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 [Tgs1][1] 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°, [tgs1][1] ? 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 [Cbc2][2] mutations that weaken cap binding. Here, we show that normal growth of [tgs1][1] ? cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the [Snp1][3] 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 [tgs1][1] ? cells at 18°. We thereby recovered [RPO26][4] (encoding a shared subunit of all three nuclear RNA polymerases) and [RPO31][5] (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of [tgs1][1] ? cold sensitivity, respectively. A structure-guided mutagenesis of [Rpo26][4], using [rpo26][4] ? complementation and [tgs1][1] ? suppression as activity readouts, defined [Rpo26][4]-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for [rpo26][4] ? complementation. The E124A and R135A alleles retained [tgs1][1] ? suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component. [1]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000006078 [2]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000006099 [3]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000001323 [4]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000006391 [5]: http://www.yeastgenome.org/cgi-bin/locus.fpl?dbid=S000005642