The Discovery Of Fibrillarin Centrosome Localization And Its Biological Significance

Objective: The nucleolus is the largest membrane-free organelle in eukaryotic cells and is primarily involved in ribosome biogenesis.The nucleolus is also an important hub for cellular stress response and plays an important role in individual development,aging,and the development of human diseases.In recent years,anti-tumor strategies targeting the nucleolus have become a biomedical research direction of great interest.Fibrillarin(FBL)is one of the most abundant proteins within the nucleolus.FBL is an important member of the conserved small nucleolar ribonucleoproteins(sno RNPs).FBL is guided by class C/D sno RNAs to participate in pre-r RNA site-specific 2’-O-methylation modification.2’-O-methylation modification occurs in all organisms and is believed to promote proper r RNA folding and stability.Although FBL is an essential gene,the 2’-Omethylation modification is nonessential for cell survival.The lethal phenotype of FBL knockdown is not fully explained by its methyltransferase activity,suggesting that FBL must have an essential function other than methylation modification.It is important to explore and discover other functions of FBL.FBL is abnormally expressed in a variety of tumors.FBL m RNA levels have been reported to be twice as high in leukemia and lymphoma cells as in normal cells.In the present study,indirect immunofluorescence observation of FBL in the human T lymphoma cell line Jurkat E6-1 unexpectedly revealed that FBL was not only localized to the nucleolus,but was also characterized by a distinct centrosome-like localization.Application of the centrosome marker protein γ-tubulin for immuno-co-staining confirmed this speculation.We confirmed this phenomenon in several human cell lines of normal and tumor origin.Notably,centrosome localization was most pronounced in hematological tumor cells Jurkat E6-1,BALL-1,and U937.FBL is a typical nucleolus RNA-binding protein,and its RNA-binding domain is thought to be critical for nucleolus localization.There are few reports on the extra-nucleolar localization of FBL.It has been suggested that FBL may be involved in the processing of various small nuclear RNAs(sn RNAs)in the Cajal bodies(CBs)surrounding the nucleolus.Recently,FBL was identified in a spatial proteomics combined with mass spectrometry study to be present in the human neuronal centrosome protein complex.However,this study did not demonstrate microscopic evidence of FBL centrosome localization,and there have been no follow-up studies on the mechanism of localization and its biological significance.Exploring the mechanism of FBL centrosome localization is one of the key questions to be addressed in this study.The centrosome is the microtubular organizing center of the cell that directs the formation of the spindle during division.The centrosome consists of two perpendicular centrioles(Centriole)and pericentrioles material(PCM).Abnormal centrosome function is an important cause of cellular multipolarization.Genomic instability such as chromosomal aneuploidy in tumor cells is thought to be closely related to abnormal centrosome function.To clarify the biological significance of FBL centrosome localization in Jurkat E6-1 cells,we performed loss-of-function studies on FBL using si RNA-mediated knockdown as well as CRISPR/Cas9-mediated knockdown techniques,respectively.The results showed that weakened FBL expression resulted in an abnormal increase in the number of centrosomes in Jurkat E6-1 cells,with a significant increase in multipolar cells and multinucleated cells.This suggests that deletion of FBL disrupts the function of centrosomes,leading to mitotic abnormalities and abnormal cellular karyotypes.It has also been reported earlier that antagonizing endogenous FBL with an antibody to FBL resulted in phenotypes such as weakened chromatin condensation and abnormal cell nuclear morphology,but the mechanism is unknown.There have been no reports of FBL affecting hematological tumor cell division at centrosomes,and exploring the molecular mechanism of how FBL affects centrosome number is another key question to be addressed in this study In summary,the present study demonstrates the first conclusive evidence of centrosome localization of human FBL protein and found to be more prominent in human T-lymphoma cells Jurkat.The composition of centrosomes varies by cell type and is characterized by diversity in individual development and tumorigenesis.We