Double life of mitochondrial ribosomal protein L7 12

Mitochondria have their own transcriptional and translational apparatus, even though they produce only a handful of proteins, therefore most of the proteins are imported from the cytoplasm. Trancription, translation and protein insertion into the membrane are interconnected: translational activators regulating mitochondrial translation are interacting with mitochondrial RNA polymerase via Nam1p and Sls1p proteins (Bryan et al. Genetics 2002), Puf proteins connect cytoplasmic translation and protein import into mitochondria by direct interaction with Tom20 subunit of the TOM protein import channel (Saint-Georges et al. PLoS ONE 2008).

But this seems not tight enough interaction for mitochondrial translation and transcription. It turnes out what mitohondrial ribosomal protein L7 12 (the one that brings translational GTPases to the ribosome), has a double life. Apart from doing its normal job as a part of the ribosome, it doubles as a transctiptional factor, selectively associating with human mitochondrial RNA polymerase and activating it (Surovtseva et al. PNAS 2011). And as if it is not enough, there are several paralogues of L7 12 in mitochondria, both in plants (Delage et al. Biochimie 2007) and in mammals (Koc et al. JBC 2001). 

Single molecule tracking fluorescence microscopy in mitochondria reveals highly dynamic but confined movement of Tom40

Most of the mitochondrial proteins are imported from the cytoplasm, with only a small fraction (about 1%) encoded in the mitochondrial genome. Import is mediated by two complexes: TOM (transporter outer membrane) and TIM (transporter inner membrane). We have a pretty good idea about the players involved in mitochondrial protein import, but we have very little idea about the dynamics of TOM/TIM movement in the mitochondrial membrane.

We tried addressing this question using single molecule fluorescent microscopy in isolated yeast mitochondria. What we see is that Tom40, the central component of TOM complex, is highly confined (i.e. restricted in terms of aerea it can sample) but within its confinement it moves pretty rapidly.

References:

Kuzmenko et al., Scientific Reports (2011) 1:95

Regulation of mitochondrial protein transport

Mitochondria have their own genome, own translational machinery, own ribosomes, but still, most of the proteins they import from the cytosole. And this they do using two protein complexes in the outer and inner membranes: TOM (Transporter Outer Membrane) and TIM (Transporter Inner Membrane). TOM itself consists of several subunits: Tom40 forms a pore through which proteins get transported, Tom20 and Tom70 work as receptors recognizing the mitochondrial proteins in the cytoplasm, and several more proteins helping out.

TOM and TIM, figure lifted from Chacinska at al., 2009

And now joint effort of Pfanner and Meisinger labs lead to a discovery that in yeast TOM-mediated protein transport is regulated by kinases casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes TOM biogenesis, and PKA phosphorylates Tom70 component of TOM under nonrespiring conditions, inhibiting it. This finding basically opens a new field: regulation of mitochondrial protein transport. Just like that.

References:

Chacinska A, Koehler CM, Milenkovic D, Lithgow T, & Pfanner N (2009). Importing mitochondrial proteins: machineries and mechanisms. Cell, 138 (4), 628-44 PMID: 19703392

Schmidt O, Harbauer AB, Rao S, Eyrich B, Zahedi RP, Stojanovski D, Schönfisch B, Guiard B, Sickmann A, Pfanner N, & Meisinger C (2011). Regulation of mitochondrial protein import by cytosolic kinases. Cell, 144 (2), 227-39 PMID: 21215441

Observer effect in biology: Schrödinger's cat mitochondria

All quantum physicists know that observation itself changes the object of observation. We will never know what things are actually doing when we are not looking, just because if in order to figure out what they do, we need to look; it's catch-22. But that's quantum physics, you say. How about molecular biology?

Well, here is an example. Mitochondria, as you know, have their own genome, and they translate it, and they do so in a very funky way. Ever translation termination is peculiar. It is a variation of bacterial translation termination, but different. There are two mitochondrial class-1 release factors (the ones which actually recognize the stop codon and cleave off the peptide): mtRF1a and mtRF1. mtRF1a is an omnipotent release factor and it recognizes normal stop codons UAA and UAG, as it was proved biochemically in vitro. mtRF1... this one is a bit tricky.

First idea is that it recognizes funky stop codons like AGA and AGG (together - AGA/G), which are indeed present in mitochondria. Biochemistry in heterologous system seems to support this one.

