Many OX PHOS and replication factor mRNAs target mitochondria through specific binding to the organelle surface, independent of co-translational protein import

Abstract

A large number of nucleus-encoded messenger RNAs (mRNAs) encoding proteins involved in oxidative phosphorylation have been found to be associated with mitochondria in vivo, indicating organelle-specific mRNA targeting. However, the identification of mitochondrion-bound mRNA (Mtb-RNA) has traditionally relied on cumbersome isolations of polysomes from a large number of input cells and is therefore biased in favour of mRNAs associated through nascent targeting peptides emerging from the polysome during cotranslational import of their encoded proteins, and tends to ignore sequence-directed mRNA targeting. We have, therefore, sought to identify and quantify Mtb-RNAs rapidly in small numbers of cells, independently of their polysomal status. We isolated Mtb-RNAs from tissue-cultured cells under different conditions and assayed them by endpoint or real-time polymerase chain reaction (RT-PCR). We observed that (i) different Mtb-RNAs are differentially affected by cycloheximide-induced polysome arrest, indicating possible artifacts of the use of this translation elongation inhibitor; (ii) several Mtb-RNAs have direct affinity for the mitochondrial surface in vitro, indicating the possibility of targeting through mRNA recognition by surface-bound RNA-binding proteins (RBP); and (iii) mRNA–mitochondrion interactions are stabilized by formaldehyde crosslinking. Our results reveal the importance of sequence-directed targeting of mRNAs to mitochondria.