Jack of all trades

One of the more intriguing enzymes that handles RNA is polynucleotide phosphorylase (PNPase).  This enzyme is a phosphorolytic 3’->5’ exonuclease; that means that it acts on the 3’ end of an RNA chain and moves towards the 5’ end, and that it adds phosphate (as opposed to water) to the broken phosphodiester bond.  This means that the products of the nucleolytic reaction are a shortened RNA chain and a nucleotide 5′-diphosphate.  The nucleolytic activity is appropriate, as the enzyme is a principal exonuclease component of the RNA-degrading machine known as the degradosome.

But RNA breakdown is not the only enzymatic activity possessed by PNPase.  As I noted in an earlier essay, PNPase was a first (perhaps THE first) nucleotidyltransferase, or RNA polymerase.  Indeed, it was an early candidate for the RNA polymerase (you know, the DNA-dependent RNA polymerases that are responsible for transcription).  This activity reflects the fact that the nucleolytic activity, when reversed, is actually a nucleotidyl transferase activity, in which RNA chains can be extended (in a template-independent fashion) using nucleotide diphosphates as substrates.  The clearest in vivo manifestation of this activity is evident in the many reports that show that PNPase can act as a poly(A) polymerase in vivo [see the review by Slomovic et al. for more on this]; this is true in bacteria and in organelles such as the chloroplast or mitochondria.

It turns out that we can add another possible function or activity to the list of properties of PNPase.  A recent paper (by Wang and coauthors; the citation and link given below) presents results that suggest that PNPase can serve as a cofactor (role not entirely clear) that facilitates the transport of RNA across the mitochondrial envelope.  That RNAs must be imported into mitochondria has been known for many years.  Mitochondria, although they are autonomous genetic systems (that is, they possess genomes apart from the nuclear genome that are transcribed, and the RNAs translated, by systems present in the mitochondria), need RNA import because their genomes do not encode the complete suite of required tRNAs or other structured RNAs (such as the RNA component of RNAse P or of the so-called MRP RNA).  However, the means by which RNAs are taken up by mitochondria remain largely unknown.  Wang et al. provide evidence that PNPase is part of the picture for RNA import in mitochondria.

In a brief nutshell, what Wang et al. did was take advantage of the fact that yeast does not possess PNPase.  Thus, they were able to add human PNPase to yeast mitochondria in vitro or yeast cells in vivo, and study the uptake andor functioning of RNAs that bind with rather high specificity to the human PNPase.  The in vitro assays were pretty standard transport assays – mix PNPase, organelle, and RNA substrate, and show that the substrate is taken up into a compartment (the organelle) that cannot be accessed by ribonucleases.  A similar in vitro system was developed by generating hepatocyte cell lines from mice that carried a liver-specific knockout of the PNPase gene, expressing human PNPase in these cells, and assaying purified mitochondria from these lines.  The in vivo assays involved expression of human PNPase and of an appropriate human RNA substrate (such as the RNAse P RNA) in yeast cells, and assessment of the effects of PNPase expression on the RNA contents of mitochondria.  The bottom line for all of this – the in vitro assays showed a clear stimulation of RNA import into yeast and mouse mitochondria by human PNPase, and the in vivo assays showed a similar effect on the RNA contents of mitochondria in yeast.

There is more to this paper, yet it still leaves open many questions as to the means by which PNPase stimulates RNA uptake by mitochondria.  But one of the interesting sidelights is the observation that RNA binding and import by PNPase is promoted by stem-loop structures in the RNA.  (One cute demonstration of this was to add an import-promoting stem-loop to an RNA that is otherwise degraded by PNPase, and show that the hybrid RNA was now a good import substrate.) This observation has ramifications for the in vivo functioning of the degradosome in bacteria and organelles, and also for the operation of the related exosome in the cytoplasm of eukaryotic cells.  (Recall that the exosome is a relative of PNPase; indeed, the core of the mammalian exosome has many of the structural features of a trimeric PNPase complex, albeit one in which the nuclease catalytic sites are no longer active.)

The citation:  Wang et al., PNPase regulates RNA import into mitochondria. Cell 142, 456-467, 2010.

The abstract:

RNA import into mammalian mitochondria is considered essential for replication, transcription, and translation of the mitochondrial genome but the pathway(s) and factors that control this import are poorly understood. Previously, we localized polynucleotide phosphorylase (PNPASE), a 3′ –> 5′ exoribonuclease and poly-A polymerase, in the mitochondrial intermembrane space, a location lacking resident RNAs. Here, we show a new role for PNPASE in regulating the import of nuclear-encoded RNAs 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. 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.

The graphical abstract (from the journal web page):

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2 Responses to Jack of all trades

  1. Clem Weidenbenner says:

    Somewhat off topic to this particular post, but have you seen Anna Marie Pyle’s piece in the September edition of The Scientist? I’ve not finished reading it yet, but it is fascinating so far.

  2. Arthur Hunt says:

    Hi Clem,

    Sorry for being slow in getting back to this.

    Pyle’s article was interesting. But I am biased – I find most articles that show an interest in RNA.

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