<b>Pub1 links </b><b><i>ATG1</i></b><b> mRNA export and translation</b>

(A) Npl3 and Pub1 interaction is RNA dependent: An Npl3-PA tagged strain expressing a centromeric Pub1-sfGFP was subjected to nitrogen starvation and harvested after 4 h. Npl3-PA was affinity isolated using IgG Sepharose beads with or without RNase in +N and -N conditions and immunoblotted to determine Npl3 and Pub1 interaction. (B) Pub1 localizes to different polysome compartments: Pub1-GFP (SPY069+pADH1 PUB1-GFP) and Rpl15-GFP (SPY069) strains were subjected to nitrogen starvation for 4 h and equal fractions were collected by polysome profiling following ultracentrifugation in a sucrose gradient. Two consecutive fractions were combined, and proteins were analyzed by immunoblotting. (C) Npl3 is predominantly localized to the nucleus: Npl3 was tagged with mNeonGreen (SPY067) and imaged under rich medium (+N) and nitrogen starvation medium (-N) conditions. DAPI was used to stain DNA. Data are representative of 200 cells analyzed. Scale bar: 5 µm. (D) Nuclear localization of Npl3 is required for it to bind to ATG1 mRNA: RNA immunoprecipitation was performed using WT (untagged, SEY6210), Npl3-PA (SPY060) and Npl3E409K-PA (SPY057) strains and ATG1 mRNA abundance was determined by qRT-PCR. Data are representative of three independent replicates, showing mean +/-SD. **** p value &lt;0.0001. (E) Loss of Pub1 causes defects in ATG1 mRNA export, similar to npl3∆: WT (WLY176), npl3∆ (SPY077) and pub1∆ (SPY006) strains were subjected to nitrogen starvation for 4 h and fractionated to obtain nuclear and cytoplasmic fractions. ATG1 abundance was determined by qRT-PCR. PGK1 was used as a control. Two-way ANOVA was performed to determine statistical significance. **** p value = 0.0005, *** p value = 0.0097. (F) Npl3 is required for Pub1-ATG1 interaction during nitrogen starvation: A centromeric plasmid harboring Pub1-sfGFP was expressed in WT (WLY176) and npl3∆ (SPY077) strains and subjected to nitrogen starvation for 4 h, following which GFP-trap nanobeads were used to immunoprecipitate Pub1. Untagged WLY176 was used as a control. ATG1 3' UTR abundance was determined by qRT-PCR. Data are representative of three independent biological replicates, showing mean +/- SD. Two-way ANOVA was performed to determine statistical significance. ** p value = 0.0095. (G) Pub1 interacts with ribosomal components and translation factors during nitrogen starvation: A Pub1-sf-GFP-expressing and untagged WT (SEY6210) strain were subjected to nitrogen starvation for 4 h and immunoprecipitated using GFP-trap nanobeads. The interactome of Pub1 was identified by mass spectrometry. Data are representative of two independent replicates. Student’s t test was used to determine statistical significance. A p-value above 0.05 was considered significant.