Skip to main content
European Commission logo print header

Spatial organization and dynamics of Escherichia coli RNA degradation machinery

Final Report Summary - RNASEDYNAMICS (Spatial organisation and dynamics of Escherichia coli RNA degradation machinery)

In the first part of the project we explored goals connected with the spatial organisation and dynamics of Escherichia coli Ribonucleic acid (RNA) degradation machinery. We prepared a set of E. coli strains with different ribonuclease (RNase)s and RNA metabolism proteins fused with green fluorescent protein (GFP) and tandem affinity purification (TAP) tag and we investigated their localisation and interactions. The main achievement of this part of the project was the discovery that RNase R is connected with the ribosomes, which will be shortly submitted to an international peer-reviewed publication.

We also wanted to investigate in detail the location, mobility, and interactions of RNA degradation machinery in E. coli. Preliminary results obtained in Prof. Cecilia Arraiano laboratory clearly showed that RNase R and RNase II, the two E. coli exonucleases belonging to the same RNase II family, are differently localised. RNase II was citoplasmatic while RNase R was mainly located near the nucleoid. To investigate this peculiarity in more details we prepared E. coli strains with endogenous RNase R fused to GFP (to investigate its localisation) or TAP tag (to investigate its interactions). We investigated the localisation of RNase R in different growth conditions and compared it in context of the nucleoid localisation or the localisation of the RNA polymerase subunit RpoC. The localisation study did not bring validated results, probably due to the low copy number of RNase R in the cell which results in weak fluorescence and brings difficulties in data interpretation. On the other hand the construction of the RNase R TAP tag fusion suggested a connection between RNase R and cellular ribosomes. To confirm these results we decided to use sucrose gradient ribosome profiling and western blots. We proved that RNase R in the different growth conditions tested was always connected to with the ribosomal fraction. Our data also showed that differently to RNase R, RNase II is not connected with the ribosomes what explains to some extent its different localisation. Our data are in agreement with the possible involvement of RNase R in the degradation of ribosomal RNAs function.

The second part of the project focused on work on the eukaryotic model Schizosaccharomyces pombe. This research started in parallel with the bacterial project. However the results were so promising that the main part of the subsequent work was concentrated on this topic. Our work unrevealed a new eukaryotic RNA degradation pathway in the cytoplasm, and the detailed results are accepted with minor modifications in the high impact international peer-reviewed journal - The EMBO Journal. The objective was the investigation of the function of a newly discovered S. pombe nuclease, which appeared to be a homologue of human DIS3L2, and belonged to the RNase II family of enzymes. Two general pathways of messenger RNA (Mrna) degradation were previously identified in eukaryotes. It has been widely established that the final step of RNA decay proceeds in either 5'-3' direction catalysed by Xrn1 exoribonuclease or 3'-5' direction catalysed by the exosome complex. The active subunit of the exosome complex is Dis3/Rrp44 is a 3'-5' exonuclease which also contains endonucleolytic activity. Recently it was shown that in humans, in contrast to budding yeast, there are three DIS3 isoforms - DIS3, DIS3L and DIS3L2. In this part of the project we identified a novel S. pombe RNA exonuclease. Our extended phylogenetic analysis proves that identified nuclease belongs to the same protein family as human DIS3L2, and that members of this family are conserved throughout eukaryotes. Our work demonstrates that in S. pombe Dis3L2 protein is an important novel factor in cytoplasmic RNA decay, degrading RNAs independently of the exosome. Therefore a conserved, 3'-5' cytoplasmic RNA degradation pathway involving Dis3L2, should be always considered as an alternative to Xrn1 and exosome degradation.