Fig. 1: Identification of RNAseK in a screen for autophagy regulators. | Nature Communications

Fig. 1: Identification of RNAseK in a screen for autophagy regulators.

From: The V-ATPase complex component RNAseK is required for lysosomal hydrolase delivery and autophagosome degradation

Fig. 1

a Validation of the GFP-LC3 reporter MEF cell line. Cells expressing Cas9 alone (sgControl) or sgRNA targeting ATG7 (sgATG7) were left untreated or amino acid (AA) starved for 2 h in the presence or absence of Baf A1 or VPS34 inhibitor (VPS34i). Cell lysates were subjected to western blot analyses using the indicated antibodies. N = 1. b Work flow for loss of function screen. MEFs stably expressing GFP-LC3 and Cas9 were transduced with sgRNA library and subjected to AA starvation (16 h) to induce autophagy and FACS sorting into GFP positive (autophagy deficient) and GFP negative (autophagy competent) populations. Genomic extraction and amplification was used to identify sgRNA sequences. b was created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. c Deep sequencing analysis of sorted GFP positive cells. Highlighted are top 100 identified hits, including Atg genes and Rnasek. d Western blot analyses of sgControl MEF cells and RNAseK knockout cells using the indicated antibodies. Cells were left untreated or AA starved for 2 h in the presence or absence of Baf A1. N = 3 biologically independent experiments. e Quantification of LC3-II levels normalised to actin in (d). f Quantification of p62 levels normalised to actin in (d). g Quantitative DNA assessment of Rnasek gene editing efficiency in C57BL/6 mice interscapular brown adipose tissue (iBAT). DNA was extracted from iBAT 2 weeks after AAV injection and analysed using online tools. Gene editing in sgRNaseK#3 was normalised to AAV SaCas9 control (sgControl). N = 3 animals. h Western blot analyses of iBAT derived from C57BL/6 mice 2 weeks after administration with the indicated AAV vectors using the indicated antibodies. N = 3 animals per condition. i Quantification of LC3-II and p62 protein levels normalised to GAPDH, as shown in (h). In all panels, mean + SEM is assessed by unpaired two-tailed Student’s t test. Source data are provided with this paper.

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