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 Journal Article
| Authors: | Qiu, Z. R.; Schwer, B.; Shuman, S. |
| Article Title: | 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 |
| Abstract: | The trimethylguanosine (TMG) caps of small nuclear (sn) RNAs are synthesized by the enzyme Tgs1 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°, tgs1Δ 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 mutations that weaken cap binding. Here, we show that normal growth of tgs1Δ cells at 18° is also restored by a C-terminal deletion of 77 amino acids from the Snp1 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 tgs1Δ cells at 18°. We thereby recovered RPO26 (encoding a shared subunit of all three nuclear RNA polymerases) and RPO31 (encoding the largest subunit of RNA polymerase III) as moderate and weak suppressors of tgs1Δ cold sensitivity, respectively. A structureguided mutagenesis of Rpo26, using rpo26Δ complementation and tgs1Δ suppression as activity readouts, defined Rpo26-(78-155) as a minimized functional domain. Alanine scanning identified Glu89, Glu124, Arg135, and Arg136 as essential for rpo26Δ complementation. The E124A and R135A alleles retained tgs1Δ suppressor activity, thereby establishing a separation-of-function. These results illuminate the structure activity profile of an essential RNA polymerase component. © 2015 Qiu et al. |
| Keywords: | controlled study; unclassified drug; nonhuman; protein domain; cell survival; gene overexpression; cell growth; carboxy terminal sequence; in vivo study; guanosine derivative; saccharomyces cerevisiae; gene repression; alanine; site directed mutagenesis; glutamic acid; small nuclear ribonucleoprotein; genetic complementation; rna polymerase; trimethylguanosine synthase; rna capping; u1 snrnp; dna directed rna polymerase iii; trimethylguanosine; article; rna polymerase subunit; rpo26; rna polymerase iii subunit rpo26; rna polymerase subunit rpo26; u1 small nuclear ribonucleoprotein subunit snp1; trimethylguanosine rna cap |
| Journal Title: | G3: Genes, Genomes, Genetics |
| Volume: | 5 |
| Issue: | 7 |
| ISSN: | 2160-1836 |
| Publisher: | Genetics Society of America |
| Date Published: | 2015-07-01 |
| Start Page: | 1361 |
| End Page: | 1370 |
| Language: | English |
| DOI: | 10.1534/g3.115.016675 |
| PROVIDER: | scopus |
| PMCID: | PMC4502370 |
| PUBMED: | 25911228 |
| DOI/URL: | |
| Notes: | Export Date: 2 September 2015 -- Source: Scopus |
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