biostudies-literatureJu JMEXT | Japan Society for the Promotion of ScienceMEXT | Japan Society for the Promotion of Science (JSPS)Japan Agency for Medical Research and DevelopmentJapan Agency for Medical Research and Development (AMED)7708https://www.ebi.ac.uk/biostudies/studies/S-EPMC12371086biostudies-literatureUnknown16(1)The N6-methyladenosine (m6A) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing. However, the mechanism by which METTL16 modifies U6 snRNA with m6A remains elusive. Here, we present cryo-EM structures of METTL16 in complex with U6 snRNA, providing insights into the METTL16-mediated modification of U6 snRNA with m6A. The structures reveal that U6 snRNA is recruited to METTL16 through specific interactions between the C-terminal kinase-associated 1 (KA-1) domain of METTL16 and the internal stem-loop (ISL) of U6 snRNA. Upon SAM binding to the catalytic pocket within the N-terminal methyltransferase domain (MTD), U6 snRNA undergoes a structural rearrangement that positions the target adenine-containing motif at the catalytic site. This conformational change is followed by an additional structural adjustment of U6 snRNA into a productive conformation, bringing the target adenosine closer to SAM within the catalytic pocket and thereby ensuring efficient m6A modification. The KA-1 domain functions as a scaffold for initial substrate recognition and facilitates the subsequent dynamic methylation process within the MTD, highlighting the cooperative roles of METTL16 domains for U6 snRNA modification.Nature communicationsStructures and mechanisms of U6 snRNA m⁶A modification by METTL16.PMC123710862611300218H0398023H00368JP23ama121002Ju JTomita KfalseStructures and mechanisms of U6 snRNA m⁶A modification by METTL16.The N6-methyladenosine (m6A) modification in U6 snRNA, catalyzed by METTL16 using S-adenosylmethionine (SAM) as the methyl donor, is required for efficient and accurate pre-mRNA splicing. However, the mechanism by which METTL16 modifies U6 snRNA with m6A remains elusive. Here, we present cryo-EM structures of METTL16 in complex with U6 snRNA, providing insights into the METTL16-mediated modification of U6 snRNA with m6A. The structures reveal that U6 snRNA is recruited to METTL16 through specific interactions between the C-terminal kinase-associated 1 (KA-1) domain of METTL16 and the internal stem-loop (ISL) of U6 snRNA. Upon SAM binding to the catalytic pocket within the N-terminal methyltransferase domain (MTD), U6 snRNA undergoes a structural rearrangement that positions the target adenine-containing motif at the catalytic site. This conformational change is followed by an additional structural adjustment of U6 snRNA into a productive conformation, bringing the target adenosine closer to SAM within the catalytic pocket and thereby ensuring efficient m6A modification. The KA-1 domain functions as a scaffold for initial substrate recognition and facilitates the subsequent dynamic methylation process within the MTD, highlighting the cooperative roles of METTL16 domains for U6 snRNA modification.2025-01-01T00:00:00Z2025 Aug2026-05-08T06:54:09.166Z2026-04-07T23:31:37.856ZS-EPMC123710864084156110.1038/s41467-025-63021-0