FIG. 5.

Pub1p is required for MFA2 3′-UTR-mediated translation regulation. A. Pub1p binds the MFA2 3′-UTR and the AU-rich domain I in a carbon source-regulated manner, in yeast extracts from glucose-grown cultures but not in yeast extracts from glycerol-grown cultures. UV-cross-linking analysis was performed with [α-³²P]UTP-labeled uncapped MFA2 3′-UTR (U*) (lanes 3 to 5 and 8 to 10) or with domain I (sequence as in Fig. 1, lanes 1 and 2 and lanes 6 and 7) as described in Materials and Methods. The uncapped MFA2 3′-UTR does not bind Pab1p (data not shown), and therefore Pub1p binding can be easily visualized around the 60-kDa marker. Shown is competition either with a 100-fold excess of MFA2 mRNA (5′-UTR and coding region without the 3′-UTR, M, lanes 3 and 8), with a 100-fold excess of unlabeled MFA2 3′-UTR (+) (lanes 2, 4, 7, and 9), or with nonspecific unlabeled polylinker RNA (−) (lanes 1, 5, 6, and 10) using extracts from glucose-grown cultures (left panel) or extracts from glycerol-grown cultures (right panel). The lower panel depicts Pub1p levels in the above extracts determined by Western analysis using 4C3 monoclonal anti-Pub1p antibody. B. Half-life analysis was performed in a pub1Δ strain grown in glucose or in glycerol growth medium as described in Materials and Methods. The Northern blot depicted was probed for MFA2 using an RNA probe as described in Materials and Methods and then normalized against U3 for loading and quantitation. C. First panel from top, Luc-MFA2 reporter translation compared to the Luc-PGK1 reporter in wild-type and pub1Δ strains grown in glucose and glycerol cultures; second panel, Luc-TNF-α ARE reporter translation; third panel, Luc-domain II reporter translation; fourth panel, translation of the control PGK1 reporter in wild-type and pub1Δ strains in glucose and glycerol media. All values are compared to the Luc-PGK1 reporter activity in the WT strain grown in glucose, are normalized for luciferase RNA levels, and were reproducible three times. D. Pab1p binds the MFA2 3′-UTR only in a PUB1 strain. UV-cross-linking analysis followed by immunoprecipitation was performed with [α-³²P]UTP-labeled capped MFA2 3′-UTR (U*) as described in Materials and Methods. Each reaction mixture included a 100-fold excess of nonspecific unlabeled capped polylinker RNA, Cap A0, and was followed by immunoprecipitation using anti-Pab1p antibody or as a control anti-Tdh/GAPDH antibody using extracts from glucose-grown cultures from a PUB1 strain (left panel) or a pub1Δ strain (right panel). This experiment was reproduced five times.