Nol9, a novel RNA-DNA 5´-kinase in ribosomal RNA processing

A large variety of ribosomal and non-ribosomal proteins participate in the biogenesis of ribosomal RNAs (rRNAs), a well-studied multistep process that generates mature rRNAs from a long polycistronic RNA precursor. We have discovered that the non-ribosomal protein Nol9 is essential for this process. Nol9 is a nucleolar polynucleotide 5`- kinase that associates with the pre-60S ribosomal particles. Depletion of Nol9 leads to a severe impairment of ribosome biogenesis. Interestingly, it is the enzymatic activity of Nol9 that is required for the efficient generation of the 5.8S and 28S rRNAs from a 32S precursor. Upon Nol9 knock down, we also observe a specific maturation defect at the 5` end of the predominant 5.8S short form rRNA (5.8SS), possibly due to the requirement of Nol9 for 5` to 3` exonucleolytic degradation. There is a dual novelty in these findings: Nol9 is the first nucleolar RNA/DNA kinase reported, and the first enzyme of this type involved in the processing of ribosomal RNA molecules. Within this grant proposal, we aim to extend these studies in order to dissect function(s) of Nol9 in human cells, both in vitro and in vivo. We will use different biochemical approaches to identify RNA substrates and interacting proteins, and proceed to establish in vitro systems to understand why is Nol9 critical for the processing of 32S rRNA precursors. To study the function of Nol9 in vivo we will generate mouse conditional knockout models in collaboration with Josef Penninger at IMBA.

The cell is the smallest unit of life. Its amazingly precise functioning is the result of billions of years of evolution. At the core of the cell are macromolecules called proteins: some of them are inert while others are alive, meaning that they catalyze reactions that in their absence would otherwise require much too long time periods incompatible with life. Alive proteins are known as enzymes. Proteins are produced in factories called ribosomes. Composed of four long RNA (RiboNucleic Acid) molecules and a plethora of associated proteins, the ribosome organizes in a large and a small subunit. The ribosome decodes the language within every single messenger RNA and translates it into a protein sequence. RNA molecules within the ribosome are initially transcribed as long precursors that then undergo an extremely complex processing before being assembled into a functional ribosome. Due to its relative simplicity this process has been largely studied in yeast, but seldom addressed in human cells. In this project we have characterized NOL9, an enzyme that is very important for the processing of ribosomal RNA precursors. NOL9 can add a phosphate group to the terminus of DNA or RNA molecules. We could show that its activity is required for the efficient processing of the ribosomal RNA. We also revealed that NOL9 acts in association with other proteins.Ongoing experiments are addressing the role of NOL9 in vivo, which means working with mice that are unable to produce NOL9 because its gene has been removed.