Determination of protein‐only RNase P interactome in Arabidopsis mitochondria and chloroplasts identifies a complex between PRORP1 and another NYN domain nuclease

Abstract

Summary The essential type of endonuclease that removes 5′ leader sequences from transfer RNA precursors is called RNase P. While ribonucleoprotein RNase P enzymes containing a ribozyme are found in all domains of life, another type of RNase P called ‘PRORP’, for ‘PROtein‐only RNase P’, is composed of protein that occurs only in a wide variety of eukaryotes, in organelles and in the nucleus. Here, to find how PRORP functions integrate with other cell processes, we explored the protein interaction network of PRORP1 in Arabidopsis mitochondria and chloroplasts. Although PRORP proteins function as single subunit enzymes in vitro, we found that PRORP1 occurs in protein complexes and is present in high‐molecular‐weight fractions that contain mitochondrial ribosomes. The analysis of immunoprecipitated protein complexes identified proteins involved in organellar gene expression processes. In particular, direct interaction was established between PRORP1 and MNU2 a mitochondrial nuclease. A specific domain of MNU2 and a conserved signature of PRORP1 were found to be directly accountable for this protein interaction. Altogether, results revealed the existence of an RNA maturation complex in Arabidopsis mitochondria and suggested that PRORP proteins cooperated with other gene expression factors for RNA maturation in vivo.

Organellar and Secretory Ribonucleases: Major Players in Plant RNA Homeostasis

“…This was, however, not true for most mRNAs and the endoribonuclease activities have not been tested in vitro (Stoll and Binder 2016). Coimmunoprecipitation and yeast two-hybrid assays recently showed that PRORP1 physically interacts with MNU2, and the latter also plays a role in tRNA accumulation (Bouchoucha et al, 2019). Because several sequence specific PPR proteins are known or predicted to bind upstream of mitochondrial mRNA 59 mature ends (Ruwe et al, 2016), the current model is that they direct unspecific endoribonucleases to the correct site of processing (Binder et al, 2016).…”

A unique C‐terminal domain contributes to the molecular function of Restorer‐of‐fertility proteins in plant mitochondria

“…This is our favoured hypothesis, as it fits with the observations that natural Rf proteins with the RfCTD domain are associated with RNA cleavage, whereas those that lack this domain are not. Knowledge about the interaction between RFLs and other partners is limited, but RFL2 is presumed to associate with the endonuclease PRORP1 (Fujii et al ., 2016; Stoll & Binder, 2016), and more recently PRORP1 was reported to directly interact with MNU2 via a specific domain of MNU2 and a proline‐rich motif of PRORP1 (Bouchoucha et al ., 2019).…”

A unique C-terminal domain contributes to the molecular function of restorer-of-fertility proteins in plant mitochondria

“…This is our favoured hypothesis, as it fits with the observations that natural Rf proteins with the RfCTD domain are associated with RNA cleavage, whereas those that lack this domain are not. Knowledge about the interaction between RFLs and other partners is limited, but RFL2 is presumed to associate with the endonuclease PRORP1 (Stoll and Binder 2016;Fujii et al 2016), and more recently PRORP1 was reported to directly interact with MNU2 via a specific domain of MNU2 and a proline-rich motif of PRORP1 (Bouchoucha et al 2019).…”