Membrane-bound RNase E: role in RNA processing, RNA surveillance and mRNA degradation – memRNase

This project is based on the collaboration of three Partners. Systematic approaches will be used to decipher the global regulatory functions of RNase E in a whole genome expression profile, in the measurement of stability of cellular transcripts, and in the mapping of RNA processing events. Our strategy will produce data with resolution at the nucleotide level. Global maps of RNA targets when RNase E is localized to either the membrane or 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. A mutant strain expressing cytoplasmic RNase E will be characterized at the molecular level. The complementary expertise of each of the partners is essential for the achievement of the project. Partner 1, a leader in the field of bacterial mRNA degradation, recently discovered that RNase E is localized to the inner cytoplasmic membrane. Partner 2 has extensive experience using systematic approaches to decipher bacterial physiology and metabolism. Partner 3 has recognized know-how in informatics analyses of RNA sequence, structure and function.

We have performed genome-wide surveys of transcript levels and stability using DNA microarrays. In these experiments, strains expressing cytoplasmic RNase E or disrupted for the genes encoding CsrA and CsrD were grown in batch and continuous cultures. CsrD is a membrane associated protein that acts in concert with RNase E to regulate the stabilities of CsrB and CsrC, which are small RNAs involved in Carbon Storage Regulation. CsrA is an RNA binding protein that interacts with CsrB, CsrC and mRNA to control the translation and stability of a large regulon of mRNAs. In the microarray analyses, transcript levels and stability were determined for approximately 2000 genes. Our 2016 publication in Science Reports presents the results of a genome-wide analysis of transcript levels and mRNA stability of strains in which the genes encoding CsrA and CsrD were disrupted. A key finding of this work is that CsrA is a global positive regulator of mRNA stability. This result suggests that CsrA binding to mRNA could have a general role in the formation of mRNP (messenger ribonucleoprotein) that is important for translation and stability. Currently, a much larger ensemble of strains and conditions are being surveyed to determine transcript levels and mRNA stability by a high-density sequencing approach. This analysis includes RNA samples that were previously analyzed by DNA microarrays as controls for the high-density sequencing analysis. Expected results include global mapping of RNase E cleavage sites at nucleotide resolution in wild type and mutant strains.

The stability of mRNA in bacteria is determined using protocols in which transcription is inhibited by rifampicin and mRNA levels are measured at times after addition of the drug to determine a rate of decay. Historically, transcript levels were determined for an individual mRNA by Northern blotting. More recently, a genome-wide approach (stabilome) was developed by making cDNA from total RNA and hybridizing to DNA microarrays. This protocol has been used routinely to accurately determine the level and decay rates of thousands of transcripts in a single experiment. One advantage of this protocol is that it does not require normalization to an internal standard. Over the past five years, microarrays have been supplanted by analyses in which cDNA prepared from RNA is analyzed by high-density sequencing. Although potentially more powerful than DNA microarrays because it permits mapping at nucleotide resolution, the normalization of high-density sequencing reads to accurately determine transcript levels is a technical challenge. We are in the midst of large-scale analyses to determine stabilomes by high-density sequencing. Once a pipeline for mapping the reads and normalizing to internal standards is validated, we will then turn our attention to the biological question that motivated this project, namely, what is the physiological role of the association of RNase E to the inner cytoplasmic membrane and how does membrane-associated RNase E interact with the CSR system.

Esquerré et al. (2016) The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli. Sci Rep 6: 25057.

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.