Functional Analysis Of M~6A Methylome Members On Rice Messenger RNA

Messenger RNA is the core molecule for the translation of genetic information.The epigenetic nucleoside modifications on m RNA play important role on of pre-m RNA splicing,the stability of mature m RNA and translation efficiency.Among more than 170nucleoside modifications,there are only 18 present on m RNA,mostly of them are methylated nucleosides.m~6A is a methylation occuring on the 6th nitrogen of the adenosine ring,and it is the most abundant nucleoside modification on m RNA.m~6A is widely found in bacteria,viruses,yeast,mammals and plants.Many studies in mammals have shown that m~6A modification on m RNAs can influence stem cell fate,fertility,and biological rhythms.m~6A is a reversible methylation,it can be synthesized by the“m~6A writer”,it can be recognized by the“m~6A reader”,and it can be removed by the“m~6A eraser”proteins.Those three types of proteins form the m~6A methylome.m~6A writer is a large multi-protein complex,the main components identified so far include Methyltransferase-like3(METTL3),METTL14,Wilm's tumor 1-associating protein(WTAP),KIAA1429/VIRMA,HAKAI,RNA binding motif protein15(RBM15)and Zinc finger CCCH domain-containing protein 13(ZC3H13).METTL3-METTL14 is the catalytic center of m~6A writer,while WTAP is a structural protein stabilizing the interaction between METTL proteins.In this work we focus on the function of rice METTL3,METTL14 and WTAP orthologs,including METTL3 ortholog OsMT?A(LOC?Os02g45110);METTL14orthologs OsMT?C(LOC?Os01g16180),OsMT?G(LOC?Os03g05420),OsMT?H(LOC?Os01g31030),and WTAP ortholog Os WTAP(LOC?Os06g27970).We constructed CRISPR-Cas9 transgenic material,and obtained loss-of-function mutants by gene editting.Those mutants were used to study the functions of m~6A writer genes.The results are stated as following:1.OsMT?A mutant showed longer growth period than wild type,the flower organs were abnormal,the pollen sterility rate was significantly higher,the tiller number was significantly reduced.The total biomass was also reduced,and the seed setting rate was extremely low(10.2%,compared to 82.1%in wild type).2.OsMT?C mutant showed longer growth period,the plant height at mature stage was significantly lower than that of wild type.3.OsMT?H mutant had reduced plant height at tillering stage,the seed setting rate decreased significantly,with the lowest seed setting rate as 0.5%.4.OsWTAP mutant did not show any significant growth phenotype.