The Mechanism Of METTL5-Mediated RRNA Adenine Methylation In Pancreatic Cancer

As the most prevalent internal modification of eukaryotic RNA,N6methyladenosine（m⁶A）modification of mRNA exerts a notable influence on multiple biological process,while m⁶A dysregulation was implicated in various diseases,including cancer.m⁶A modifications also occur on non-coding RNA,including ribosomal,transfer and small nuclearRNA.Methyltransferase N6-adenosine（METTL5）is a methyltransferase that specifically catalyzes 18S rRNA N6 methylation at adenosine 1832(m⁶A₁₈₃₂),which is located in a critical position in the decoding center,therefore suggesting its potential importance in the regulation of translation.However,the underlying mechanism of METTL5-mediated translation regulation of specific genes is still unclear.In addition,the revealed biological functions of METTL5are largely undefined,particularly in cancer.Therefore,in the present study,the potential oncogenic activity and the underlying mechanisms of METTL5 in pancreatic cancer were evaluated.This study revealed for the first time a distinct oncogenic role of METTL5 in pancreatic cancer.METTL5 expression was significantly elevated in PDAC tissues compared with adjacent non-tumor tissues,and the high level of METTL5 was associated with a poor overall survival rate.Meanwhile,the elevated 18S rRNA m⁶A methylation was detected in PDAC cells.In addition,METTL5 overexpression significantly stimulated cell proliferation,migration,invasion and tumorigenesis in pancreatic cancer.Notably,cells overexpressing catalytically inactive METTL5(METTL5^mut)showed little effect on PDAC progression,indicating that the oncogenic function of METTL5 in PDAC was dependent on catalytic activity.Furthermore,METTL5 enhanced c-Myc translation,and the oncogenic function of METTL5 may involve an increase in c-Myc translation,as evidenced by the fact that the oncogenic effect caused by METTL5 overexpression could be abolished by c-Myc knockdown.METTL5-mediated m⁶A₁₈₃₂ modification has been indicated its importance in mRNA translation processes.Previous studies have mentioned that METTL5-mediated 18S rRNA m⁶A may fine-tune the translation of a particular subset of mRNA.However,the mechanism via which METTL5 regulates the translation of a particular target mRNA is not clear.c-Myc has more broadly distributed m⁶A across multiple exons,including the four m⁶A modification sites near the terminal region of the CDS and two distinct m⁶A modification sites near the translation initiation site,one in the 5’UTR and another in the CDS region near the 5’UTR.To gain insight into the involvement of m⁶A modifications on c-Myc mRNA in METTL5-mediated c-Myc translation regulation,stably transfected cell lines were established with dCas13d-ALKBH5 system.Afterwards,the m⁶A modifications in the 5’UTR,CDS region（close to the 5’UTR）and the one near the stop codon of c-Myc mRNA were directly removed respectively and to investigate the effect of METTL5 on c-Myc translation.Our results showed that m⁶A modifications at the 5’UTR andCDS region（near the 5’UTR）of c-Myc mRNA played a key role in the specific regulation of c-Myc translation by METTL5.TRMT112 acts as a cofactor of METTL5,is essential for the stabilization and enzymatic activity of METTL5.This study demonstrated that TRMT112 and METTL5synergistically promote pancreatic cancer progression.In addition,TRMT112knockdown significantly abolished oncogenic function of METTL5 overexpression in PDAC,and vice versa.These results suggested that METTL5 and TRMT112 may function together in pancreatic cancer.Furthermore,the significant effect of TRMT112interference on the carcinogenic function of METTL5 suggested that the role of METTL5 in cancer may depend on its catalytic activity.To conclude,to the best of our knowledge,the present study revealed for the first time a distinct oncogenic role of METTL5 in pancreatic cancer.It was demonstrated that METTL5 promoted c-Myc translation,while the location and abundance of c-Myc mRNA m⁶A modifications played a critical role in the specific translation regulation by METTL5.It was further validated that TRMT112,as a cofactor of METTL5,is required for the oncogenic functions of METTL5 in pancreatic cancer.