PMC:4502370 / 25135-29005 JSONTXT

{"project":"2_test","denotations":[{"id":"25911228-15340060-43354270","span":{"begin":596,"end":600},"obj":"15340060"},{"id":"25911228-21749979-43354272","span":{"begin":3521,"end":3525},"obj":"21749979"},{"id":"25911228-21749978-43354273","span":{"begin":3542,"end":3546},"obj":"21749978"}],"text":"RPO26 and RPO31 suppress tgs1Δ at low gene dosage\nThe identification of two RNA polymerase subunits as dosage suppressors of tgs1∆ suggested a novel connection between TMG caps and transcription. The connection via Rpo31 to RNA polymerase III, which is responsible for the synthesis of many essential noncoding RNAs (5S rRNA, U6 snRNA, tRNAs), was particularly puzzling insofar as none of the known Pol III transcripts have 5′ TMG (or m7G) caps. One scenario that might explain the genetic suppressor results is that the loss of TMG caps affects nucleolar architecture and function (Colau et al. 2004) such that the assembly or activity of Pol III is compromised at cold temperature, and this defect can be overcome, in part, by overexpressing either RPO31 or RPO26. If this is the case, then we might expect that simultaneously overexpressing RPO31 and RPO26 would afford better growth of tgs1∆ cells at 18° than increasing the gene dosage of either gene alone. We tested this by introducing RPO31 and RPO26 on the same 2-µ plasmid, but observed no better rescue of tgs1∆ growth in the cold than that afforded by 2-µ RPO26 (data not shown). Another prediction of the above scenario is that tgs1∆ suppression should require high gene dosage. To address this issue, we placed the RPO31 and RPO26 genes on CEN plasmids and transformed them into tgs1∆ cells. The striking finding was that provision of RPO26 or RPO31 on a CEN plasmid was just as effective as the 2-µ RPO26 or RPO31 plasmids in restoring tgs1∆ growth at restrictive temperature (Figure 4).\nFigure 4 RPO26 and RPO31, at low gene dosage, are capable of restoring growth of tgs1Δ at 18°. (Left) Yeast tgs1Δ cells were transformed with a CEN URA3 plasmid bearing wild-type TGS1 (positive control), an empty 2-μ URA3 vector (negative control), and 2-μ URA3 plasmids expressing wild-type RPO26, intron-less RPO26 cDNA (RPO26*), or RPO31. Ura+ transformants were selected at 30° and then tested for growth at 18° by spotting serial 10-fold dilutions of liquid cultures (grown at 30° in SD–Ura medium) on Ura− agar plates. The plates were photographed after incubation for 7 d at 18°. (Right) Yeast tgs1Δ cells were transformed with a CEN LEU2 plasmid bearing wild-type TGS1 (positive control), an empty CEN LEU2 vector (negative control), and CEN LEU2 plasmids expressing wild-type RPO26, RPO26*, or RPO31. Leu+ transformants were selected at 30° and then tested for growth at 18° by spotting serial 10-fold dilutions of liquid cultures (grown at 30° in SD-Leu medium) on SD-Leu agar plates. The plates were photographed after incubation for 7 d at 18°. The aforementioned results point toward an alternative explanation for tgs1∆ suppression, whereby the lack of TMG caps selectively impacts the expression of RPO26 and/or RPO31, such that even one extra copy of these genes allows for growth in the cold. RPO26 seemed to us the more plausible target of such an effect, because: (i) TMG caps are certainly implicated genetically in pre-mRNA splicing; (ii) the RPO26 gene contains an intron, whereas RPO31 does not; and (iii) prior studies had shown that a 60% reduction in the level of mature RPO26 mRNA (caused by a mutation in the RPO26 promoter) resulted in a cold-sensitive growth defect (Nouraini et al. 1996b). We initially considered a scenario in which adequate Rpo26 expression might somehow require the presence of an intron in the pre-mRNA, akin to what has been described for the yeast Sus1 and the intron-containing SUS1 pre-mRNA (Cuenca-Bono et al. 2011; Hossain et al. 2011). In that case, we would expect that an intron-less cDNA version of RPO26 would not be able to suppress tgs1∆. However, we found that the RPO26 cDNA (designated RPO26* in Figure 3) was just as effective as the native RPO26 gene in promoting tgs1∆ growth at 18°, whether delivered on a 2-µ vector or a CEN vector (Figure 4)."}