 90S and is proposed to cleave at sites A1 and A2 of pre-rRNA.The crystal structure of Utp24 from Schizosaccharomyces pombe was determined at 2.1 angstrom resolution in this study.The structure contains the PIN domain,but lacks the unstructured N-terminal tail.Utp24 structurally resembles the ribosome assembly factor Utp23 and both contain a CCHC type Zn-finger motif.The function of Utp24 was analyzed in S.cerevisiae with depletion of wild-type protein and expression of mutant proteins.Mutation of the Zn-coordinating residues inhibited yeast growth and 18S rRNA processing,indicating that the Zn-finger motif is an essential element of Utp24.Depletion of Utp24 disturbs the assembly of 90S and abolishes pre-rRNA cleavage at sites A0,A1 and A2.The 90S assembled with inactivated Utp24 contained strongly accumulated of 22S pre-rRNA and enriched nuclear exosome,which is recruited for degradation of 5'ETS,indicating that the 90S is arrested at a post-AO-cleavage state.In addition,the 22S pre-rRNA was highly polyadenylated at the 3' end,indicating that it was subjected to TRAMP-mediated RNA surveillance.Despite of high sequence conservation,Utp24 proteins from H.sapiens and S.pombe were assembled with 90S in S.cerevisiae,but unable to form an active 90S,suggesting that Utp24 needs to be precisely positioned in 90S.Our study provides biochemical and structural insight into the role of Utp24 in 90S assembly and activity.This research also analyzed the function of the RNA helicase Mtr4 in small ribosomal subunit assembly.The MTR4 gene was placed under a GAL promoter and the RNA and protein components of 90S pre-ribosomes were analyzed after depletion of Mtr4 in glucose medium.Depletion of Mtr4 caused a strong accumulation of 22S pre-rRNA in 90S and blocked the association of the exosome,which is recruited to degrade the cleaved 5' ETS.As a result,the 90S failed to develop into the pre-40S.The active site of Mtr4 was required for yeast growth and small subunit assembly.A cryo-EM structure of Mtr4-depleted 90S was determined at 4.5 angstrom resolution.This study reveal that Mtr4 is required for exosome recruitment and small subunit assembly.This study investigated the functions of Utp24 and Mtr4 thoroughly.Dissection of the assembly of 90S in different mutants of these two genes enhances the understanding to 90S assembly process.the 3' end of accumulated pre-rRNA was highly polyadenylated,investigation of the function and polyadenylation mechanisms of these polyA is a new scientific question proposed by this study.