Epigenetic modifications on individual bases in DNA and RNA can encode inheritable genetic information beyond the canonical bases. Among the nucleic acid modifications, DNA N6-methadenine (6mA) and RNA N6-methyladenosine (m6A) have recently been well-studied due to the technological development of detection strategies and the functional identification of modification enzymes. The current findings demonstrate a wide spectrum of 6mA and m6A distributions from prokaryotes to eukaryotes and critical roles in multiple cellular processes. It is interesting that the processes of modification in which the methyl group is added to adenine and adenosine are the same, but the outcomes of these modifications in terms of their physiological impacts in organisms are quite different. In this review, we summarize the latest progress in the study of enzymes involved in the 6mA and m6A methylation machinery, including methyltransferases and demethylases, and their functions in various biological pathways. In particular, we focus on the mechanisms by which 6mA and m6A regulate the expression of target genes, and we highlight the future challenges in epigenetic regulation.

DNA N6-methyladenine (6mA) has recently been found to play regulatory roles in gene expression that links to various biological processes in eukaryotic species. The functional identification of 6mA methyltransferase will be important for understanding the underlying molecular mechanism of epigenetic 6mA methylation. It has been reported that the methyltransferase METTL4 can catalyze the methylation of 6mA; however, the function of METTL4 remains largely unknown. In this study, we aim to investigate the role of the Bombyx mori homolog METTL4 (BmMETTL4) in silkworm, a lepidopteran model insect. By using CRISPR-Cas9 system, we somatically mutated BmMETTL4 in silkworm individuates and found that disruption of BmMETTL4 caused the developmental defect of late silkworm embryo and subsequent lethality. We performed RNA-Seq and identified that there were 3192 differentially expressed genes in BmMETTL4 mutant including 1743 up-regulated and 1449 down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that genes involved in molecular structure, chitin binding, and serine hydrolase activity were significantly affected by BmMETTL4 mutation. We further found that the expression of cuticular protein genes and collagens were clearly decreased while collagenases were highly increased, which had great contributions to the abnormal embryo and decreased hatchability of silkworm. Taken together, these results demonstrated a critical role of 6mA methyltransferase BmMETTL4 in regulating embryonic development of silkworm.

Translationally controlled tumor protein (TCTP) is a highly conserved protein possessing numerous biological functions and molecular interactions, ranging from cell growth to immune responses. However, the molecular mechanism by which TCTP regulates immune function is largely unknown. Here, we found that knockdown of Bombyx mori translationally controlled tumor protein (BmTCTP) led to the increased susceptibility of silkworm cells to virus infection, whereas overexpression of BmTCTP significantly decreased the virus replication. We further demonstrated that BmTCTP could be modified by SUMOylation molecular BmSMT3 at the lysine 164 via the conjugating enzyme BmUBC9, and the stable SUMOylation of BmTCTP by expressing BmTCTP-BmSMT3 fusion protein exhibited strong antiviral activity, which confirmed that the SUMOylation of BmTCTP would contribute to its immune responses. Further work indicated that BmTCTP is able to physically interact with interleukin enhancer binding factor (ILF), one immune molecular, involved in antivirus, and also induce the expression of BmILF in response to virus infection, which in turn enhanced antiviral activity of BmTCTP. Altogether, our present study has provided a novel insight into defending against virus via BmTCTP SUMOylation signaling pathway and interacting with key immune molecular in silkworm.

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