Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• MicroRNA (miRNA) biogenesis is crucial for gene regulation.
• The microprocessor complex (MC), comprising Drosha and DGCR8, processes primary miRNAs (pri-miRNAs) into pre-miRNAs.
• Understanding DGCR8 regulation is key to controlling miRNA production.

Purpose of the Study:

• To investigate the role of DGCR8 phosphorylation in miRNA biogenesis.
• To identify phosphorylation sites on human DGCR8.
• To determine the functional consequences of DGCR8 phosphorylation on MC activity and cellular processes.

Main Methods:

• Phosphoproteomic analysis to map DGCR8 phosphorylation sites.
• Expression of wild-type, phosphomimetic, and phosphomutant DGCR8 in mammalian and insect cells.
• Assessment of protein levels, mRNA levels, localization, self-association, and protein stability.
• Analysis of miRNA processing activity and cellular proliferation assays.

Main Results:

• Identified 23 phosphorylation sites on human DGCR8.
• DGCR8 is phosphorylated by ERK/MAPK, suggesting a link to extracellular signaling.
• Phosphomimetic DGCR8 increased DGCR8 and Drosha protein stability and cellular levels without affecting mRNA.
• MC processing activity was not altered by DGCR8 phosphorylation status.
• Cells expressing phosphomimetic DGCR8 showed enhanced proliferation and a progrowth miRNA profile.

Conclusions:

• DGCR8 phosphorylation by ERK/MAPK enhances its protein stability, leading to increased cellular levels.
• Phosphorylation of DGCR8 influences cellular proliferation and miRNA expression profiles.
• DGCR8 phosphorylation serves as a regulatory mechanism integrating extracellular signals to modulate miRNA biogenesis and cell behavior.