Investigating the role of RNA 5- methylcytosine on ribosomal and transfer RNAs in Arabidopsis thaliana

Abstract

RNA 5-methylcytosine (m5C) is implicated to have multiple biological and molecular roles in single cell and multicellular organisms, including regulation of mRNA translation and plant growth. However, there are still many questions waiting to be answered. In my research, I addressed the roles of m5C on rRNAs and tRNAs in Arabidopsis thaliana. In Chapter 2, NOP2, a putative rRNA m5C methyltransferase was shown to be genetically redundant and essential for ovule development. Three NOP2 orthologues, NOP2A, NOP2B and NOP2C were identified in the A. thaliana genome and are genetically redundant. I found nop2a nop2b mutants were lethal due to female gametophyte abortion at the two to eight-nucleate stages. Reduction of NOP2A in nop2b nop2c mutants using an artificial miRNA led to a range of post-embryo phenotypes, impaired ribosome activity and reduced m5C methylation of C2268 on the 25S rRNA. In Chapter 3, I tested tRNA cleavage in T2 RNase mutants, rns1, rns2, rns4 and wild type under oxidative stress by northern blotting. Small RNA sequencing suggested that loss of RNS2 results in changes of both pre-tRNA and mature tRNA profiles under oxidative stress. I also tested tRNA cleavage without m5C methyltransferase TRM4B, and without ribonuclease RNS2, and found m5C appears to protect tRNAAsp(GTC) from RNS2 cleavage. In Chapter 4, I explored the processing and molecular targets of tRNA halves originating from cleaved tRNAAsp(GTC). When a 35 nt 5’-half tRNAAsp fragment was expressed from a strong polymerase III promoter and the transgene was transformed into plants, no transgenic plants were recovered. Hence, we named this transgene, Killer. Subsequent transient expression of Killer in Nicotiana benthamiana leaves followed by small RNA sequencing and a targeted genetic suppressor screen in Arabidopsis indicated that the 35 nt 5’-half tRNAAsp in Killer was processed into several sRNAs, and involved RNA silencing proteins RDR6, DCL2, DCL3, DCL4, AGO1, AGO3, AGO4, AGO5, AGO7, AGO9 and AGO10. Two sRNAs from 5’-half tRNAAsp appear to silence embryo-defective genes, At1g67490 and At1g32490, thereby leading to embryo lethality. Taken together, my research addressed functions of m5C methyltransferases NOP2 and TRM4B, and revealed a role in the processing of tRNAAsp5’ in A. thaliana.