Debunking myths on genetics and DNA

Monday, April 30, 2012

Fixing mitochindrial mutations with targeted mitochondrial RNA import


If you've been reading the blog for a while now, you've heard this many times: not all mutations are deleterious, but the ones that are can increase the risk of certain cancers and diseases. Numerous genetic defects have been attributed to mutations, and mutation in the mtDNA, the mitochondrial DNA, are no exceptions:
"Specific mutations in mtDNA have been implicated in muscular and neuronal disease and in the decline of organ function with aging. Despite a significant need, there are currently no effective treatments for mtDNA alterations [1]".
I've often talked about gene therapy as a way to try and fix defects caused by genetic mutations. The idea behind gene therapy is to introduce the non-mutated gene into the cells so that the production of the healthy protein can be restored. So, a natural question to ask would be: can we use gene therapy to fix mtDNA mutations as well?

Unfortunately the answer is no: if a nuclear encoded protein is mutated, it may fail to assemble correctly in the cytosol and thus it may be prevented from entering the mitochondria correctly.  Fortunately, though, Wang et al. [1] had a different idea.

Back in February I discussed the migration of genes from the organelles to the nucleus and, in some cases, back to the nucleus, a phenomenon that took place throughout evolution. One of the consequences is that in the human genome the majority of mitochondrial proteins are encoded in nuclear genes. I recently discovered that this import/export activity is also true for RNA, tRNAs in particular.

Transfer RNA, or tRNA, is the type of RNA that pairs a complementary triplet (anti-codon) with each of the mRNA coding triplets (codon) as specified by the set of rules of the genetic code. This helps to place the corresponding amino acid into the right position of the protein sequence, as illustrated in the following figure from Wikipedia.

In their March PNAS paper, Wang et al. note:
"A subset of nucleus-encoded tRNAs is imported into the mitochondria of almost every organism. The number of imported tRNAs ranges from one in yeast to all in trypanosomes [protozoa] [. . .] The nucleus-encoded RNAs in the mitochondrion have potentially diverse import pathways, but the details of these pathways and import mechanisms are still being revealed."
Wang and colleagues asked the following question: could we use this naturally happening import activity to import healthy RNA into the mitochondria and "fix" mitochondrial mutations?

First, they made sure that the tRNAs they were going to use to "fix" the mutations were correctly imported into the mitochondria. In order to do so, the researchers appended a sequence, called RP sequence, to the 5' end of the RNAs. They observed, both in vitro and in vivo in mouse models, that
"Only mt-RNA precursors with the appended RP sequence were efficiently imported into isolated mitochondria."
In vivo, they generated expressions of the hCOX2 gene, with and without the RP import sequence, and introduced them in mouse cells.
"Cells expressing RP-hCOX2, but not hCOX2, nucleus-encoded mtRNA showed mitochondrial transcript import and hCOX2 protein translation within mitochondria, indicating that the RP import sequence also is required and functions with coding mtRNA in vivo."
Summarizing, the RP sequence functions as a "driver" that directs the RNA into the mitochondria and, once there, the RNA is indeed translated and the corresponding protein expressed.

Wang et al. applied all of the above to a cell line that harbors two mutations. Both mutations cause an inefficiency in tRNA translation, which results in defective cell respiration. Remember, tRNA is what defines the protein primary structure out of the "instructions" handed over by the mRNA. So, if there's not enough tRNA, not enough proteins are made, and that usually isn't a good thing. Though a complete recovery was not expected, as the mutant mt-tRNAs are still present, not substituted, the imported wild-type mt-tRNAs with three elements expressed (the extended stem, the RP sequence, and MRPS12 3' UTR -- see paper for details) were able to partly restore the respiratory defect caused by the mutations.

Compared to previous methods, this approach is innovative because it doesn't require the introduction of non-native TRNAs with foreign protein factors.
"Rationally engineered human mitochondrial tRNAs and mRNAs can both be efficiently targeted and functional. The fusion RNA presequences are encoded in the nuclear genome and can be imported into mitochondria where they are processed, restore translation, and are degraded via normal pathways in the mitochondrion."

[1] Wang, G., Shimada, E., Zhang, J., Hong, J., Smith, G., Teitell, M., & Koehler, C. (2012). Correcting human mitochondrial mutations with targeted RNA import Proceedings of the National Academy of Sciences, 109 (13), 4840-4845 DOI: 10.1073/pnas.1116792109

ResearchBlogging.org



5 comments:

  1. antisocialbutterflieMay 1, 2012 at 4:41 AM

    I second Steve's comment.

    ReplyDelete
  2. Interesting. there was a paper a while back using a nuclear-encoded mitochondrial tRNA, or maybe just the mitochondrial import sequence to deliver a ribozyme (an enzyme made of RNA for the kids out there) to the mitochondria. The tRNA-ribozyme chimera was imported to and functioned. Encoding both the tRNA-mRNA above with a tRNA-ribozyme targeting the defective version of the mtmRNA might be more efficient in attenuating the disease.

    One very nice thing about RNA-based constructs is that they're not immunogenic, unlike proteins.

    Rob

    ReplyDelete
    Replies
    1. That's pretty cool, thanks for the comment, Rob!
      If you happen to come across the reference for that paper, I'd like to read it. Thanks!

      Delete
  3. Better late than never. It was done in plants, but should be transferable to most eukaryotes.

    "Organelle trafficking of chimeric ribozymes
    and genetic manipulation of mitochondria"

    http://nar.oxfordjournals.org/content/early/2011/07/17/nar.gkr580.full-text-lowres.pdf

    ReplyDelete

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