1 Department of Oral Pathology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai, China.
2 Department of Stomatology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
3 Department of Gynecology, First Hospital of Shanxi Medical University, Taiyuan, China.
4 Concordia Institute for Information Systems Engineering, Concordia University, Montreal, QC, Canada.
5 Department of Endodontics, Central Laboratory of Jinan Stomatological Hospital, Jinan Key Laboratory of Oral Tissue Regeneration, Shandong Provincial Health Commission Key Laboratory of Oral Diseases and Tissue Regeneration, Jinan, Shandong, People's Republic of China.
6 Department of Oral Maxillofacial-Head Neck Oncology, Shanghai Ninth People's Hospital

Background: The distribution of the m6A methylation modification on the transcriptome is highly regionally selective and is mainly concentrated in abnormally long exons and stop codons. However, in-depth research on the selective mechanism of m6A methylation is still lacking.

Methods: In this research, meRIP sequencing, mRNA sequencing, meRIP, luciferase reporter assays and CRISPR/Cas9 conditional knockout mice were used to elucidate the distribution characteristics of NFATc1 m6A.

Results: METTL14 controls osteoclast-mediated bone resorption by means of the methylation (4249 A) of the NFATc1 gene during osteoclast differentiation. Exon junction complexes (EJCs) selectively protect the m6A methylation sites of the NFATc1 gene. When the methylation sites are located within short exon fragments (50-200 nt), EJCs prevent their hypermethylation and degradation through the "shield effect"; when the methylation sites are located in the 3' UTR region or long exon fragments (greater than 300 nt), the "shield effect" disappears. Downstream, YTHDF2 induced the degradation of hypermethylation NFATc1 transcripts without site restriction.

Conclusions: EJCs act as "shields" to regulate the m6A region selectivity of the NFATc1 gene, thereby determining the characteristics of m6A distribution in the gene. Importantly, EJCs can raise the level of m6A methylation of NFATc1 and degrade its mRNA, thereby inhibiting osteoclast differentiation and preserving bone mass. These results will be helpful for identifying potential molecular targets for osteoporosis treatment.

Key points: METTL14 controls osteoclast-mediated bone resorption by means of the methylation (4249 A) of the NFATc1 gene during osteoclast differentiation. Exon junction complexes (EJCs) protect the remaining methylation sites of the NFATc1 gene (located in the inner exon fragment of 50-200 nt) from hypermethylation and degradation. The "shield effect" disappears when the exon fragment is extended to 300 nt. Downstream, YTHDF2 induced the degradation of hypermethylation NFATc1 transcripts without site restriction.