The novel regulatory influence of Argonaute 1 on messenger RNA 5-methylcytosine modification in Arabidopsis thaliana.

5-Methylcytidine (m5C) modifications play a critical role in RNA metabolism. TRM4B (tRNA-specific methytransferase 4B) is the only known component of the plant m5C machinery that mediates mRNA (messenger RNA) methylation. To date, no regulators governing site-specific m5C modifications have been identified, apart from several degenerated sequence motifs. Drawing on relevant findings in animals, we investigated the involvement of AGO1 (Argonaute 1) in m5C regulation using an ago1 mutant of Arabidopsis. m5C-RIP-seq (m5C RNA immunoprecipitation followed by deep sequencing) identified high-confidence 5981 and 4559 m5C peaks in WT (wild type) and ago1, respectively. A substantial proportion of these peaks overlapped between WT and ago1, indicating their high gene specificity. Compared to WT, ago1 exhibited 366 hyper-methylated and 168 hypo-methylated peaks. While both hyper- and hypo-methylation could correlate with either up- or down-regulated gene expression, actively transcribed genes with relatively complex structures were preferentially targeted by m5C modification. A significant fraction of m5C-associated genes are functionally involved in mRNA surveillance and ribosome biogenesis. Intriguingly, over 60% of m5C peaks identified in WT were associated with the miRNA (microRNA) binding sites. Furthermore, AGO1 binding signals could be detected on the corresponding transcripts for >40% of the m5C peaks based on the AGO1-RIP-seq experiment. Immunoprecipitation coupled to mass spectrometry validated the interaction between TRM4B and TRANSPORTIN1 (TRN1)-a known AGO1-interacting protein that is also implicated in the miRNA pathway. These findings support a model in which the miRNA-guided AGO1 complex plays a critical role in mediating site-specific m5C modifications.