FIG. 6.

Stabilization of RPS16B by Nab2 is dependent on the presence of the ARE-like sequence. (A) Schematic of integration at the RPS16B locus demonstrating how the integration of RPS16B-ΔARE was accomplished. The location of the primer pairs used to simultaneously amplify both endogenous RPS16B and RPS16B-ΔARE by qRT-PCR is also indicated. The F primer, which is used to amplify both endogenous RPS16B and the integrated RPS16B-ΔARE allele, hybridizes within the ORF region of both RPS16B sequences. The R primer, which specifically amplifies endogenous RPS16B, hybridizes with the ARE element. The R′ primer, which specifically amplifies RPS16B-ΔARE, hybridizes to the junction created by the deletion of the ARE element. (B) The half-lives of both the RPS16B and RPS16B-ΔARE transcripts were simultaneously determined in wild-type (WT), Δpub1, nab2-1, and nab2-67 cells harboring the rpb1-1 allele. Cells were grown to mid-log phase in YPD medium and shifted to the nonpermissive temperature for 0, 2, 5, 10, 15, and 20 min. Total mRNA was isolated from each sample, reverse transcribed to cDNA, and subjected to qRT-PCR to determine RPS16B, RPS16B-ΔARE, and PGK1 (control) transcript half-lives. Standard deviations in the data are indicated.