<b>Pub1 promotes the translation</b><b> of </b><b><i>ATG1</i></b><b> mRNA and </b><b>autophagy</b>

(A) An analysis of ATG1 3' UTR interaction network reveals novel interactors: Volcano plot showing RBPs interacting with the ATG1 3' UTR compared to PGK1 as a control. The data show Npl3 interacting partners such as the previously identified Gbp2, and novel ATG1 binding partners such as Xrn1 and Pub1 (highlighted in salmon). Data are representative of two independent biological replicates, mean +/- SD. Student’s t test was performed to determine statistical significance. P values above 0.05 were considered significant. (B) Pub1 interacts with the 3' UTR of ATG1 during nitrogen starvation: Pub1-sfGFP was expressed using a centromeric plasmid in a WT (SEY6210) strain and subjected to RNA immunoprecipitation following nitrogen starvation for 4 h. ATG1 mRNA bound to Pub1 was quantified through qRT-PCR using primers specific to different regions on the transcript. Data are representative of two independent biological replicates, showing mean+/- SD. One-way ANOVA was performed to determine statistical significance. **** p value = 0.0003, ** p value = 0.0043, * p value = 0.0164. (C) Loss of Pub1 reduces Atg1 protein expression: WT (WLY176) and pub1∆ (SPY006) strains were subjected to nitrogen starvation for 3 h and Atg1 protein levels were determined by immunoblotting. Pgk1 was used as a loading control. (D) Loss of Pub1 does not affect ATG1 transcription: WT (WLY176) and pub1∆ (SPY006) cells were subjected to nitrogen starvation for 3 h and the abundance of ATG1 mRNA was determined by qRT-PCR. Data are representative of three independent biological replicates, showing mean +/- SD. Student’s t test was performed to determine statistical significance. (E) Pub1 recruits polysomes onto the ATG1 transcript during nitrogen starvation: WT (SEY6210) and pub1∆ (SPY006) strains were subjected to nitrogen starvation for 3 h and the abundance of ATG1 transcript in the different polysome fractions were determined using qRT-PCR. The data for the WT strain (SEY6210) are the same as in Figure 2B. Data are representative from two independent biological replicates, showing SD+/- mean. Two-way ANOVA was used to determine statistical significance. *** p value = 0.0004, ** p value = 0.0022, * p value &lt; 0.05. (F) Pub1 is a positive regulator of autophagy during nitrogen starvation: WT (WLY176) and pub1∆ (SPY006) strains were subjected to nitrogen starvation for 4 h and assayed to determine Pho8∆60 activity. WT and pub1∆ strains were transformed with a plasmid overexpression ATG1 under copper promoter. Overexpression of ATG1 rescues the autophagy defect in pub1∆. Data are representative of three independent biological replicates, showing mean +/- SD. One-way ANOVA was performed to determine statistical significance. ** p value &lt;0.0067. (G) Loss of Pub1 reduces viability during long-term nitrogen starvation: WT (WLY176) and pub1∆ (SPY006) strains were subjected to nitrogen starvation for the indicated times and the indicated dilutions were spotted on YPD plates and grown for 3 days at 30°C.