NonO is a multifunctional protein that associates with RNA polymerase II and induces senescence in malignant cell lines

Gene expression in eukaryotic cells is a complex, multistep process. Numerous studies have suggested that transcriptional and post-transcriptional processes are coordinated. An important player in this coordination is the Cterminal domain (CTD) of the largest subunit of RNA polymerase II. This thesis describes studies to characterize two CTD-associated proteins: NonO/p54nrb and PSF. These two proteins share 71% identity within a 320 amino acid region that includes two tandem RNP-type RNA binding domains, a putative helix-turn-helix motif, and a highly charged domain. Previous studies on NonO/p54nrb and PSF implicate them as two multifunctional proteins. Here I show that NonO physically interacts with PSF. NonO and PSF, either alone or in a heteromeric complex, interact with both hypo- and hyper-phosphorylated CTD and associates with RNA pol II in vivo. This suggests a role for both NonO and PSF in the coupling of transcription and RNA processing. Unlike most splicing factors, NonO does not localize to classically defined “speckles”. Instead it localizes both in a diffuse non-nucleolar pattern, and within multiple discrete nuclear ‘dots’ of unknown origin that are distinct from classic speckles. The localization of NonO is transcription-dependent and is regulated by phosphorylation. Over-expression of the C-terminal half of NonO relocalizes both RNA pol II and NonO to highly concentrated dots of apparent identity. PSF, while colocalizes with NonO in transcription-competent cells, accumulates in different nuclear structures when transcription is blocked. The appropriate nuclear localization of NonO may be important for growth regulation. Stable transfection showed that altered expression of NonO and PSF induces senescence in several highly transformed primate cell lines, but not in murine cell lines or in primary murine or human fibroblasts. NonO redistributes to the cytoplasm in cell lines having undergone senescence. A C-terminally truncated NonO localizes predominantly to the cytoplasm and accelerates the senescence process, suggesting that cytoplasm localization may be the trigger of senescence. The senescence caused by NonOPSF is dependent on the activation of Rb pathway and is telomere-independent. While the mechanism of NonO-PSF induced senescence remains to be determined, we suggest that their differential localization properties may play a significant role.