Functional Study Of Bms1 And Ttf1 Interaction At RDNA Loci In Controlling Cell Cycle Progression

Nucleolus harbors machineries responsible for rRNAs maturation and ribosome small and large subunits assembly.As a component of the small subunit(SSU)processome,Bms1,a nucleolar GTPase,partners with Rcll to cleave pre-rRNA at the A2-site.Studies have shown that mutation in zebrafish Bms1l displays a hypoplastic liver and malfunctioned BMS1 in human causes aplasia cutis congenital,both due to cell cycle arrest.However,researches about Bms1l have mostly been focusing on its function on assembling ribosome small subunit,how Bms1l/BMS1 controls cell cycle progression is unknown.Successful completion of cell cycle needs to pass at least four checkpoints:the G1/S,S/G2,G2/M and metaphase-to-anaphase transition checkpoints.All of these checkpoints are regulated in a complicated and sophisticated manner,in order to ensure efficient cell proliferation,rDNA loci harbors hundreds of copies of tandemLy arrayed rDNA genes.The pre-rRNA is transcribed from the rDNA loci by RNA Polymerase I(Pol-I)whose activity is vigorous during the S-phase,leading to a conflict with rDNA replication.Replication-fork-barrier(RFB)sequences downstream of each rDNA gene play a specific role in resolving this conflict.Fork blocking protein(Fob1)in yeast and RNA Pol-I transcription termination factor 1(TTF1)in mice and human function as RFB factors to terminate rDNA transcription and mediate replication-fork arrest.Therefore,the on-and-off of TTF1 at the RFB-sites might serve as a checkpoint for S-to-G2 transition.However,how the TTF1 is displaced from the RFB-sites remains elusive.In this study,methodologies molecular biology,genetics,developmental biology,cell biology and bioinformatics are comprehensively applied to explore the new functions of nucleolar protein Bms 1,especially its interaction with Ttf1 in rDNA region,in regulating cell cycle progression by using zebrafish and human cell lines as models.Zebrafish genome contains two closely linked homologous ttf1 genes on chromosome 5,namely ttf1 a and ttf1b.A chromatin-immunoprecipitation sequencing(ChIP-Seq)analysis revealed that Ttfl bound to four homologous regions 3'-downstream of the rDNA gene.Endogenous Ttfl was significantly enriched on its binding-sites in both bms1lsq163 and bms1l=jul when compared with that in WT.Binding of the purified Ttfl a to the downstream region of the rDNA gene was depended on the presence of GTP in a dosage-dependent manner.Further more,Ttf1 level was obviously elevated in both GTPase-activity compromised bms1lsq163 mutant and bmsll knockout bmsll=jul mutants and was accumulated in the nucleoli.Co-immunoprecipitation(Co-IP)showed that Bms 11 interacted with both Ttf1a and Ttf1b.Proteins extracted from 3dpf WT embryos showed that endogenous Ttfl was successfully pulled down by Bmsll,and both endogenous and overexpressed Bms11sq163 mutant protein could interact with Ttf1.Finally,we found that Bms1 interacted with Ttf1 to disassociate the Ttfl-rDNA complex in a GTPase-dependent manner.GTPase-activity compromised zebrafish Bmsl mutants almost lost ability to dissaociate Ttf1 from the‘Sal-boxes'.This result nicely explains the accumulation of Ttf1 in bmsllsq 163 and bmsllzjul mutants.Abberant accumulation of Ttf1 at the rDNA loci causes replication stress that leads to genomic DNA over-replication thus abnormal DNA content in the mutant cells.DNA over-replication triggers DNA damage response that in turn arrests cell cycle at the S-phase,causing a smaller liver in bms1 mutants.As observed in zebrafish,knockdown of human BMS1 arrests cells at the S-phase coupled with nucleolar replication stress and genomic DNA over-replication,suggesting Bms1 has conserved function in different species.We therefore propose that Ttf1 and Bms1 together impose a nucleolar checkpoint on cell cycle progression through balancing S-phase rDNA transcription and replication.Our findings not only unravel the novel function of nucleolar factors in regulating cell cycle progression but also help us to gain new understanding of genetic control of the development of liver and other endoderm organs.Our findings also provide novel insignts in identifying diagnostic therapeutic targets on human diseases.