Second is that there is no need for mtRF1 at all, and AGA/G stop codons actually never get read at all, therefore there is no need to recognize these! Wow, that's radical and this is why it is published in Science. This story is the subject of this post.

So... how did they figure it out. They check for ribosomal positioning on the termination codon and they figure out that it seems to slip (frame-shift) from the non-standart uAGA/G codon backward and ends up with classical UGAa/g in the A-site. Bang, problem solved, we do not need to recognize the strange stop codon and thus there is no need for mtRF1 at all. Clever. But how do they see it?

They use bacterial toxin RelE. This peculiar molecule binds in the ribosomal A-site and cleaves mRNA there. It works in bacteria, eucaryotes and, obviously, mitochondria because the ribosome is so darn conserved. However, RelE does not cleave all the codons with the same efficiency, it has very strong preferences for certain sequences - such as regular stop codons, UGA or UGG!

Fig. 1 RelE efficiency is different for different codons, lifted from Pedersen at al. 2003

Looking at the x-ray structure of RelE in the complex with mRNA and 70S ribosome we can see why: it is all down to the interactions between the specific residues in RelE and mRNA. If these residues are not there, there will be no interaction and no cleavage - see Fig. 2.

Fig. 2 Proposed reaction mechanism for RelE-mediated cleavage, lifted from Neubauer at al., 2009.

And now - back to the Schrödinger's cat. When researchers used RelE to probe for position of the mitochondrial ribosome on the mRNA, all the cleavages detected were with UAG in the A-site. Why? Well, because this is where RelE can cut, so it cleaved there. It may have even caused this frame-shift. Why didn't they see any ribosomes on the AGG? well, because RelA does not want to cleave there!

So... may be the tool used for observation changed the system and told us something about itself (something that we already knew). Not about the system! Still, it's a Science paper, hey. And the idea is very, very cute!

And, of course, I can be completely wrong!

Fig. 3 Schrödinger's cat. Not really related to RelE at all.


PS: as it turnes out, the problem of affecting the biological system while studying it was discussed by at length here: Bridson EY, & Gould GW, Quantal microbiology.


References:

Neubauer C, Gao YG, Andersen KR, Dunham CM, Kelley AC, Hentschel J, Gerdes K, Ramakrishnan V, & Brodersen DE (2009). The structural basis for mRNA recognition and cleavage by the ribosome-dependent endonuclease RelE. Cell, 139 (6), 1084-95 PMID: 20005802

Andreev D, Hauryliuk V, Terenin I, Dmitriev S, Ehrenberg M, & Shatsky I (2008). The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome. RNA (New York, N.Y.), 14 (2), 233-9 PMID: 18083838

Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, & Ehrenberg M (2003). The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell, 112 (1), 131-40 PMID: 12526800

Young DJ, Edgar CD, Murphy J, Fredebohm J, Poole ES, & Tate WP (2010). Bioinformatic, structural, and functional analyses support release factor-like MTRF1 as a protein able to decode nonstandard stop codons beginning with adenine in vertebrate mitochondria. RNA (New York, N.Y.), 16 (6), 1146-55 PMID: 20421313

Soleimanpour-Lichaei HR, Kühl I, Gaisne M, Passos JF, Wydro M, Rorbach J, Temperley R, Bonnefoy N, Tate W, Lightowlers R, & Chrzanowska-Lightowlers Z (2007). mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG. Molecular cell, 27 (5), 745-57 PMID: 17803939

Temperley R, Richter R, Dennerlein S, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2010). Hungry codons promote frameshifting in human mitochondrial ribosomes. Science (New York, N.Y.), 327 (5963) PMID: 20075246

Lekomtsev SA (2007). Non-standard genetic codes and translation termination. Molekuliarnaia biologiia, 41 (6), 964-72 PMID: 18318113

Bridson EY, & Gould GW (2000). Quantal microbiology. Letters in applied microbiology, 30 (2), 95-8 PMID: 10736007

Viral nature of the mitochondrial RNA polymerase

Mitochondria contain their own genome, and they transcribe it. Since mitochondria are of bacterial origin, one would expect that their polymerase would be similar to that of bacteria. And it is so the case for chloroplasts, which are also of bacterial origin.

However, mitochondrial polymerase not homologous to that of bacteria, or, for that matter, to cytosolic eukariotic polymerases. It is homologous to... polymerases of T phages, T3 and T7!

