Molecular insights into THUMPD2-mediated m2G methylation of the U6 snRNA

ABSTRACT: THUMPD2 installs N2-methylguanosine (m2G) at position 72 of the U6 snRNA, and this modification, which lies at the catalytic core of the spliceosome, is required for efficient splicing of weak splice sites. However, when during the U6 lifecycle this modification is installed and what features of the U6 snRNA and THUMPD2 are required for methylation of G72 remain elusive. Here, we show that U6 associated with THUMPD2 is not oligouridylated and that late-acting U6 snRNP biogenesis factors are enriched with THUMPD2. We map RNA crosslinking sites within the N-terminal region and C-terminal Rossman-fold methyltransferase domain of THUMPD2, and show the requirement of these domains for THUMPD2 interaction with U6 in cells. Using a newly developed m2G-sensitive deoxyribozyme to monitor U6-m2G72 levels in cellular RNAs, we demonstrate the requirement of SAM binding and interaction with the THUMPD2 cofactor TRMT112 for efficient methylation. Our data further reveal that 2’-O-methylation of C62, C63 and A70 do not increase the affinity of THUMPD2 for the U6 internal stem-loop (ISL), but enhance m2G methylation of G72. Nucleotide substitutions within the loop region of the ISL that extend the stem similarly increase m2G72 installation, demonstrating that the stability of the ISL is key for methylation by THUMPD2. Together these results provide a new layer of understanding of how an RNA modification that fine-tunes pre-mRNA splicing is introduced.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Olexandr Dybkov  

LAB HEAD: Henning Urlaub

PROVIDER: PXD053510 | Pride | 2026-05-25

REPOSITORIES: Pride