Early Advance Toward Allotopically-Expressed Mitochondrial RNA

Posted by Michael Rae on August 14, 2010 | Chief Science Officer's Team

Age-related accumulation of mutations in mitochondrial DNA (mtDNA) are widely suspected to play an important role in the degenerative aging process, albeit that controversy remains as to the mechanism(s) linking the two. Large deletions in mtDNA seem an especially likely culprit, due to (a) the clear, but focal, accumulation of cells homoplasmic for mtDNA deletions in postmitotic tissues; (b) their association with age-related pathology (the substantia nigra accumulates deletion-homoplasmic cells in aging and Parkinson's disease, as do skeletal muscle fiber segments in association with fiber breakage and sarcopenia, etc); and (c) their ability to completely abrogate oxidative phosphorylation (OXPHOS), principally because even the smallest mtDNA deletion encompasses several genes encoding mitochondrial tRNAs.

A number of credible proposals have been advanced for rejuvenation biotechnology to restore youthful mitochondrial function in such cells, reverting their abnormal metabolism and allowing them to resume participation in normal tissue function. The lead candidate approach, first proposed by SENS Foundation Chief Scientific Officer de Grey,(1) is the placement of functioning "backup copies" of the protein-coding mtDNA genes in the cell nucleus ("allotopic expression" (AE)). There has been substantial progress in this area since then,(eg (5-9)), and in recent years SENS Foundation has prioritized funding of AE research beginning with early work by Mark Hamalainen in Ian Holt's lab at Cambridge, and later in both the SENS Foundation Research Center and in the lab of Dr. Marisol Corral-Debrinski at the Institut de la Vision at Pierre and Marie Curie University, Paris(8). Active investigation of AE is soon to resume in the latter two centers.

But other potential routes to mitochondrial rejuvenation do exist and should also be developed, including the wholescale intraorganellar replacement of mtDNA using "protofection" (2) and the delivery of allotopic RNA to the organelle. The latter possibility was highligted by work targeting tRNA human cell mitochondria with the transgenic use of the transfer RNA import complex adapted from the parasitic protozoon Leishmania tropica.(3) Working with Newcastle University's Dr. Robert Lightowlers and others,  UCLA's Carla Koehler and Michael Teitella have now identified and begun to characterize a mammalian-specific mitochondrial system for the import of nuclear-encoded RNA, which could well be exploited to meet this biomedical challenge.

Much is understood about the mechanisms that regulate nuclear-encoded protein import into mitochondria. By contrast, much less is known about the factors that regulate mitochondrial RNA import. Almost every organism with mitochondria imports tRNAs and aminoacyl-tRNA  ... with mammalian mitochondria importing several different tRNAs both in vitro and in vivo  ...  RNase MRP and RNase P enzyme complexes localize and function in mammalian mitochondria and may contain RNAs that are encoded within the nucleus. ... [T]he RNA component of RNase P was imported into mammalian mitochondria and the RNase P RNA and processing activity has been copurified from mitochondria. By contrast, RNase P RNA is encoded in the mitochondrial genome in Saccharomyces cerevisiae. Recently, an additional RNase P enzyme, consisting of three protein subunits, has been purified from human mitochondria. This alternative RNase P enzyme processes single tRNA 5′ precursor sequences in vitro without an RNA component...

Previously, we localized polynucleotide phosphorylase (PNPASE), a 3′ → 5′ exoribonuclease and poly-A polymerase  ["that uses phosphorolysis to degrade RNA"], in the mitochondrial intermembrane space, a location lacking resident RNAs. ... This was a surprise because we expected that PNPASE would instead localize in the RNA-abundant mitochondrial matrix. ... 

Here, we show that PNPASE has a central role in augmenting the import of small RNA components required for DNA replication and RNA processing into the mitochondrial matrix.  PNPASE reduction impaired mitochondrial RNA processing and polycistronic transcripts accumulated. Augmented import of RNase P, 5S rRNA, and MRP RNAs depended on PNPASE expression and PNPASE–imported RNA interactions were identified. ...

To establish a direct PNPASE role, a systematic search was used to identify PNPASE-dependent RNA import sequences. Primers were designed to generate distinct segments of the 340 nucleotide (nt) RNase P RNA full length sequence. ... Import assays were performed using full length or truncated in vitro transcribed RNase P RNAs. ... [A series of truncations and importation experiments] implicat[ed] an import signal between nt 103 and 140. The most obvious, predicted secondary structure of RNase P RNA in this region was a 20 nt stem-loop. Interestingly, a similarly-predicted stem-loop structure was also identified in MRP RNA. ... [E]ach 20 nt stem-loop sequence was fused to the 5′-terminus of the GAPDH RNA, which is not imported. Strikingly, the RNase P and MRP stem-loop structures licensed the PNPASE-dependent import of GAPDH RNA into yeast mitochondria. By contrast, a control random 20nt sequence could not mediate this import. Human mitochondrial tRNA(trp) with the RNase P RNA step-loop structure, but not tRNA(trp) itself, was imported into isolated mouse liver mitochondria ... Finally, replacement of the human RNase P RNA stem-loop sequence with the 20nt random sequence blocked augmented RNase P RNA import into yeast mitochondria in vivo, confirming the role of the stem-loop in PNPASE-regulated import.

