Nucleolar GTPase Bms1 displaces Ttf1 from RFB-sites to balance progression of rDNA transcription and replication

18S,5.8S,and 28S ribosomal RNAs(rRNAs)are cotranscribed as a pre-ribosomal RNA(pre-rRNA)from the rDNA by RNA polymerase I whose activity is vigorous during the S-phase,leading to a conflict with rDNA replication.This conflict is resolved partly by replication-fork-barrier(RFB)-sites sequences located downstream of the rDNA and RFB-binding proteins such as Ttf1.However,how Ttf1 is displaced from RFB-sites to allow replication fork progression remains elusive.Here,we reported that loss-of-function of Bms1l,a nucleolar GTPase,upregulates rDNA transcription,causes replication-fork stall,and arrests cell cycle at the S-to-G2 transition;however,the G1-to-S transition is constitutively active characterized by persisting DNA synthesis.Concomitantly,ubf,tif-IA,and taf1b marking rDNA transcription,Chk2,Rad51,and p53 marking DNA-damage response,and Rpa2,PCNA,Fen1,and Ttf1 marking replication fork stall are all highly elevated in bms1l mutants.We found that Bms1 interacts with Ttf1 in addition to Rc1l.Finally,we identified RFB-sites for zebrafish Ttf1 through chromatin immunoprecipitation sequencing and showed that Bms1 disassociates the Ttf1–RFB complex with its GTPase activity.We propose that Bms1 functions to balance rDNA transcription and replication at the S-phase through interaction with Rcl1 and Ttf1,respectively.TTF1 and Bms1 together might impose an S-phase checkpoint at the rDNA loci.

Interaction between Bms1 and Rcl1,two ribosome biogenesis factors,is evolutionally conserved in zebrafish and human

Ribosomes are large RNA and protein complexes that function as the machinery for translation protein synthesis （Boisvert et al., 2007; Ben-Shem et al., 2011： Henras et al., 2015： Kbatter et al., 2015; McCann et al., 2015）. The eukaryotic ribosome is composed of two subunits, the 60S large subunit （LSU） and the 40S small sub- unit （SSU）, which collectively comprise of four different ribosomal RNA （rRNA） species and more than 70 proteins （Ben-Sbem et al., 2011： Henras et al., 2015; Khatter et al., 2015）. The LSU contains the 28S, 5.8S and 5S rRNAs and the SSU contains the 18S rRNA. The assembly of each subunit is initiated in the nucleolus using the respective rRNAs as backbones （Ben-Shem et al., 2011; Henras et al., 2015; Khatter et al., 2015）.