Figure 6.

Alteration of U1 snRNA 5′-end dinucleotide impacts on CBC binding to U1 snRNA. (A) Rescue of the GG/tgs1Δ synthetic lethality by CBC2-Y24A mutation at 30°C. Yeast strains bearing tgs1Δ in conjunction with various combinations of SNR19 and CBC2 alleles were examined for their growth phenotypes at different temperatures. The GU allele that can rescue tgs1Δ cs phenotype was included as a control. (B) The U1 snRNA 5′-end GG mutant provides an environment for hyperstable interaction between its m⁷G cap and CBC. U1 snRNP variants differing at the 5′-end dinucleotide of the U1 snRNA (GG, CU, and wild-type) were affinity purified and probed the abundance of Cbp80p using anti-FLAG M2 antibody. Anti-Prp40p and anti-Smd1p were respectively used to detect the Prp40p and Smd1p, which are integral to U1 snRNP. (C) The identity of the U1 snRNA 5′-end dinucleotide may impact on the TMG cap formation. More U1 snRNA from the CU and GU mutants (Group I) could be precipitated by anti-TMG antibody than that of the control strains (WT and ΔAU). In contrast, less U1 snRNA could be recovered from the GG and GA mutants (Group III). The precipitated U1 snRNA was quantitated by RT-qPCR and normalized against U2 snRNA in the same RNA sample. Data represent the mean ± S.E.M. of at least three independent biological replicates.