Phosphorylation by G1-specific cdk-cyclin complexes activates the nucleolar transcription factor UBF

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Ribosomal RNA (rRNA) gene transcription is essential for cell growth and is tightly regulated during the cell cycle.
• Serum stimulation of quiescent cells triggers progression through the G1 phase, accompanied by increased rRNA synthesis.
• The nucleolar transcription factor UBF (upstream binding factor) is a key regulator of rRNA gene transcription by RNA polymerase I.

Purpose of the Study:

• To investigate the mechanism of transcriptional activation of rRNA genes during G1 phase progression.
• To determine the role of UBF phosphorylation in regulating rDNA transcription.
• To identify specific kinases and phosphorylation sites involved in UBF regulation.

Main Methods:

• Analysis of UBF activity and phosphorylation patterns in NIH 3T3 fibroblasts.
• Tryptic phosphopeptide mapping to identify phosphorylation sites on UBF.
• Site-directed mutagenesis to alter specific phosphorylation sites, including Ser484.
• In vivo and in vitro assays to assess the impact of mutations on rDNA transcription.

Main Results:

• Serum stimulation leads to increased rRNA gene transcription in NIH 3T3 fibroblasts.
• UBF phosphorylation pattern changes during cell cycle progression.
• Serine 484 (Ser484) of UBF is identified as a direct phosphorylation target for cyclin-dependent kinase 4 (cdk4)-cyclin D1 and cdk2-cyclin E.
• Mutation of Ser484 to alanine significantly impairs both in vivo and in vitro rDNA transcription.

Conclusions:

• UBF is regulated in a cell cycle-dependent manner.
• Phosphorylation of UBF at Ser484 by G1 cdks-cyclins is a critical step in the activation of rDNA transcription.
• This study establishes a direct link between G1 cell cycle regulators, UBF phosphorylation, and the control of rRNA synthesis.