Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A Flow chart of the genome‐wide screening for novel essential factors of autophagy in yeast cells. For the screen performance, the knockout library (4,857 non‐essential genes) strains were grown on YPD plates followed by starvation on nitrogen‐lacking plates for 7 days. After starvation, the yeast cells were re‐grown on YPD plates to detect their viability. The potential candidates from this screen were selected based on the starvation‐resistance observation (deletion of an autophagy gene causes cell death after starvation). B N‐terminally GFP‐tagged 50Q was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 1, 4, and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls. C WT and Rpb9‐deleted yeast cells were starved for 3 days in SD‐N medium and then detected for cell viability by serial dilution spotting and growth on YPD plates. D, E Deficiency of Rpb9 caused blockage of autophagosome formation in yeast cells. Autophagosome marker Cherry‐Atg8 expressed in indicated cells was observed by fluorescence microscope before and after nitrogen starvation. In WT yeast cells, Cherry‐Atg8 was transferred to vacuoles by autophagy leading to the detection of Cherry signal in vacuoles. The Cherry‐Atg8 dots in vam3∆ cells showed blockage of autophagosome fusion and diffused cytosolic Cherry‐Atg8 in rpb9∆ or in atg1∆ cells showed blockage of autophagosome formation. Experiments were conducted for at least three times and representative images were shown. Scale bars: 5 μm. F Autophagic transport of substrate Ape1 was blocked in Rpb9‐deficient cells. Autophagy substrate Ape1 was labeled with RFP and expressed in WT and rpb9Δ cells. RFP‐Ape1 translocation to vacuoles by the autophagy process leading to the detection of RFP signal in vacuoles and blockage of autophagy in rpb9∆ cells caused cytosolic retention of Ape1. Experiments were conducted for at least three times and representative images were shown. Scale bars: 5 μm. G Detection of Rpb9‐dependent transport of autophagic substrate Pho8Δ60 by ALP assays. Yeast cells were starved for 4 h. Activated Pho8Δ60 activity in vacuoles was measured with indicated cells. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, **indicates P < 0.01, ***indicates P < 0.001 ( n = 5 biological replicates). H Vacuole localization and degradation of endocytosis substrate GFP‐Sna3 in WT, atg1∆ , and rpb9∆ cells. Scale bars: 5 μm. I Carboxypeptidases Y (Cpy1), a vacuolar‐resident hydrolase, was analyzed for its transporting to vacuoles by the secretory pathway in WT, atg1∆ , and rpb9∆ cells. Scale bars: 5 μm.

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Genome Wide, Knock-Out, Serial Dilution, Marker, Fluorescence, Microscopy, Labeling, Translocation Assay, Activity Assay, Standard Deviation, Comparison

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A N‐terminally GFP‐tagged Atg8 was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 0, 4, and 16 h starvation. Yeast cells with ATG1 deletion were used as positive controls. B C‐terminally GFP‐tagged Pgk1 was checked for its autophagic degradation in indicated yeast cells by GFP processing assays after 0, 4, and 16 h starvation. The PVDF membrane was subject to Ponceau staining and used as a loading control. C–E Expression determination of HA‐Rpb9 in experiments Fig . F Autophagic transfer of endogenous Ape1 in indicated yeast cells was analyzed at rich medium conditions. G Atg1 puncta was regulated by Rpb9. Atg1 was C‐terminally GFP tagged in chromosome and its puncta in indicated yeast cells after 4‐h SD‐N starvation was observed and quantified. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, **indicates P < 0.01 ( n = 5 biological replicates). Scale bars: 5 μm. H Atg13 puncta was regulated by Rpb9. Atg13 was C‐terminally GFP tagged in chromosome and its puncta in indicated yeast cells after 4‐h SD‐N starvation was observed and quantified. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, **indicates P < 0.01 ( n = 5 biological replicates). Scale bars: 5 μm. I Atg9 recruitment to Atg8 puncta was not regulated by Rpb9. C‐terminal Cherry tagged Atg9 and N‐terminal GFP tagged Atg8 were expressed in indicated yeast cells and their puncta co‐localization was observed and quantified. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test ( n = 5 biological replicates). Scale bars: 5 μm. J Atg12‐Atg5 conjugation was not regulated by Rpb9. C‐terminal HA‐tagged Atg5 and N‐terminal HA‐tagged Atg12 were expressed in indicated yeast cells and the Atg12‐Atg5 conjugation levels were analyzed. K Vacuole transfer of Carboxypeptidases Y (Cpy1) by the secretory pathway was analyzed in WT, vps15∆ , and vps34∆ cells. Scale bars: 5 μm.

