2′-O-methylation-dependent installation of N²-methylguanosine in the U6 internal stem loop facilitates efficient spliceosome assembly

The internal stem loop (ISL) of the human U6 snRNA, which catalyzes pre-mRNA splicing, contains LARP7-dependent, snoRNA-guided 2′-O-methylations and an N²-methylguanosine (m²G) that is required for splicing of weak splice sites. Here, we show that installation of m²G₇₂ by the THUMPD2-TRMT112 methyltransferase complex is one of the last maturation events during U6 snRNP biogenesis. We dissect features of THUMPD2 required for association with U6 and present an experimentally validated model of the THUMPD2-TRMT112-U6 complex. Using in vitro methylation assays as well as a newly developed m²G-sensitive deoxyribozyme to monitor U6-m²G₇₂ levels in cellular RNAs, we reveal that 2′-O-methylations within the U6 ISL enhance methylation of G₇₂. We show that m²G₇₂ and the 2′-O-methylations in U6 independently and interdependently influence alternative splicing. Furthermore, our data demonstrate that 2′-O-methylations in the ISL are required for incorporation of U6 into snRNPs whereas m²G₇₂ influences the progression of the U6 snRNP into larger assemblies, highlighting distinct roles of these modifications during spliceosome assembly.