Physiological role of the ß-CASP Ribonucleases in Archaeal RNA metabolism – CASPAR

RNA processing, which involves ribonucleases and ancillary enzymes such as RNA helicases, has a pivotal role in living cells and constitutes a crucial step in the regulation of gene expression. Most of our knowledge on RNA processing comes from only two of the three domains of life, Eukarya and Bacteria. However, in Archaea, exploration of RNA maturation and decay pathways is still in its early stages. First hints towards identifying RNA maturation and degradation pathways came from our recent phylogenomic studies and biochemical characterizations of two major archaeal ß-CASP ribonucleases, aCPSF1 and aRNase J. The ß-CASP nucleases have emerged as central to RNA metabolism over the last decade. These enzymes, catalysing both endo- and 5’-to-3’ exo-ribonucleolytic degradation, are unique among all known ribonucleases. The ß-CASP enzymes acting on RNA form two separated groups, one related to the eukaryal termination factor CPSF73 and the other to the bacterial RNase J ribonuclease. aCPSF1 and aRNase J are orthologs of CPSF73 and RNase J, respectively, showing that Archaea possess an original RNA processing system together with mosaic features. This highlights the advantage of an archaeal model to gain further mechanistic and evolutionary information of fundamental processes across the three domains of life.
The proposal focuses on understanding the physiological role of aCPSF1 and aRNase J, the two major phylogenetically conserved archaeal ß-CASP ribonucleases, in the Thermococcales with Pyrococcus abyssi and Thermococcus barophilus as models. Our working hypothesis is that aCPSF1 and aRNase J are at the centre of protein interaction networks (our unpublished data) which includes two putative RNA helicases of superfamily 2, aSki2b and aLhr2, which probably cooperate in essential RNA metabolic pathways. Our strategy to decipher the role of aCPSF1 and aRNase J in RNA cellular metabolism and identify aCPSF1 and aRNase J partners and multi-protein complexes is based on multiple approaches combining genetics, biochemistry, transcriptomics, proteomics and phylogenomics. The first part of the project will be dedicated to understanding the physiological relevance of the two major ß-CASP ribonucleases, aCPSF1 and aRNase J, and of the two putative SF2 RNA helicases, aSki2b and aLhr2, in archaeal cells. In a second part, we will ask if aCPSF1 and aRNase J work in concert with protein partners and will decipher which categories of archaeal RNA are in contact with aCPSF1, aRNase J, aSki2b and aLhr2. We will map the pairwise interactions between partners holding together ß-CASP complex(es) and characterize the ribonucleolytic activities of the reconstituted complex as well as the ATPase and unwinding activity of the putative SF2 RNA helicases. Finally, in the third part, the evolutionary links of aCPSF1 and aRNase J protein partners will be explored.
To our knowledge, this work will provide, the first identification of interacting networks and RNA processing machinery(ies) containing ß-CASP ribonucleases and SF2 RNA helicases in Archaea as well as the first evolutionary study identifying RNA metabolism core components in archaeal genomes. Together CASPAR will offer a major step towards the understanding of fundamental RNA processing pathways as mRNA decay, ribosomal RNA maturation and transcription termination in Archaea. The approaches will deliver a solid base in a largely overlooked but extremely important field of archaeal RNA metabolism by elucidating the fundamental building blocks of the network and its topology as well as the evolutionary history of RNA metabolism across the three domains of life.