Drosophila melanogaster miPEP8 Regulates Cell Size Through its Interaction With ref(2)P/p62.

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.