CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor

Abstract

The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource ( http://crispr.deniclab.com ) for further mining of novel autophagy mechanisms.

Negative Regulation of Autophagy by UBA6-BIRC6–Mediated Ubiquitination of LC3

“…Several recent studies also used genome-wide CRISPR/Cas9 screens to identify novel components and regulators of the autophagy machinery in mammalian cells [67] [68] [69] [70].…”

“…As in our study, these screens were performed using cells expressing fluorescently-tagged LC3 or autophagy receptors such as SQSTM1, NPD52, TAX1BP1 or NBR1. All of these screens resulted in the identification of novel candidates, most notably the ER protein TMEM41B, which was shown to participate in autophagosome biogenesis [68] [69] [70]. Our screen differed from previous ones in that we used cells expressing endogenously-tagged LC3B, thus avoiding artifacts of overexpression.…”

“…It is noteworthy that neither UBA6 nor BIRC6 ranked high in previous screens. This could be due to the overexpression of LC3B from a CMV promoter [70], which likely overwhelmed the ability of UBA6 and BIRC6 to target LC3B for degradation. Indeed, we observed that CMV-driven overexpression of FLAG-tagged LC3B resulted in smaller differences in the levels of this protein in WT, UBA6-KO and BIRC6-KO cells ( Figures 3D, 6A).…”

“…Indeed, we observed that CMV-driven overexpression of FLAG-tagged LC3B resulted in smaller differences in the levels of this protein in WT, UBA6-KO and BIRC6-KO cells ( Figures 3D, 6A). Furthermore, UBA6 and BIRC6 could have been missed in previous screens using autophagy receptors as reporters [67] [68] [70] because the levels of these proteins were unaltered under regular culture conditions, as observed for SQSTM1 and NBR1 in BIRC6-KO cells ( Figure 7A). Only when cells were treated with cycloheximide did differences in the turnover of these proteins become apparent ( Figure 7A-C).…”

Autophagy dark genes: Can we find them with machine learning?

“…However, recent studies have increasingly emphasized the existence of systems controlling and executing autophagy in mammalian cells that are quite different from those in yeast [3]. Among wellaccepted examples, are several autophagy factors absent in yeast that have been identified in organisms from C. elegans to H. sapiens, including ATG101 [4], FIP200 [5], VMP1 [6], EPG5 [7], Stx17 [8,9], and TMEM41B [10][11][12].…”