Human tumor suppressor p14ARF negatively regulates rRNA transcription and inhibits UBF1 transcription factor phosphorylation

The nucleolar Arf protein has been shown to regulate cell cycle through both p53-dependent and -independent pathways. In addition to the well-characterized Arf-mdm2-p53 pathway, several partners of Arf have recently been described that could participate in alternative regulation process. Among those is the nucleolar protein B23/NPM, involved in the sequential maturation of rRNA. p19(ARF) can interact with B23/NPM in highmolecular complexes and partially inhibit the cleavage of the 32S rRNA, whereas the human p14(ARF) protein has been shown to participate in the degradation of NPM/B23 by the proteasome. These data led to define Arf as a negative regulator of ribosomal RNA maturation. Our recent finding that the human p14ARF protein was able to specifically interact with the rRNA promoter in a p53-independent context, led us to analyse in vitro and in vivo the consequences of this interaction. Luciferase assay and pulse-chase experiments demonstrated that the rRNA transcription was strongly reduced upon p14(ARF) overexpression. Investigations on potential interactions between p14(ARF) and the transcription machinery proteins demonstrated that the upstream binding factor (UBF), required for the initiation of the transcriptional complex, was a new partner of the p14(ARF) protein. We next examined the phosphorylation status of UBF as UBF phosphorylation is required to recruit on the promoter factors involved in the transcriptional complex. Upon p14(ARF) overexpression, UBF was found hypophosphorylated, thus unable to efficiently recruit the transcription complex. Taken together, these data de. ne a new p53-independent pathway that could regulate cell cycle through the negative control of rRNA transcription.