Functional Study Of The Nucleolar Protein Rcl1 In Zebrafish Liver Development

Rcll is a highly conserved nucleolar protein and is first discovered and studied in yeast.These studies have shown that Rcll participates in the rRNA processing as a component of SSU processome.In human cells,depleting Rcll also causes defectiveness in rRNA processing.In this study,we explore the function of Rcll in vertebrates with zebrafish as a model organism.In zebrafish,the rcll gene is maternally expressed and rcll transcripts are accumulated in the unfertilized eggs.After fertilization,the level of the maternal rcll transcripts is decreased.The zygotic rcll transcripts reach a peak at?12 hours post ferlization and then is gradually decreased.Whole miunt in situ hybridization shows that the expression of rcll is enriched in the digestive organs including the liver,exocrine pancreas and intestinal bulb.Immunostaining experiment shows that Rcll is localized in the nucleoli in the liver.We obtained two independent zebrafish rcl1 mutant alleles,namely rcl1lacZ which was obtained from an insertional mutagenesis screen and another rcl1cas9 obtained using CRISPR/Cas9 technique for investigating the role of rcll gene during zebrafish early embryogensis.We found that these two rcll null mutants are defective in the development of endoderm derived organs including the liver,intestine,pancreas,gallbladder,swimming bladder and mandibular pharyngeal arch.Besides,mesoderm-derived organs also suffer from mild defects.Immunostaining experiment shows that when Rcll is absent,hepatocytes cannot enter into the M phase from the late stage of G2 phase,leading to a cell cycle arrest.We found that the absence of Rcll in zebrafish affected the processing and maturation of 18S rRNA.During the processing of 47S pre-rRNA,there were an obvious accumulation of the intermediate products failure to remove the 5'ETS nucleotides,which could not be processed into mature 18S rRNA,resulting in a significant reduction of 18S rRNA content.Further experiments are needed to determine the mechanism of this phenomenon.Finally,we obtained the WT and rcll mutant transcriptomes at 4.5 days post fertilization and analyzed abnormally expressed genes in rcll mutant.We found that the up-regulated genes mainly enriched in the rRNA metabolic process and processing,ribonucleoprotein complex biogenesis and ribosome biogenesis,etc.This may be due to the depletion of Rcll affecting ribosome biogenesis,and thereby upregulated the expression of other genes involved in ribosome biogeniesis through negative feedback.On the other hand,products of the down-regulatied genes are involved in various catalytic activity.Meanwhile,the deletion of rcll gene resulted in a total of 549 alternative splicing events in 429 genes.Interestingly,among the 429 genes,the expression levels of 387 genes did not show significant changes.