Tracking the elusive 5′ exonuclease activity of Chlamydomonas reinhardtii RNase J
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Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5′ exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation.
RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5′ end maturation is thought to be achieved by the combined action of a 5′ exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5′ exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5′ exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5′ exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.
KeywordsRNase J Chlamydomonas reinhardtii Endoribonuclease Exoribonuclease RNA metabolism
We thank Saravuth Ngo for efficient technical assistance. We are grateful to Jacqueline Plumbridge for useful discussions and critical reading of the manuscript. CNRS and University Paris Diderot, Sorbonne Paris Cité provided funding to the Research Unit UMR8261. A.L. received a doctoral fellowship from the “Initiative d’Excellence” program from the French State (Grant DYNAMO, ANR-11-LABX-0011) and the Edmond de Rothschild Foundations.
AL did the cloning, protein purification and in vitro and in vivo analysis of RNase J; AJ carried out cloning of certain RNase J variants and their in vitro tests; RK and LS did the chloroplast isolation and RNase J localization experiment; EG purified and tested potential RNase J partner proteins; FAW, SL and HP were involved in experimental design, data analysis and supervision of the project. HP wrote the manuscript with support from SL.
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