Interaction Between Fibrillarin And LYAR And Its Effects On RRNA Methylation

?-thalassemia is a hereditary hemolytic anemia caused by a mutation in the ?-globin gene that impairs ?-globin synthesis.The activation of endogenous ?-globin is an important strategy for ?-thalassemia disease caused by insufficient synthesis of ?-globin.Our previous study found that nucleolar protein LYAR transcriptionally regulates the expression of y-globin.Studies have also shown that LYAR binds to the ribosome and promotes the ribosome biogenesis at the processing level of pre-rRNA.It is speculated that LYAR can regulate gene expression not only at the transcriptional level but also at the translational level.However,the mechanism of gene regulation by LYAR at translation level remains unclear.In order to fully understand the regulatory mechanism of LYAR on ?-globin expression,we constructed a LYAR-FLAG over-expressed erythroleukemia K562 stable cell line and immunoprecipitated the LYAR-containing protein complex.After protein mass spectrometry analysis,the LYAR protein complex contains many ribosomal proteins and some nucleolar proteins,one of which in the higher content is nucleolar protein Fibrillarin(FBL).FBL is a ribose 2'-O-methylase that catalyzes the methylation modification of pre-rRNA and plays an important role in the modification of ribosomal RNA and ribosome assembly.High levels of FBL are accompanied by modifications of the rRNA methylation pattern,impairment of translational fidelity,and an increase of internal ribosome entry site(IRES)-dependent translation initiation of key cancer genes.Through in vivo and in vitro interaction experiments,we confirmed the direct interaction between LYAR and FBL.We found that the interference of LYAR protein expression reduces the level of rRNA methylation,although FBL mRNA and protein levels are not affected.We interfered with the expression of FBL and found that the levels of methylation of rRNA are reduced.The interaction of LYAR with FBL affects rRNA methylation.Previous studies have shown that LYAR is a transcription factor with a specific DNA binding motif(GGTTAT/G)that specifically binds to the 5'UTR region of ?-globin and recruits the methyltransferase PRMT5,which catalyzes the symmetric double methylation of histone H4R3(H4R3me2s),thereby inhibiting the expression of y-globin.We confirmed that y-globin expression was upregulated after interfering with LYAR protein expression.However,the levels of ?-globin mRNA did not change,but the protein level decreased,after interfering with the expression of FBL,indicating that FBL regulate the expression of ?-globin at the translational level potentially.The detailed mechanism of interaction between LYAR and FBL in translation regulation remains to be elucidated in the future.