Exon junction complexes regulate osteoclast-induced bone resorption by influencing the NFATc1 m6A distribution through the "shield effect".

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.