Chemistry

RNA binding to p62 impacts selective autophagy

Position of RNA regulation in autophagy was relatively unexplored till Horos et al. not too long ago confirmed that vault RNA1-1 binds on to p62/SQSTM1, a well-established autophagy receptor. This binding results in inhibition of p62 oligomerization and damaging regulation of selective autophagy pathways.

Autophagy is a conserved eukaryotic mechanism for the clearance of undesirable mobile elements like protein aggregates (aggrephagy), broken organelles (e.g., mitophagy) and pathogens (xenophagy).1 On this course of, a membrane bilayer termed autophagosome encloses the disposable rubbish. Autophagosomes fuse with lysosomes for the decomposition and recycling. There are two modes of autophagy: selective and nonselective. Whereas selective autophagy identifies ubiquitin chains on the cargo for the focused supply, nonselective autophagy randomly sequesters cytoplasmic elements for lysosomal degradation.2

To harbor the cargo, selective autophagy makes use of a number of autophagy receptors, e.g., p62, OPTN, NDP52 and NBR1.three p62/SQSTM1, one of many best-characterized autophagy receptor, comprises LIR (LC3 interacting area) motif and UBA (ubiquitin related) area at its C-terminus which can be required for the binding to Atg8 members of the family (LC3/GABARAPs) and ubiquitin chains, respectively.three With the assistance of those interactions, p62 achieves binding to the ubiquitinated cargo on one hand and to the LC3-decorated phagosomes alternatively. p62 performs a essential function in aggrephagy, mitophagy and xenophagy.Four p62 is itself degraded in autophagolysosomes along with the cargo and can also be distinguished as a consequence of its capability to type higher-order oligomers by way of its N-terminal PB1 (Phox and Bem1p) area, which assist to achieve high-avidity interactions with ubiquitin and LC3-coated surfaces.5 Oligomerization depends on ubiquitin chains (primarily Ok63- or M1-linked ubiquitin chains) and results in section separated our bodies earlier than subsequent clearance by autophagy.6,7

In a latest research, Horos et al., recognized p62 as a RNA-binding protein utilizing a proteome-wide methodology named RBDmap (RNA-binding domains map).eight,9 On this methodology, UV-crosslinked protein-RNA complexes are purified utilizing magnetic oligo(dT) beads in two steps after which subjected to mass spectrometric evaluation. The peptide depth ratios between the RNA-bound and -released fractions are then used to establish RNA-binding websites. Subsequently, p62-RNA interplay was confirmed utilizing northern blotting, western blotting, PNK (polynucleotide kinase) assay and in vitro binding assays. Among the many RNAs recognized to bind p62, vault RNAs (vtRNAs) turned out to be probably the most outstanding hit. vtRNAs are ~88–100 nt lengthy non-coding RNAs transcribed by RNA polymerase III.10 All vtRNAs are predicted to fold into attribute stem-loop constructions. The function of stem loop in vtRNAs remains to be unknown, nonetheless, they doubtlessly take part in interactions with RNAs and proteins. Curiously, the evaluation of iCLIP experiments instructed that the binding of vtRNAs to p62 is pushed by structural signatures like loops relatively than a consensus binding motif. The authors additional recognized vtRNA1-1 as a primary p62-interacting RNA. The vtRNA1-1 interacting area of p62 was mapped to the zinc cluster 1 of the Zinc Finger (ZZ) area, which was additional characterised by lack of binding of the R139A, Ok141A (RK/A) double mutant of p62. Nonetheless, ZZ area doesn’t appear to be the one interplay floor because the oligomerization-deficient mutant of p62 (R21A, D69A, D73A triple mutant known as p62 PB1m) additionally confirmed diminished binding to vtRNA1-1.11

