RNase E is an essential enzyme that has a global role in RNA metabolism. It functions as part of a large macromolecular complex known as the RNA degradosome. In 2008, the group of A.J. Carpousis (coordinator) published experimental work demonstrating that RNase E is localized at the periphery of the cell and bound to the inner cytoplasmic membrane by a membrane targeting sequence (MTS). RNase E localization is important for normal cell growth. The slow growth of the rne-delta-MTS strain suggests an alteration of RNase E activity in the cell that could involve accessibility to substrates or interactions with other membrane-bound machinery. The aim of our proposal is to explore the physiological role of the localization of RNase E to the inner cytoplasmic membrane. Over the past decade, the application of fluorescence microscopy to localize bacterial machineries involved in transcription, translation, RNA processing and mRNA degradation has toppled the long held myth that these processes occur in an aqueous ‘soup’ of freely diffusible macromolecules. Despite the lack of internal membranes, the machineries involved in transcription, translation and RNA processing and degradation are separated spatially. RNA polymerase is associated with the nucleoid at the center of the cell, freely diffusible polyribosomes are localized to a cytoplasmic space between the nucleoid and the inner cytoplasmic membrane, and key components in RNA processing and degradation are localized to the inner cytoplasmic membrane. Corollaries to the spatial separation of the transcription and degradation machineries are the prediction that mRNA degradation is initiated at the inner cytoplasmic membrane and that key steps in the maturation of transfer and ribosomal RNA also occur there. Our working hypothesis is that the membrane localization of the RNase E limits destructive interactions with functional RNA. We will focus mainly on the influence of RNase E membrane localization on the degradation of mRNA and the action of regulatory noncoding RNAs, but this work will also impact our understanding of RNA processing and RNA surveillance. Systematic approaches will be used to decipher the global regulatory functions of RNase E in a whole genome expression profile, in the stability of every cellular transcript, and in the processing events within the whole transcriptome. Our strategy will produce data with resolution at the nucleotide level. Global maps of RNase E direct targets when localized either at the membrane or displaced in the cytoplasm will be produced. A comparison of both data sets will give information on substrate accessibility depending on the cellular localization of RNase E. The rne-delta-MTS strain will be characterized at the molecular level. The physiological role of the localization of RNase E will also be addressed by a classical approach genetic approach involving a screen for suppressors of the growth defect in the rne-delta-MTS strain. This work will have major impact on our understanding the spatial organization of the bacterial cell regarding RNA processing, RNA surveillance and mRNA degradation. The complementary expertise of each of the 3 partners is essential for the achievement of the project. Partner 1, the group of A.J. Carpousis, has extensive experience in the field of bacterial mRNA degradation and was at the origin of the discovery of a multienzyme mRNA degrading complex known as the RNA degradosome. Partner 2, the group of M. Cocaign-Bousquet, has extensive expertise using systematic approaches to decipher bacterial physiology and metabolism. Partner 3, the group of C. Gaspin, has recognized expertise for their work on bioinformatic studies in the RNA field.