The LC3-conjugation machinery specifies the loading of RNA- binding proteins into extracellular vesicles (Nature Cell Biology 2020)

報告日期: 2020/04/21
報告時間: 17:10/18:00
報告學生: 古雅竺(英文報告)
講評老師: 王憶卿
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The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles

Leidal AM, Huang HH, Marsh T, et al.

Nat Cell Biol. 2020;22(2):187–99.

Presenter: 古雅竺                         

Advisor: 吳華林/余建泓 老師                         Date/Time: 2020/04/21 17:10-18:00

Commentator: 王憶卿 老師                    Location: Room 602, Med. College Building

Abstract

Beyond the cellular self-eating function, autophagy also promotes the secretion of cellular components including inflammatory cytokines, lysozyme and proteins lacking signal peptides. These untraditional processes are referred to as secretory autophagy. Although recent studies revealed autophagy-related proteins (ATGs) are involved in cellular secretion, the underlying mechanisms are still unclear. Moreover, the cargoes specified in this autophagy-dependent secretion also remain unclarified. Here, the authors identified LC3-mediated secretome by using proximity-dependent biotinylation (BioID) strategy. These secreted candidates were enriched in RNA-binding proteins (RBPs) and proteins released within extracellular vesicles (EVs). Further isolating the EVs from the conditioned medium of biotin ligase (BirA*)-LC3 cells, they found LC3II is co-fractioned with EVs. Fluorescence micrographs also showed intracellular LC3 was localized within multivesicular bodies (MVBs), suggesting LC3 specifically packaged targets into EVs to secrete outside the cells. Using Tandem Mass Tag (TMT)-based quantitative proteomics to analyze LC3-dependent EV proteome, they demonstrated many RBPs such as SAFB, HNRNPK, LARP1, SF3A1 and G3BP1 were also included in the BirA*–LC3B-labelled secretome. Focusing on SAFB and HNRNPK for following investigation, they revealed these two RBPs required LC3-conjugation machinery to release, which pathway was termed as LC3- dependent EV loading and secretion (LDELS). Since secretion of several RBPs mainly depended on LDELS, they further evaluated how LDELS influences extracellular RNA secretion by performing RNA-seq. The results showed significant differences in the species of small noncoding RNA which secreted within EVs. In addition, they identified neutral sphingomyelinase 2 (nSMase2) and factor associated with nSMase2 activity (FAN) were required for LDELS. Overall, this study depicted a mechanism of secretory autophagy depending on LC3-conjugation machinery, which loads RBPs and RNA cargoes into EVs to secrete outside the cells.

Reference:

  1. Cadwell, K. & Debnath, J. Beyond self-eating: the control of nonautophagic functions and signaling pathways by autophagy-related proteins. J. Cell Biol. 217, 813–822 (2018).