Termination of protein synthesis in mammalian mitochondria.
Protein synthesis in mammalian mitochondria produces 13 ..
Does this additional density derive from 5S rRNA? If so then it seems likely that this density will correspond to only a fragment of the entire 5S, necessitating a form of processing. Is an alternative possibility that this RNA species is only transiently associated with the mt-LSU and is not present in the fully assembled 55S particle? Although plentiful, the copies of cytosolic rRNAs vastly outnumber those of the mt-rRNAs. Moreover due to nuclear encoded but mitochondrially destined proteins that are co-translationally translocated across the outer mitochondrial membrane (OMM), it is difficult to purify mitochondria without cytosolic ribosomes anchored as co-contaminants. Protease shaving and RNase treatment with or without disruption of the OMM can significantly reduce but rarely eliminates all the 18S, 28S and 5S present in preparations, making qPCR analysis unreliable. So how might it be possible to convincingly discriminate between bono fide mitochondrial 5S and a contaminating population? We have performed a simple RNA isolation from fractions following isokinetic sucrose density gradients, which separates the mitoribosomal and cytosolic ribosomal subunits in total cell preparations. If 5S rRNA were present in fully assembled mt-LSU, it would be found in stoichiometric amounts relative to 16S rRNA species. A northern blot following such a fractionation of HEK293 cells is shown in . Probing for major rRNA components (5S, 12S, 16S, 18S and 28S) indicated that a small fraction of the 5S is incorporated into a low density particle as has been well described, previously , whilst the vast majority is associated with the 80S particle as indicated by the co-migration of 18S and 28S rRNA (). Clearly, there is no significant pool of 5S rRNA co-migrating with the16S rRNA, a marker of the mt-LSU, precluding any possibility that the 5S rRNA is present in stoichiometric amounts within the mt-LSU. Further, if the rRNA present is a processed shorter form of 5S, then it avoids detection by standard northern blot using the entire 5S species as probe. It is intriguing that a second RNA species is present in the porcine mt-LSU, but this simple fractionation experiment shows it is unlikely to be the 5S rRNA, unless it is weakly bound and/or subject to degradation.
Organization and Regulation of Mitochondrial Protein Synthesis
Returning briefly to the issue of 5S rRNA import into mammalian mitochondria, this is an issue that has evoked much discussion. Mitochondria from many organisms require RNA species to be imported from the cytosol to support protein synthesis. This is particularly well recognised for transfer RNAs (reviewed in ), where 2 import systems have been characterised . In contrast, PNPase has been implicated in augmenting the import of various other RNA species into human mitochondria, including the 5S rRNA. This surprising observation is supported in part by the main submitochondrial location of PNPase being between the two membranes. Although this protein is better known for its ability to degrade rather than transport RNA , Wang et al. performed a large number of in vitro and in vivo experiments in various species to support the claim, including the use of mutated PNPase to separate import and enzymatic functions. Interested readers are recommended to consult this work .