Antibodies specific for N6-methyladenosine react with intact snRNPs U2 and U4/U6

Abstract

Antibodies specific for N6-methyladenosine (m6A) were elicited in rabbits and used to study the accessibility in intact snRNPs of the m6A residues present in the snRNAs U2, U4 and U6. The antibody quantitatively precipitates snRNPs U2 and U4/U6 from total nucleoplasmic snRNPs U1-U6 isolated from HeLa cells, which demonstrates that the m6A residues of the respective snRNAs are not protected by snRNP proteins in the snRNP particles. While the anti-m6A IgG does not react at all with U5 RNPs lacking m6A, a significant amount of U1 RNPs was co-precipitated despite the fact that U1 RNA does not contain m6A either. Since anti-m6A IgG does not react with purified U1 RNPs and co-precipitation of U1 RNPs is dependent on the presence of U2 RNPs but not of U4/U6 RNPs, these data indicate an interaction between snRNPs U1 and U2 in vitro. The anti-m6A precipitation pattern described above was also observed with snRNPs isolation from mouse Ehrlich ascites tumor cells, indicating similar three-dimensional arrangements of snRNAs in homologous snRNP particles from different organisms.