The authors additionally examined the useful penalties of vtRNA1-1 binding on p62 exercise and autophagy. Knockdown of vtRNA1-1 by antisense LNAs (locked nucleic acids) induced the shift in LC3B-II/LC3B-I ratio, indicating elevation in autophagy flux. Underneath hunger circumstances, the degrees of vtRNA1-1 are diminished, which facilitates p62-mediated autophagy induction. This raised the questions: how does RNA binding have an effect on autophagy by way of p62? What’s the underlying mechanism? Oligomerization of p62 and the binding of p62 to ATG8-like proteins (LC3B and GABARAP used on this research) seems to be prevented within the presence of vtRNA1-1. In a so-called ‘laddering assay’ the authors confirmed that p62 oligomerization may very well be visualized in cellulo after UV remedy and this oligomerization was visualized by western blot within the type of p62 higher-order oligomers/ladders. On this assay the ZZ area RK/A mutant seems to have the tendency to type extra higher-order oligomers in comparison with the wild-type (WT) p62 at basal circumstances, suggesting that vtRNA1-1 prevents p62 oligomerization.

To additional tackle this subject, the authors utilized a chemical compound XIE62-1004-Four (XIE) that has been proven to bind ZZ area of p62, inducing p62 oligomerization and additional p62-dependent autophagy. In comparison with the WT cells, vtRNA1-1 knockout cells confirmed elevated ranges of LC3B lipidation beneath XIE remedy, underlining the truth that vtRNA1-1 prevents p62-mediated autophagosome formation.

This research properly exhibits that p62 is a bona fide RNA-binding protein and its bodily interplay with vtRNA1-1 blocks oligomerization of p62 and disables efficient clearance of the gathered ubiquitinated cargo by selective autophagy (Fig. 1). But, a number of intriguing questions comply with this research. First, how does hunger downregulate the degrees of vtRNA1-1? The authors examined the impact of elevated expression of vtRNA1-1 by transfection on autophagy and located that elevated vtRNA1-1 led to prevention of p62-dependent autophagy and p62 accumulation itself. Since p62 accumulation has been implicated in a wide range of most cancers varieties, the query is whether or not the most cancers cells additionally use vtRNA1-1 as a device to protect p62 ranges that in flip promotes tumor progress by mTORC1, Nrf2 and NF-κB signaling.Four Malfunctions in autophagic equipment or p62 mutations have been proven to end in p62 aggregates, which type inclusion our bodies attribute of a number of pathological circumstances in mind and liver.12 Thus, it’s fascinating to find out the structural foundation of the interactions of vtRNA1-1 with the PB1 and ZZ domains of p62, which can assist design novel therapeutic interventions to inhibit p62 multimerization in such illnesses. Aside from vtRNAs, tRNAs additionally appeared as a success for p62 binding. The physiological penalties of the binding of different vtRNA paralogs and tRNA to p62 want additional investigation. Notably, the research raises the query of whether or not binding of different RNA molecules can regulate autophagy at a number of ranges of this multi-step course of or it is vitally distinctive to just one autophagy receptor as proven for p62. Maybe that is solely the tip of the iceberg and we have now quite a bit to find out about riboregulation in mobile catabolism.

Fig. 1Fig. 1

RNA binding negatively regulates p62-mediated autophagy. a p62 comprises an N-terminal PB1 area that’s chargeable for its oligomeric character adopted by a zinc finger area. C-terminal LIR motif and UBA area are chargeable for binding to Atg8 members of the family and ubiquitin (Ub) chains, respectively. Vault RNA1-1, a small non-coding RNA, blocks p62 oligomerization by way of interplay with the PB1 and ZZ domains. This leads to the inhibition of p62 exercise as autophagy receptor. b Starved cells present low ranges of vtRNA1-1, which allows p62 multimerization. Thus, Ub-dependent selective autophagy is favored beneath hunger in a p62-mediated method. The members of ATG8 household (LC3s/GABARAPs) are coloured in purple

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Institute of Biochemistry II, Goethe College – Medical School, College Hospital Frankfurt, Theodor-Stern-Kai 7, Frankfurt am Major, 60590, Germany

Mohit Misra & Ivan Dikic

Buchmann Institute for Molecular Life Sciences, Goethe College Frankfurt, Riedberg Campus, Max-von-Laue-Straße 15, Frankfurt am Major, 60438, Germany

Mohit Misra & Ivan Dikic

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https://doi.org/10.1038/s41422-019-0167-2


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