PNPASE augmented import of RNase P RNA into yeast mitochondria is nonphysiologic. Therefore, we developed WT, PNPASE KO, WT expressing human PNPASE, and PNPASE KO expressing human PNPASE MEFs for import assays.  ... . Radiolabeled RNase P RNA was not imported into mitochondria from the PNPASE KO MEFs, but was imported into mitochondria that contained mouse and/or human PNPASE. The in vitro import of RNase P, MRP, 5S rRNA, and GAPDH RNAs was also tested [to similar effect], ... whereas cytosolic GAPDH RNA was not imported. As expected, more than half of the imported MRP RNA was processed into the mature ∼130 nt form. By contrast, mitochondrial RNA import was severely compromised in HepKO liver mitochondria. Combined, these results strongly support PNPASE as the first RNA import factor that mediates the translocation of specific RNAs into the mammalian mitochondrial matrix.

These results strongly implicate the structural specificity of mitochondrial RNA import and the direct involvement of PNPASE in this process. ... PNPASE RNA processing and import activities were separable and a mitochondrial RNA targeting signal was isolated that enabled RNA import in a PNPASE-dependent manner. Combined, these data strongly support an unanticipated role for PNPASE in mediating the translocation of RNAs into mitochondria. (4)

This study makes a significant contribution to our understanding of the mechanisms of mitochondrial RNA import of nuclear-encoded RNA in mammals. And although it is just the first step along the way, in principle this discovery also suggests a way to bypass the paralysis of mitochondrial protein synthesis secondary to large mtDNA deletions.  Dr. Adhya's work with the Leishmania transfer RNA import complex (3) for which reason he was given a prominent place at the 3rd SENS Scientific Conference, had already suggested the possibility of delivering the full spectrum of mitochondrially-encoded RNAs into the mitochondrion from "backup copies" of the underlying genes housed in the nucleus.  Pursuit of the same strategy through the exploitation of the native mammalian RNA import machinery would be expected to achieve the same outcome in a more elegant and facile manner. In principle, all that might be required is the fusion of the RNase P stem-loop structure on to the allotopically-expressed genes for the full spectrum of mitochondrially-encoded RNA species. Such constructs would be designed to allow for their transcription without cytosolic translation by noninclusion of a 5′UTR. Conceivably, the efficiency of mitochondrial delivery could be enhanced if a segment of the 5′UTR exploited by Dr. Corral-Debrinski's group could be identified that would not allow for translation, but would still preserve its ability to facilitate translocation to the mitochondrial surface. 

And of course, in principle there is no exclusivity between AE of protein and AE of mRNA: provided that at minimum the mitochondrial tRNAs can be allotopically expressed and imported, regenerative engineers could deliver some fully-translated AE proteins and some mRNA precursors, depending on the facility and efficiency of either approach for a given protein, and on the burden on the relevant import machinery.

This is a preliminary sketch of a possible future rejuvenation biotechnology, and one that the investigators evidently had not contemplated. Their own attention at this time is focused on the more immediate basic science questions of "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."(4) But it opens up the potential of a new way to achieve one of the goals necessary for the function and structure of tissues, organs, cells, and (yes) organelles to be restored to youthful norms.

References

1. de Grey AD. A mechanism proposed to explain the rise in oxidative stress during aging. J Anti-Aging Med 1998;1(1):53-66. 

2.Khan SM, Bennett JP Jr. Development of mitochondrial gene replacement therapy. J Bioenerg Biomembr. 2004 Aug;36(4):387-93. Review. PubMed PMID: 15377877.

3. Mahata B, Mukherjee S, Mishra S, Bandyopadhyay A, Adhya S. Functional delivery of a cytosolic tRNA into mutant mitochondria of human cells. Science. 2006 Oct 20;314(5798):471-4. PubMed PMID: 17053148.

4. Wang G, Chen HW, Oktay Y, Zhang J, Allen EL, Smith GM, Fan KC, Hong JS, French SW, McCaffery JM, Lightowlers RN, Morse HC 3rd, Koehler CM, Teitell MA. PNPASE Regulates RNA Import into Mitochondria. Cell. 2010 Aug 6;142(3):456-467. PubMed PMID: 20691904.

5.  Zullo SJ, Parks WT, Chloupkova M, Wei B, Weiner H, Fenton WA, Eisenstadt JM, Merril CR. Stable transformation of CHO Cells and human NARP cybrids confers oligomycin resistance (oli(r)) following transfer of a mitochondrial DNA-encoded oli(r) ATPase6 gene to the nuclear genome: a model system for mtDNA gene therapy. Rejuvenation Res. 2005 Spring;8(1):18-28. PubMed PMID: 15798371.

6. Manfredi G, Fu J, Ojaimi J, Sadlock JE, Kwong JQ, Guy J, Schon EA. Rescue of a deficiency in ATP synthesis by transfer of MTATP6, a mitochondrial DNA-encoded gene, to the nucleus. Nat Genet. 2002 Apr;30(4):394-9. Epub 2002 Feb 25. PubMed PMID: 11925565.

7. Guy J, Qi X, Pallotti F, Schon EA, Manfredi G, Carelli V, Martinuzzi A, Hauswirth WW, Lewin AS. Rescue of a mitochondrial deficiency causing Leber Hereditary Optic Neuropathy. Ann Neurol. 2002 Nov;52(5):534-42. PubMed PMID: 12402249.

8. Bonnet C, Augustin S, Ellouze S, Bénit P, Bouaita A, Rustin P, Sahel JA, Corral-Debrinski M. The optimized allotopic expression of ND1 or ND4 genes restores respiratory chain complex I activity in fibroblasts harboring mutations in these genes. Biochim Biophys Acta. 2008 Oct;1783(10):1707-17. Epub 2008 May 6. PubMed PMID: 18513491.

9. Supekova L, Supek F, Greer JE, Schultz PG. A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression. Proc Natl Acad Sci U S A. 2010 Mar 16;107(11):5047-52. Epub 2010 Mar 1. PubMed PMID: 20194738.