hypothesize that the centrosome localization of FBL may be related to the genetic background of Jurkat cells(e.g.,abundant centrosomal RNA)or the properties of the FBL protein itself(e.g.,high expression,RNA-binding ability,and the ability to drive phase-separated GAR domains).how FBL is localized in centrosomes and how it affects centrosome function are two of the most interesting central scientific Question.This study will expand the understanding of the non-ribosome generating function of the nucleolin FBL and may provide new clues for future cancer therapies targeting nucleolar.Methods: 1.The subcellular localization of human FBL protein in normal and tumorderived cells or the intracellular localization of EGFP-FBL fusion expressing green fluorescent protein was observed by indirect immunofluorescence method combined with light and confocal microscopy imaging.Density gradient centrifugation was applied to isolate and purify centrosome complexes and to detect FBL,microtubules,and centrosome-associated protein expression by protein immunoblotting(Western-blot).2.siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout were applied to study the loss-of-function of FBL.Indirect immunofluorescence and fluorescence microscopy were applied to observe and count the number of centrosomes;transmission electron microscopy was applied to observe the ultrastructure of centrosomes.Flow cytometry was applied to detect the cell cycle,cell counting was applied to draw growth curves,and cell proliferation was assessed.Apply Tet-on inducible expression system to transiently overexpress FBL.3.Detection of FBL-bound centrosomal protein fractions by immunoprecipitation Co-IP combined with WB.Apply RNA immunoprecipitation(RIP)combined with q-PCR to detect centrosomal FBL-interacting RNA;explore the key structural domains of FBL centrosomal localization by constructing GST-FBL full-length and truncated mutants in vitro.4.Application of the phase separation disruptor 1,6-hexanediol(1,6-HD)to assess whether Jurkat centromeric PCM has a phase separation environment and the effect on FBL localization.Application of ribonuclease A(RNase A)treatment as a means to assess the effect of centrosomal RNA on FBL centrosome enrichment.Results:(1)In addition to being located primarily in the nucleolus,FBL has centrosomal localization in a variety of human cells.Stronger FBL centrosome localization signals were seen in the hematological tumor cell lines U937,BALL-1 and Jurkat E6-1 cells.Exogenously expressed EGFP-FBL co-localized with the microtubule-binding protein Nu MA at centrosomes during division.FBL protein was detected in centrosome complexes isolated by sucrose density gradient centrifugation.(2)Centrosome localization of FBL was significantly attenuated after 1,6-hexanediol treatment of Jurkat in a time-and concentration-dependent manner.The centrosome localization signal of FBL was significantly attenuated after ribonuclease A(RNase A)treatment of Jurkat E6-1 cells.(3)The percentage of cells containing multiple(≥3)centrosomes was more than twofold higher in knockdown as well as heterozygous deletion Jurkat E6-1 cells.and were statistically significant.Knockdown of FBL expression inhibited Jurkat cell growth.Transient overexpression of FBL did not show an abnormal number of centrosomes,but increased micronucleated cells.(4)Transmission electron microscopy results showed that the electron density around centrosomes of FBL heterozygous deletion cells was elevated and unevenly distributed.The expression of PCM-related proteins was decreased in FBL heterozygous deletion cells.These included Nu MA,PCNT and γ-tubulin.(5)γ-tubulin is a major component of FBL Co-IP in vivo.In vitro GST-Pull down results showed that the N-terminus and C-terminus of FBL help bind the centrosomal microtubule protein γ-tubulin.(6)RIP-q PCR results showed that m RNA levels of centrosomal PCM members(Nu MA,PCNT,CEP350,ASPM,HMMR,and ccdc88c)were down-regulated in FBL heterozygous deletion cells.Conclusions:(1)Nucleolar protein FBL has centrosomal localization in several tested human normal and cancer cell lines,and is particularly evident in blood-derived tumor cells.(2)Centrosomal localization of FBL requires centrosomal RNA,which is facilitated by the GAR structural domain at the N-terminus and the methyltransferase structural domain at the C-terminus of FBL.(3)The centrosome FBL-mediated PCM phase separation environment plays an important role in ensuring centrosome function and maintaining genome stability.