One of the major goals of WP1 is to study the role of the dual function endo/5’-exoribonuclease J in mRNA maturation and turnover in the Chlamydomonas cp and in B. subtilis. In a new collaboration between Partners 1 and 3, we showed that CrRNase J has endo- but no significant intrinsic 5′-exoribonuclease activity that would be compatible with its proposed role in mRNA maturation (Liponska, Plant Mol Biol, 2018). This is the first reported example of an RNase J ortholog that does not possess a 5′-exoribonuclease activity and suggests that it acts mainly as an endoribonuclease in Chlamydomonas cp RNA metabolism. We also studied the roles of other RNases conserved between the cp and B. subtilis. Partner 3 showed that YacP, renamed Rae1 for ribosome-associated endoribonuclease 1, is a translation dependent RNase. Based on the crystal structure of the enzyme, we propose that Rae1 enters the ribosome A-site to cleave mRNA in a specific codon context (Fig. 1), whose consensus remains to be determined (Leroy, EMBO J, 2017). Another major task of WP1 was to study the role of M-factors in the maturation of Chlamydomonas cp mRNAs. These proteins are typically members of the tetra-, penta- or octotricopeptide repeat (TPR, PPR or OPR) families that carry tandem degenerated helical repeats of 34, 35 and 38 residues, respectively, each recognising a different RNA base. Partner 1 identified clusters of sRNAs (cosRNAs) corresponding to M-factor protected fragments from the 5’ end of almost all mature mRNAs (Cavaiuolo, Nucleic Acids Res, 2017). We additionally identified 32-34 nt ribosome-protected fragments in vivo allowing us to identify the function and molecular targets of two specific trans-acting factors: the M-factor OPR56 and the T-factor PPR1.

Our main ambition with WP2 is to ask whether the 5’-UTRs from a cp gene could drive the expression of a reporter gene in bacteria in the presence of its cognate M and T-factors and whether we could demonstrate SD-dependent translation in the cp. Partners 1 and 3 collaborated to show that the cp petA mRNA, which lacks a canonical SD sequence, only drives a very limited gene expression in both E. coli and B. subtilis, even in the presence of its cognate M and T-factors, suggesting that only the cp ribosome can function with cp T-factors. Introduction of SD sequences of increasing strength into this mRNA resulted in increased PetA accumulation in Chlamydomonas. However, even petA mRNAs with strong SD sequences were not translated in the absence of its cognate T-factor, suggesting that its interaction with the cp ribosome is necessary even for SD-dependent translation initiation (Rütgers et al., 2018, in preparation). Lastly, in all three kingdoms of life, including organelles, tRNAs carry a large number of nucleotide modifications that are essential for translation accuracy, reading frame maintenance and tRNA structure. Partner 6 solved several crystal structures of TtuA, a tRNA thiouridine synthetase, and showed that it uses an intriguing new catalytic mechanism involving stealing a sulphur atom from an iron-sulfur cluster to transfer it to the target uridine (Arragain, Proc Natl Acad Sci U S A, 2017). The discovery of this new mechanism of sulfur transfer has significance far beyond bacterial and organellar translation.

The initial survival of a bacterium in a new host cell cytoplasm requires the development of resistance to host cell defence mechanisms, notably antimicrobial peptides (AMP). Partner 1 has noticed a remarkable similarity between the amino acid composition and amphiphilic a-helical nature of AMPs and the transit peptides (TP) used to bring proteins into modern day organelles. A three-step pathway for the reappropriation of AMPs to function as TPs has been proposed to allow the establishment of a stable endosymbiont (Figure) (Wollman, Traffic, 2016).