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Coregulation of Pol I, Pol II and Pol III in ribosome biogenesis in mammalian cells

Final Activity Report Summary - Pol I transcription (Coregulation of Pol I, Pol II and Pol III in ribosome biogenesis in mammalian cells)

Cells need ribosomes in order to create proteins. Ribosomes consist of ribonucleic acids (RNAs) and proteins. The ribosomal RNAs are produced by RNA polymerases (Pol) I and III during a process called transcription. The ribosomal proteins are produced in a two-step process, the first being transcription by Pol II. Thus, the making of a ribosome requires the action of three different Pols. It is believed that, in order to obtain a balanced amount of ribosomal components, the activities of the three Pols are co-regulated. It was shown in yeast, in consistence with this idea, that deliberate deregulation of Pol I activity resulted in the deregulation of the activities of the other two Pols.

The initial objective of this study was to determine whether a co-regulation of the three Pols for synthesis of ribosomal components could be demonstrated in human cells. To approach this, I attempted to deregulate Pol I transcription in human cells in the same manner as had been done in yeast. However, in collaboration with Drs Ingrid Grummt and Holger Bierhoff from the ‘German Cancer Research Center’, Heidelberg, Germany, I found that the mechanism by which Pol I synthesised ribosomal RNA in human cells was different enough from the mechanism used in yeast cells, therefore I could not use the previously exploited approach to deregulate Pol I activity in yeast. Although I could not answer my initial question with this approach, the project revealed important clues as to the mechanism of human Pol I transcription.

I then decided to start a second project and to continue exploring deregulating human Pol I by other methods, as planned in my original proposal. However, my second project started to produce highly interesting results, and I hence quite quickly decided to focus all my efforts on it.

This second project involved the protein Maf1, which inhibited Pol III activity in yeast and mammalian cells. It also affected Pol I and II transcription. The study of Maf1 could, therefore, reveal mechanisms by which the activities of the three Pols were regulated. Maf1 itself was known to be regulated by phosphorylations, a modification which results in yeast in the inactivation of Maf1. Maf1 phosphorylation was triggered by signals coming from outside the cell, for example nutrient availability, and transferred to Maf1 through a cascade of events which were still poorly understood. Indeed, despite the importance of Maf1, the study of this protein in mammalian cells was just beginning by the time of the project elaboration.

The general objective of this study was to characterise how Maf1 was regulated, in particular to identify where and by which enzyme Maf1 was phosphorylated. I was able to show that Maf1 was rapidly dephosphorylated in response to signals such as serum starvation, which mimicked nutrient deprivation. I identified two major sites of phosphorylation and, by the time of the proposal completion, I was testing several enzymes which were likely candidates to perform the Maf1 phosphorylations.

Using cells that did not express Maf1 I could show that there was no transcription arrest induced by signals such as serum starvation, even though these signals resulted in transcription arrest in normal cells. We concluded that Maf1 was required to obtain transcription arrest by the signalling pathways transmitting nutrient starvation information.

Both projects were on transcription regulation and gave us new insights in this important cellular process. Firstly, we discovered a major difference between the regulation of Pol I in yeast and mammalian cells. Secondly, we identified important aspects of the regulation of the Pol III inhibitor Maf1. Deregulated transcription could have major consequences and eventually result in cancer. A better understanding of the transcription regulation was essential to give us new clues on how to fight this major disease.