Two Routes to Genetic Suppression of RNA Trimethylguanosine Cap Deficiency via C-Terminal Truncation of U1 snRNP Subunit Snp1 or Overexpression of RNA Polymerase Subunit Rpo26

摘要

The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme via sequential methyl additions to the N2 atom of the m⁷G cap. Whereas TMG caps are inessential for Saccharomyces cerevisiae vegetative growth at 25° to 37°, ∆ cells that lack TMG caps fail to thrive at 18°. The cold-sensitive defect correlates with ectopic stoichiometric association of nuclear cap-binding complex (CBC) with the residual m⁷G cap of the U1 snRNA and is suppressed fully by mutations that weaken cap binding. Here, we show that normal growth of ∆ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the subunit of yeast U1 snRNP. These results underscore the U1 snRNP as a focal point for TMG cap function in vivo. Casting a broader net, we conducted a dosage suppressor screen for genes that allowed survival of ∆ cells at 18°. We thereby recovered (encoding a shared subunit of all three nuclear RNA polymerases) and (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of ∆ cold sensitivity, respectively. A structure-guided mutagenesis of , using ∆ complementation and ∆ suppression as activity readouts, defined -(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for ∆ complementation. The E124A and R135A alleles retained ∆ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component.