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Membrane, Staining, Control, Expressing, Standard Deviation, Comparison, Conjugation Assay

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A WT and Rpb9‐deleted yeast cells were grown in YPD to log phase and then cultured in SD‐N media for 3 h. Yeast cells were collected and subject to genome mRNA sequencing and quantification. B Clustered analysis of differential gene sets from RNA‐seq data was shown with a heat map. C Genome‐wide analysis of differential gene expression in rpb9∆ yeast cells compared to WT cells ( P ‐value < 0.05, blue: downregulated, red: upregulated). Significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test ( n = 3 biological replicates). D WT and rpb9∆ yeast cells were grown to log phase in YPD (+N) and then shifted to nitrogen starvation (−N) for 3 h. ATG1 mRNA levels were quantified by qRT‐PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, ***indicates P < 0.001 ( n = 5 biological replicates). E–I Overexpression of ATG1 could partially restore autophagy in rpb9∆ cells. ATG1 , ATG9 , ATG13 , ATG17 , and ATG5 were overexpressed in rpb9∆ yeast cells, GFP‐50Q was checked for its autophagic degradation by GFP processing assays after 1, 4, and 16 h starvation.

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Cell Culture, Sequencing, RNA Sequencing, Genome Wide, Gene Expression, Comparison, Quantitative RT-PCR, Standard Deviation, Over Expression

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A Data from genome‐wide analysis of differential gene expression in rpb9∆ yeast cells compared to WT cells showed Rpb9 deletion caused specific downregulation of the ATG1 gene. Bars represent mean, error bars represent standard deviation ( n = 5 biological replicates). B WT, rpb9∆ , and rpb9type="InMathematical_Operators">∆ +HA‐Rpb9 yeast cells were grown to log phase in YPD (+N) and then shifted to nitrogen starvation (−N) for 3 h. mRNA levels of ATG1 , ATG5 , ATG13 , and ATG17 were quantified by qRT‐PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, *indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001 ( n = 5 biological replicates). C Protein expression levels of chromosome tagged Atg1, Atg5, Atg13, and Atg17 in WT and rpb9∆ yeast cells were analyzed before and after SD‐N starvation. D Atg1 was HA‐tagged at the C‐terminal by chromosome recombination in ypt7∆ cells and in ypt7∆ rpb9∆ cells and its protein levels were analyzed before and after nitrogen starvation in an SD‐N medium.

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Genome Wide, Gene Expression, Standard Deviation, Quantitative RT-PCR, Comparison, Expressing

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A Schematic representation of yeast Rpb9 and indicated truncates checked for function in autophagy and regulation of ATG1 transcription. B Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and autophagic degradation of GFP‐50Q was checked by GFP‐processing assays. C The truncates and mutants of the Rpb9 were expressed in rpb9∆ yeast cells and cell viability after 3‐day starvation in SD‐N medium was detected by serial dilution spotting and growth on YPD plates. D Indicated Rpb9 truncates and mutants were expressed in rpb9∆ cells and ATG1 mRNA levels were analyzed by qRT‐PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, *indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001 ( n = 5 biological replicates).

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Serial Dilution, Quantitative RT-PCR, Standard Deviation, Comparison

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A Schematic diagram of Rpb9 in RNA polymerase II. The 10 subunits of yeast RNA polymerase II were shown as ribbon diagrams (this figure was prepared with PyMOL). Rpb9 was shown with orange color while the other subunits were shown in gray. B His‐tagged Rpb9 was purified from Escherichia coli cells and subject to an electrophoretic mobility shift assay (EMSA) together with DNA probes spanning the indicated promoter regions of ATG1 .

