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Martin Koš 
Ribosomal RNA processing and modification

Groupleader: Martin Koš


Ribosome biogenesis is a major metabolic process in all organisms. Many components of the ribosome synthesis pathway have been linked to the regulation of cell growth and the cell cycle. This complex process, which is highly conserved in eukaryotes, begins with transcription of a large ribosomal RNA (rRNA) precursor that is subsequently covalently modified and processed into mature 18S, 5.8S and 25S rRNAs. These rRNAs assemble together with the independently transcribed 5S rRNA and ~80 ribosomal proteins into functional ribosomes. Pre-rRNA processing takes place in very large particles (>2MDa) called pre-ribosomes, molecular machines that ensure that the rRNA is properly processed, folded and assembled with ribosomal proteins. At least 170 non-ribosomal proteins and 70 small nucleolar RNAs (snoRNAs) have been implicated in ribosome biogenesis in budding yeast. These findings revealed that the process of ribosome maturation is surprisingly complex and the pre-ribosomal particles are highly dynamic. However, the precise functions of most of the components involved remain unclear.
The goal of the lab is to extend our understanding of molecular mechanism underlying the ribosome biogenesis. We focus on the following questions:

A) What is the role of RNA helicases in ribosome biogenesis? Among the myriad factors required for ribosome synthesis are 18 putative ATP-dependent RNA helicases. The potential need for RNA unwinding during ribosome synthesis seems obvious, as the rRNA has extensive secondary structure that appears to be incompatible with assembly. In addition, base-pairing between rRNA and snoRNAs is necessary for rRNA modification and cleavage. Initial genetic analyses showed that individual helicases have non-redundant functions but failed to reveal the specific functions of individual helicases. It has been recently shown that two RNA helicases are required for unwinding snoRNAs from pre-rRNA. However, the substrates for the other RNA helicases remain unknown. The lab is investigating the mechanisms by which RNA helicases modulate the structure of a large RNA-protein complex such as a ribosome. The gained insight will shed light on their potential functions in other processes, for example splicing or transcription.

B) The mechanims and functions of rRNA modifications. Ribosomal RNAs in all kingdoms of life are extensively covalently modified by methylation and pseudouridylation. The number of modifications increased during evolution from a handful in Bacteria to >200 in humans. It is generally believed that these modifications fine-tune the ribosomal RNA conformation for efficient and precise translation. Despite of many years of research the precise roles of individual modification and underlying mechanisms remain largely unclear. It is also unclear to which extent the process of rRNA modification is regulated and if ribosomes with different modification status and thus potentially specialized functions are produced.  

C) How is the regulation of ribosome biogenesis connected to cell growth? Systems biology of ribosome biogenesis. The rate of ribosome synthesis is precisely adjusted to the growth status of the cell. The TOR pathway was shown to regulate ribosome biogenesis in response to environmental signals, mainly at the level of transcription. However, it remains unclear how co-regulation of rDNA transcription with production of ribosomal proteins and ribosome synthesis factors is achieved. It is also unknown whether ribosome synthesis is regulated at steps downstream of rRNA transcription. One of the obstacles in the field is a lack of techniques to time-resolve the plethora of pre-ribosomal complexes and identify their precise functions. We will use a combination of techniques of systems biology (quantitative methods and mathematical modeling) and classical biochemistry to tackle these problems.

The lab uses as model organisms yeast Saccharomyces cerevisiae and mammalian cells in tissue culture.

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