An mRNA degradation complex in Bacillus subtilis
mRNA Abbau in Bacillus subtilis
by Martin Lehnik-Habrink
Date of Examination:2011-10-26
Date of issue:2011-12-14
Advisor:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Jörg Stülke
Referee:Prof. Dr. Kai Tittmann
Referee:PD Dr. Wilfried Kramer
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EnglishIn every organism the degradation of mRNAs is a fundamental process. This step not only recycles nucleotides but also adjusts gene expression due to the availability of the mRNA templates. The main players in this process are RNases which can function as single enzymes or in multiprotein complexes, the so-called RNA degradosomes. Recently, the existence such an RNA degradosome was demonstrated in the Gram-positive model organism Bacillus subtilis. The investigation of the complex and detailed analyses of some of its members was the aim of this work. The initial model of the RNA degradosome of B. subtilis consists of RNases and glycolytic enzymes. The central RNase of the complex is the endoribonuclease RNase Y. To assess the impact of RNase Y on mRNA decay we performed microarray analysis demonstrating that depletion of the enzyme affects the degradation of transcripts on global scale. Further analysis of several targets revealed that RNase Y is indeed crucial for the turnover of the transcripts resulting in remarkably increased half lives upon depletion of the enzyme. These experiments highlight RNase Y as a key player in the mRNA degradation of B. subtilis. Due to its high significance in the mRNA decay process we further investigated RNase Y for its physiological and biochemical properties. We studied its domain organization and revealed that the protein contains substantial regions of intrinsic disorder. Furthermore we demonstrated that the membrane localization of RNase Y is essential for survival. As mentioned, the initial model of the RNA degradosome contained only RNases and glycolytic enzymes. In contrast to homolog complexes in other bacteria, a DEAD-box RNA helicase was not present in our model. Therefore we re-addressed this issue and revealed that the DEAD-box RNA helicase CshA has the ability to interact with all enzymes of the complex, especially with the important RNase Y. The suggested participation of CshA in the RNA degrading complex is underlined by the fact that deletion of cshA affects the abundance of hundreds of mRNAs. In addition to its participation in the RNA degradosome, we found that CshA is implicated in the biogenesis of ribosomes and the adaption to low temperatures. In conclusion, the analysis of RNase Y and CshA as members of the RNA degradosome revealed the impact of both enzymes for the physiology of B. subtilis and pave the way for further investigations.
Keywords: Bacillus subtilis; RNA degradation; RNase; mRNA; RNA helicase; degradosome; proteincomplex; DEAD-box
Der Abbau von mRNA Molekülen ist ein fundamentaler Prozess aller Lebewesen. Dieser Schritt dient nicht nur der Rückgewinnung von Nukleotiden, sondern reguliert auch die Genexpression durch die Verfügbarkeit von mRNA Matrizen. Die wichtigsten Spieler in diesem Prozess sind RNasen. Diese können als einzelne Enzyme arbeiten oder in Multiproteinkomplexen vorkommen, den sogenannten RNA-Degradosomen. Ein solches RNA Degradosom wurde kürzlich auch für den Gram-positiven Modelorganismus Bacillus subtilis beschrieben. Die Untersuchung dieses Komplexes und detailierte Analysen der beteiligten Enzyme waren die Ziele dieser Arbeit.
Schlagwörter: Bacillus subtilis; RNA Abbau; RNase; mRNA; RNA Helikase; Degradosom; Proteinkomplex; DEAD-box