Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile.
During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8's ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.
Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06536, USA.
Kristina M
KM
Herbert
Genaro
G
Pimienta
Suzanne J
SJ
DeGregorio
Andrei
A
Alexandrov
Joan A
JA
Steitz
D002460:Cell Line; D002465:Cell Movement / Q000235:genetics; D049109:Cell Proliferation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000235:genetics*; D016601:RNA-Binding Proteins; D043244:Ribonuclease III / Q000378:metabolism*
C474043:DGCR8 protein, human; D035683:MicroRNAs; D011506:Proteins; D016601:RNA-Binding Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; C517918:DROSHA protein, human; D043244:Ribonuclease III