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Drosophila melanogaster miPEP8 Regulates Cell Size Through its Interaction With ref(2)P/p62
Carine Duboé1, Clémence Guillon1, Nathanael Jariais1
1Laboratoire de Recherche en Sciences Végétales (LRSV), CNRS/Université de Toulouse/INPT, Auzeville-Tolosane, France.
Abstract:
MiPEPs are microproteins encoded by primary transcripts of microRNAs (pri-miRNAs). Initially identified in plants, we recently characterized a miPEP in Drosophila melanogaster, named miPEP8, which is involved in the regulation of wing size. However, mechanisms at play are unknown. In the present study, we take advantage of the Drosophila cell line Schneider 2 (S2) to further investigate miPEP8 function at the molecular level. Overexpressing miPEP8 in S2 cells induced a reduction of cell size as well as an increase of the proportion of cells in the G1 phase of the cell cycle and a decrease of the autophagic flux. A proteomics analysis revealed that miPEP8 overexpression in S2 cells induces the upregulation of several proteins including the autophagosome cargo protein ref(2)P (the orthologue of the human p62/Sequestosome 1 protein). The interactome of miPEP8 was generated and revealed interactions between this miPEP8 and the mTORC1/autophagy pathway. Bioinformatics analysis identified a short linear motif (SLiM) on miPEP8 sequence. Mutation of this SLiM prevented the interaction between ref(2)P/p62 and miPEP8. Mutation of the SLiM also reverted the smaller cell size phenotype observed when overexpressing miPEP8 in S2 cells. RNA interference targeting ref(2)P/p62 reversed the cell size phenotype, suggesting that this protein plays a role in the regulation of cell size in Drosophila. Finally, the cell size phenotype was also observed in vivo on wings of flies either mutated or overexpressing miPEP8.
Insights
Micropeptides (miPEPs) like miPEP8 regulate cell size in Drosophila. miPEP8 interacts with ref(2)P/p62, impacting the mTORC1/autophagy pathway and cell cycle, ultimately affecting wing size.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- Micropeptides (miPEPs) are encoded by microRNA primary transcripts (pri-miRNAs).
- A Drosophila melanogaster miPEP, miPEP8, was previously linked to wing size regulation, but its mechanism remained unclear.
- Understanding miPEP8's function is crucial for elucidating novel regulatory pathways.
Purpose of the Study:
- To investigate the molecular mechanisms underlying miPEP8's function in Drosophila.
- To identify proteins and pathways interacting with miPEP8.
- To determine miPEP8's role in cell size and cell cycle regulation.
Main Methods:
- Overexpression of miPEP8 in Drosophila Schneider 2 (S2) cells.
- Proteomics analysis to identify upregulated proteins.
- Interactome generation and bioinformatics analysis to identify motifs and interactions.
- Mutation of a short linear motif (SLiM) on miPEP8.
- RNA interference (RNAi) targeting ref(2)P/p62.
- In vivo studies on Drosophila wings.
Main Results:
- miPEP8 overexpression reduced S2 cell size, increased G1 phase cells, and decreased autophagic flux.
- Proteomics revealed upregulation of ref(2)P (p62 orthologue) upon miPEP8 overexpression.
- miPEP8 interacts with the mTORC1/autophagy pathway.
- A SLiM on miPEP8 mediates interaction with ref(2)P/p62; its mutation reverted cell size reduction.
- RNAi against ref(2)P/p62 reversed the cell size phenotype.
- Cell size phenotypes were observed in vivo in Drosophila wings.
Conclusions:
- miPEP8 regulates cell size in Drosophila, partly through interaction with ref(2)P/p62 and the mTORC1/autophagy pathway.
- Ref(2)P/p62 is a key mediator of miPEP8's cell size regulatory function.
- These findings reveal a novel role for miPEPs in cellular homeostasis and organismal development.
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