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Purification, Electrophoretic Mobility Shift Assay

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A Chromatin immunoprecipitation (ChIP) assay was performed in Rpb9‐HA yeast cells and precipitated DNA was extracted and detected by qRT‐PCR analysis with primers spanning the indicated regions upstream and downstream of the ATG1 starting code. TFC1 was used as negative control. The dotted line indicates no enrichment compared to the control. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, *indicates P < 0.05, **indicates P < 0.01 ( n = 5 biological replicates). B His‐tagged Rpb9 and GST‐tagged Gcn4 were purified from Escherichia coli cells and subject to an electrophoretic mobility shift assay (EMSA) together with DNA probes spanning the indicated promoter regions of ATG1 . C WT, rpb9∆ , and gcn4∆ yeast cells were grown to log phase in YPD (+N) and shifted to nitrogen starvation (−N) for 3 h. ATG1 mRNA levels were quantified by qRT‐PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, *indicates P < 0.05, ***indicates P < 0.001 ( n = 5 biological replicates). D ChIP assays were performed with WT Rpb9‐HA or gcn4∆ Rpb9‐HA yeast cells followed by qRT‐PCR analysis. TFC1 was used as a negative control. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, *indicates P < 0.05, **indicates P < 0.01 ( n = 5 biological replicates). E Purified GST‐Gcn4 and His‐Rpb9 proteins from E. coli cells were mixed and GST pulldown assays were performed. F, G Neither overexpression of Gcn4 in rpb9∆ yeast cells nor overexpression of Rpb9 in gcn4∆ yeast cells can restore autophagy. Gcn4 or Rpb9 were overexpressed in rpb9∆ or gcn4∆ yeast cells respectively and autophagic degradation of GFP‐50Q was detected.

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Chromatin Immunoprecipitation, Quantitative RT-PCR, Negative Control, Control, Standard Deviation, Comparison, Purification, Electrophoretic Mobility Shift Assay, Over Expression

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A Schematic map of protein domains of Rpb9 orthologs in yeast and complex eukaryotic organisms. S. cerevisiae : Saccharomyces cerevisiae , H. sapiens : Homo sapiens , M. musculus : Mus musculus , D. melanogaster : Drosophila melanogaster , C. elegans : Caenorhabditis elegans . B Phylogenetic tree of Rpb9 orthologs from indicated eukaryotic species. C Rpb9 orthologs from complex eukaryotic species could restore autophagy in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP‐50Q was detected. D N‐terminal zinc finger domain and linker region of Rpb9 orthologs from different species could restore autophagy defects in rpb9∆ yeast cells. Indicated truncates of Rpb9 orthologs were expressed in rpb9∆ yeast cells and autophagic degradation of GFP‐50Q was detected. E Rpb9 orthologs restored starvation resistance in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ yeast cells and cell viability after SD‐N starvation was detected. F Rpb9 orthologs restored ATG1 transcription in rpb9∆ yeast cells. Indicated Rpb9 orthologs were expressed in rpb9∆ cells and ATG1 mRNA levels were analyzed by qRT‐PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, *indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001 ( n = 5 biological replicates). G POLR2I, the human ortholog of yeast Rpb9, regulates ULK1 transcription. Human HEK293T cells were transfected with siRNA targeting POLR2I and 72 h later the mRNA levels of autophagy genes were analyzed by qRT‐PCR. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, *indicates P < 0.05 ( n = 5 biological replicates).

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Quantitative RT-PCR, Standard Deviation, Comparison, Transfection

Journal: EMBO Reports

Article Title: The RNA polymerase II subunit Rpb9 activates ATG1 transcription and autophagy

doi: 10.15252/embr.202254993

Figure Lengend Snippet: A Protein alignment of Rpb9 orthologs from Saccharomyces cerevisiae , Homo sapiens , Mus musculus , Drosophila melanogaster , and Caenorhabditis elegans . B HEK293T cells were transfected with siRNA targeting POLR2I for 72 h and then the mRNA levels of POLR2I were analyzed. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, ***indicates P < 0.001 ( n = 5 biological replicates). C POLR2I knockdown increased the protein levels of autophagy receptor p62 which was subject to autophagic degradation. HEK293T cells were transfected with siRNA targeting POLR2I for 72 h and then the protein levels of p62 were analyzed. D POLR2I knockdown reduced the number of autophagosomes shown by LC3 puncta. HEK293T cells with expression of GFP‐LC3B were transfected with siRNA targeting POLR2I for 72 h and then the LC3 puncta was observed and quantified. Bars represent mean, error bars represent standard deviation, significance was determined by one‐way ANOVA (unpaired) followed by Tukey's multiple comparison test, **indicates P < 0.01 ( n = 5 biological replicates). Scale bars: 10 μm.

Article Snippet: Polyclonal POLR2I antibody (17270‐1‐AP) was from Proteintech.

Techniques: Transfection, Standard Deviation, Comparison, Knockdown, Expressing