<b>Npl3 promotes </b><b><i>ATG1</i></b><b> translation and autophagy through an RRM-motif-dependent mechanism</b>m

(A) Npl3 is required for export of ATG1 mRNA: WT (SEY6210) and npl3∆ (SPY77) strains were starved for nitrogen for 4 h and fractionated to obtain nuclear and cytoplasmic fractions. Then 20 pg of spike-in control RNA was added, and total RNA was extracted. qRT-PCR was performed to quantify the percentage of ATG1 mRNA in the two fractions. PGK1 was used as a control RNA, showing the specificity of this regulation. The spike-in RNA was used for normalization. Data are representative of three biological replicates, showing mean +/- SD. Two-way ANOVA was performed to determine the statistical significance. ** p value = 0.0388 (B) Polysome recruitment onto ATG1 mRNA is reduced when NPL3 is deleted: WT (SEY6210) and npl3∆ (SPY077) cells were subject to nitrogen starvation. Lysates were fractionated in a sucrose gradient and the abundance of ATG1 transcript was analyzed by qRT-PCR. Data are representative of two independent biological replicates, showing mean +/- SD. Two-way ANOVA was performed to determine the statistical significance. **** p value &lt;0.0001, *** p value &lt;0.0005, ** p value &lt; 0.01. (C) Npl3 targets the 5' UTR of the ATG1 transcript: Npl3-GFP tagged strains in WT ATG1 promoter (SPY113) and ATG1 mutant where the endogenous promoter is switched to the 5' UTR of ATG7 were subject to nitrogen starvation for 6 h and lysates collected were utilized to perform RNA immunoprecipitation. The abundance of the +35 fragment of ATG1 mRNA was quantified to determine the importance of 5' UTR in enabling binding of ATG1 with Npl3. Data are representative of 2 biological replicates, showing mean +/- SD. A t-Test was performed to determine the statistical significance. * p value = 0.0315 (D) Npl3 interacts with ATG1 in an RRM2-dependent manner: RNA immunoprecipitation of WT (untagged, SEY6210), Npl3-PA (SPY060) and Npl3 ∆RRM2-PA (SPY062) was performed to quantify the abundance of ATG1 mRNA interaction during nitrogen starvation. Data are representative of three biological replicates, showing mean +/- SD. Two-way ANOVA was performed to determine statistical significance. **** p value &lt;0.0001, *** p value = 0.0001. (E) The Npl3 RRM2 is required for upregulation of Atg1 protein during nitrogen starvation: WT (SEY6210), Npl3 ∆RRM2 (SPY060) and npl3∆ (SPY077) strains were subjected to nitrogen starvation for 6 h and Atg1 protein levels were determined by immunoblotting. Pgk1 was used as a loading control. (F) Quantification of D. Two-way ANOVA was performed to determine statistical significance. ** p value = 0.0043, * p value = 0.0239. (G) Npl3 RRM2 truncation does not affect ATG1 mRNA transcription: WT (SEY6210) and Npl3 ∆RRM2 (SPY060) cells were subjected to nitrogen starvation for 4 h and the abundance of ATG1 mRNA was determined. Student’s t test was performed to determine statistical significance. (H) Polysome profiling shows that ATG1 mRNA localization in cells expressing the Npl3 RRM2 deletion phenocopies npl3∆ during nitrogen starvation: ATG1 mRNA abundance in different polysome fractions was quantified in the WT (SEY6210) and Npl3 ∆RRM2 strains. The data for the WT strain (SEY6210) are the same as in Figure 2B. Two-way ANOVA was performed to determine the statistical significance. ****p value &lt;0.0001, *** p value &lt; 0.001.