The working hypothesis and implications for a future characterisation of Mybbp1a 97

Based on the experimental outcome of this thesis work and in consideration of the available literature a future working hypothesis is introduced in the following. Suggestions for potential future experiments to further explore Mybbp1a function under different aspects have been already given throughout the discussion part. Still, this final chapter aims at integrating potential future approaches to provide a concluding comprehensive picture of the actual state of experiments. The transcription of rRNA genes, subsequent pre-RNA processing and ribosome assembly processes together constitute the major energy-consuming process in the cell and therefore the rate of ribosome biogenesis needs to be tightly linked to cellular proliferation. Currently available data suggest that in proliferating cells, Mybbp1a is mainly associated with the pre-ribosomal complexes where it acts as a scaffold for rRNA processing and assembly factors and is functionally required to drive efficient ribosome biogenesis. Reduced levels of ribosome biogenesis upon stress signals and/or reduced demands of ribosomes potentially result in the disassembly of pre-ribosomal particles, which involves the partial processing of Mybbp1a and its release to the nucleoplasm.

Figure 34: A future working hypothesis - Mybbp1a is part of a feed back mechanism to co-ordinate Pol I transcription and rRNA processing with the actual conditions of the cell.

(A) Under proliferative conditions Mybbp1a (blue ellipse) is part of the pre-rRNA processing complex and supports efficient ribosome biogenesis and cellular proliferation. (B) Nucleolar stress might lead to disruption of pre- ribosomal particles, partial processing of Mybbp1a and its release to the nucleoplasm. While full length Mybbp1a would repress rDNA transcription nucleoplasmic Mybbp1a would influence Pol II-dependent gene expression and p53 stabilisation leading to cell cycle arrest and eventually apoptosis (see also text).

While nucleolar full-length Mybbp1a would repress RNA Pol I transcription, potentially by disturbing pre-initiation complex formation or recruitment of chromatin-related factors to the

B

A

rDNA promoter, processed nuclear Mybbp1a would modulate the activity of transcription regulators, such as c-Myb, PGC-1α or NF-κB, to cease cell cycle progression, proliferation and energy production. It is well established that internal and external signalling pathways target Pol I-associated factors to translate the current demand of ribosomes in pre-initiation complex formation and subsequent rRNA synthesis rates. It will be interesting to assess such a role for the heavily phosphorylated Mybbp1a protein (Beausoleil et al. 2004; Beausoleil et al. 2006; Nousiainen et al. 2006; Olsen et al. 2006; Yu et al. 2007; Dephoure et al. 2008; Wang et al. 2008; Gauci et al. 2009).

The suggested hypothesis raises two major questions for future characterisation with respect to Mybbp1a function. On the one hand a definite mode of action or even different mechanisms underlying Mybbp1a function could not be finally determined during this thesis work. Here, three different aspects would be especially interesting for future assessment. First, the detailed characterisation of direct interaction partners of Mybbp1a could allow further insights on how Mybbp1a exerts its function as rDNA transcriptional repressor or component of the rRNA processing machinery. It would be interesting to see by the help of in vitro binding studies with recombinant proteins if Mybbp1a directly interacts with a subunit of the Pol I enzyme complex or rather with one of its associated factors such as TIF-IA. In addition, a final identification of the associated factors within the RNase-resistant complex or the assessment of existing interaction of Mybbp1a with the human t-Utps would be helpful to further shape the protein’s role in rRNA processing. Second, taking into account the experimental results of this thesis work and others it seems prospective to investigate a potential involvement of chromatin-related processes in Mybbp1a’s mode of action in more detail. Besides the reassessment of Mybbp1a binding to r-chromatin by qualitatively modified and extended ChIP experiments the analysis of potential interaction with specific chromatin modifications or modifiers such as remodelling machines or histone modifiers as well as its impact on chromatin-related processes such as nucleosome remodelling by functional assays would allow further conclusions on this issue. Finally, it would be important to distinguish between the impact of Mybbp1a on rDNA transcription and rRNA processing, which is rather difficult on the endogenous level. Therefore a dissection of these processes could be better realised by transient transfection of various Pol I reporter constructs including different lengths of rRNA coding sequence to analyse Pol I transcriptional output and eventually the recruitment of the rRNA processing machinery including Mybbp1a.

The second major issue for further investigation is the potential involvement of Mybbp1a in cell cycle regulation and/or other specific signalling pathways. Different data sets from the yeast and mammalian systems point in this direction. It will be especially interesting to test any physic interaction or functional impact of Mybbp1a with or on the Rb or the p53 pathway.

Intriguingly, very recently Yanagisawa and colleagues have determined a Mybbp1a- dependent stabilisation of p53-p300 interaction upon nucleolar stress to enhance p53- mediated transcription activation (Kuroda et al. 2011). Furthermore, it will be interesting to analyse, which signalling pathways target Mybbp1a by phosphorylation and in which way this might modulate its activity and function with the of help mutation analysis and the application of small molecule inhibitors directed against these pathways.

Importantly, several of the latest Mybbp1a-related publications also put a clinical relevance to the protein. For example, Mybbp1a was found to interact with the survival of motor neurons protein (SMN), of which reduced levels cause the inherited disorder spinal muscular atrophy (SMA). Mybbp1a partially co-localised with SMN in Cajal bodies in HeLa cell nucleoplasm and, like SMN, was reduced in cells from an SMA patient (Fuller et al. 2010). Most notably in this regard is the identification of Mybbp1a interaction with PGC-1α, a key regulator of energy metabolism, as well as Prep1, a homeodomain transcription factor (Fan et al. 2004; Diaz et al. 2007). The Prep1 factor has subsequently been shown to have a role in glucose homeostasis and insulin sensitivity in mouse models mediated, at least in part, by the interactor Mybbp1a (Oriente et al. 2008). These findings thereby contribute important implications for the understanding of energybalance and the development of diabetes.

Taken together Mybbp1a integrates many interesting functional features with respect to rRNA metabolism as revealed by this thesis work and various other important cellular pathways such as regulation of cellular proliferation, cell cycle and energy metabolism. Additionally, its further characterisation is not only important with regard to its cellular role(s) but most probably also to the understanding of mechanisms underlying fundamental cellular processes such as transcription regulation and co-ordination with subsequent RNA processing.