In contrast to the protein translocation, very little is understood about the import of specific RNAs into the mitochondrion. A confounder is that the spectrum of RNAs imported and the import factors and mechanisms seem to vary greatly amongst different organisms. At an extreme is mammalian mitochondria, in which despite strong evidence for RNA import (Alfonzo and Soll, 2009
; Tarassov et al., 1995
), no factors have thus far been identified. Here we implicate PNPASE as the first RNA import factor for mammalian mitochondria. Our results show that PNPASE KO disrupts mitochondrial morphology and respiration in mouse liver cells, at least partially by inhibiting the import of RNAs that control the transcription and translation of the ETC proteins. Our data also suggest that a nucleic acid component of the RNase P RNA processing complex, possibly RNase P
RNA (Puranam and Attardi, 2001
), is imported in vivo to process linked tRNAs in long mitochondrial transcripts. PNPASE mediated RNA delivery into the mitochondrial matrix and this import was augmented over background. Strikingly, PNPASE RNA import and RNA processing functions were separable and predicted stem-loop structures were identified in two imported RNAs that could transfer PNPASE-dependent import potential to non-imported RNAs. Combined, these results open a new chapter for studies into the pathway(s) and mechanism(s) of RNA import into mammalian mitochondria.
A key question that this study presages is how PNPASE regulates the import of specific cytosolic RNAs. For this, the location of PNPASE first needs to be considered. Previously, we localized PNPASE to the IMS. Carbonate extraction studies also indicated that PNPASE was bound to the IM facing the IMS (Chen et al., 2006
; Rainey et al., 2006
). However, more recently, others have shown a weak interaction with the matrix-localized RNA helicase hSUV3, suggesting a matrix localization for PNPASE (Szczesny et al., 2009
; Wang et al., 2009
). Our sub-fractionation studies were highly reproducible and, in contrast, we favor the idea that hSUV3 bound to PNPASE after all of the mitochondrial sub-compartments were exposed during purification. The methods used to identify this interaction do not exclude this possibility and we have already shown an unanticipated interaction between PNPASE and the cytosol-localized oncoprotein TCL1 using similar limited resolution methods (French et al., 2007
). Our failure to again identify a hSUV3 and PNPASE interaction using an ultra-sensitive dual-tag expression and purification system (Claypool et al., 2008
) further supports this interpretation (Figure S1
). An alternative explanation that we cannot exclude or confirm with current methodologies is that a small amount PNPASE could get into the matrix and interact with hSUV3.
A second interesting area opened by the current results is to determine how PNPASE controls RNA import into mitochondria. In concept, PNPASE could import RNAs from the cytosol into the IMS and then pass this RNA to another protein or complex that would assist it through the IM into the matrix (). Interestingly, PNPASE augments import in yeast, which does not have a PNPASE homolog, indicating a distinct RNA import mechanism that PNPASE can augment directly or independently. In mouse liver and MEF mitochondria, PNPASE KO is incomplete because cells without PNPASE are non-viable, so it is unclear whether it is absolutely essential for all RNA import or whether the amount detected in import assays is still mediated by the minimal residual amount of PNPASE present required for cell survival. Furthermore, it is not clear if this imported RNA in PNPASE KO mitochondria is stuck in the IMS and not in the matrix, which could provide further insight for the detailed function of PNPASE in RNA import.
PNPASE has two external domains (KH and S1
) that bind RNA near the opening of a central processing pore in a trimeric complex (Carpousis, 2002
; Symmons et al., 2000
). It is not clear whether the same domains are used indiscriminately or in some distinct manner to trigger PNPASE RNA processing versus import functions. It is possible that the stem-loop structures identified in RNase P
RNAs interact with PNPASE in a manner that triggers only import rather than processing. Interestingly, GAPDH
RNA can be a target of PNPASE degradation activity in vitro (French et al., 2007
), although when either of the identified stem-loop structures is appended to the 5’-terminus, GAPDH
RNA is efficiently imported into mitochondria (). Indeed, RNA structural elements regulate PNPASE function in chloroplasts and prokaryotes (Lisitsky et al., 1996
; Yehudai-Resheff and Schuster, 2000
) and a stem-loop structure protects the RNA from degradation by PNPASE in chloroplasts (Yehudai-Resheff et al., 2001
). A detailed dissection of what constitutes a trigger sequence for processing versus import activities is clearly warranted. Finally, since the overall sequence homology between the identified stem-loop structures on RNase P
RNAs is not high, binding interactions between specific RNAs and PNPASE may be stronger or weaker, allowing or inhibiting detection by standard in vitro techniques such as IP, which could make the verification of additional candidate imported RNAs challenging without the functional import assay system for validation.
Studies and important applications that use RNA import in mammalian cells have been hampered by the inconsistent requirements between systems and their study in vitro versus in vivo. Although the exact mechanism for how PNPASE augments and licenses RNA import is not yet known, the identification of PNPASE represents the first receptor-like component that binds RNA in mammalian cells to mediate RNA import into the mitochondrial matrix. This finding, along with the identification of import signal sequences, should open up additional studies to determine what other pathway components are involved and what the RNA sequence or structure rules tell us about how PNPASE may decipher between processing and import.