The p14/p19ARF (ARF) tumor suppressor provides an important link in the activation of p53 (TP53) by inhibiting its targeted degradation via the E3 ligases MDM2/HDM2. However, ARF also limits tumor growth by directly inhibiting ribosomal RNA synthesis and processing. Initial studies of the ARF tumor suppressor were compounded by overlap between the INK4A and ARF genes encoded by the CDKN2A locus, but mouse models of pure ARF-loss and its inactivation in human cancers identified it as a distinct tumor suppressor even in the absence of p53. We previously demonstrated that both human and mouse ARF interact with Transcription Termination Factor 1 (TTF1, TTF-I), an essential factor implicated in transcription termination and silencing of the ribosomal RNA genes. Accumulation of ARF upon oncogenic stress was shown to inhibit ribosomal RNA synthesis by depleting nucleolar TTF1. Here we have mapped the functional nucleolar localization sequences (NoLS) of mouse TTF1 and the sequences responsible for interaction with ARF. We find that both sequences lie within the 25 amino acid N-terminal repeats of TTF1. Nucleolar localization depends on semi-redundant lysine-arginine motifs in each repeat and to a minor extent on binding to target DNA sequences by the Myb homology domain of TTF1. While nucleolar localization of TTF1 predominantly correlates with its interaction with ARF, NoLS activity and ARF binding are mediated by distinct sequences within each N-terminal repeat. The data suggest that the N-terminal repeats of mouse TTF1, and by analogy those of human TTF1, cooperate to mediate both nucleolar localization and ARF binding.
Keywords: Nucleolar localization sequence; RNA polymerase I; Transcription Termination Factor 1; p19/p14ARF; ribosome biogenesis; tumor suppressor.