However, it is slightly modified. It has an extension at the N-terminus, and this extension is highly variable. Using this extension yeast mitochondrial polymerase interacts with protein Nam1p, which is involved in mRNA stabilization. Nam1p in turn interacts with the whole bunch of mitochondrial membrane proteins which organise translation and following assembly of the proteins constituting the core of cytochrome c oxidase complex (COX).

In this way all the steps of COX formation are connected in the mitochondria: polimerase binds to Nam1p, Nam1p binds to membrane-bound translational enhancers, enhancers bind mRNA and the ribosome, the ribosome itself binds to membrane, and translated protein is inserted in the membrane. There is even a special term for this sort of coupled transcription, translation and insertion - transertion. Very neat system!

Amazingly, different eukaryotes reinvent this system in different ways. Most of the components are clade-specific, and since the system is so very interconnected, it is is very, very different in different eucaryotes. Therefore when yeast geneticists say that they are studying Saccharomyces cerevisiae as a model system in order to understand human mitochondrial translation they are... well... how should I say it? well, they are being over-optimistic.

PS: and phages and mitochondria are trading polymerases both ways! A cyanophage was discovered that has an ex-mitochondrial DNA (not RNA) polymerase!

References:

Masters BS, Stohl LL, & Clayton DA (1987). Yeast mitochondrial RNA polymerase is homologous to those encoded by bacteriophages T3 and T7. Cell, 51 (1), 89-99 PMID: 3308116

Rodeheffer MS, Boone BE, Bryan AC, & Shadel GS (2001). Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase. The Journal of biological chemistry, 276 (11), 8616-22 PMID: 11118450

Naithani S, Saracco SA, Butler CA, & Fox TD (2003). Interactions among COX1, COX2, and COX3 mRNA-specific translational activator proteins on the inner surface of the mitochondrial inner membrane of Saccharomyces cerevisiae. Molecular biology of the cell, 14 (1), 324-33 PMID: 12529447

Gagliardi D, Stepien PP, Temperley RJ, Lightowlers RN, & Chrzanowska-Lightowlers ZM (2004). Messenger RNA stability in mitochondria: different means to an end. Trends in genetics : TIG, 20 (6), 260-7 PMID: 15145579

Yi-Wah Chan, Remus Mohr, Andrew D. Millard, Antony B. Holmes, Anthony W. Larkum, Anna L. Whitworth, Nicholas H. Mann, David J. Scanlan, Wolfgang R. Hess and Martha R. J. Clokie. Discovery of cyanophage genomes which contain mitochondrial DNA polymerase. Mol Biol Evol (2011) doi: 10.1093/molbev/msr041

mitochondrial mRNA UTRs: insanity, lunacy and absurd

mRNA in general have 5'UTR (untranslated region) followed by ORF (open reading frame) and then comes 3'UTR. Both UTRs regulate mRNAs stability, localization, translational efficiency. In eukaryotes cytozolic mRNAs have 3'UTR polyA, which regulates mRNA stability and translational efficiency, and 5'UTR which regulates efficiency of translation.

Mitochondrial mRNAs... it's a mess.

1) 3'UTRs are regulating localization (i.e. transport into the mitochondria, (Pattini 2003 PIMD 15376913)) and in mRNA stability (Gagliardi 2004 PIMD 15145579). Mechanisms of mRNA stabilization seem to be very, very different in plants (polyA destabilizes, like it does in bacteria!), yeast (no polyA at all, just like it is in yeast cytozolic mRNAs!) and mammals (polyA stabilizes, just like it does in eukaryotic cytozolic mRNAs!). 3 different approaches?! It is about as insane as it gets, really.

In mammals 3'UTRs are short (Ojala 1981 PIMD 7219536), unlike in plants and yeast, where 3' UTRs are long.

2) 5'UTRs are regulating translation (mRNA-specific enhancers of translation bind to them and thus regulate translation initiation) and localization within the mitochondria, thus coupling translation and insertion of the mRNA into the membrane. Most of the proteins that are translated in yeast mitochondria are intermembrane proteins involved in respiration and ATP production (review Towpik 2005 PIMD 16341268), thus coupling translation and insertion is a must. And indeed, when multisubunit complexes are assembled, translation of the individual subunits is geometrically coordinated (Naithani 2003 PIMD 12529447).

All this is well and good, but there are issues. Somehow investigation of 3' and 5'UTRs is a big thing in plant mitochondria, and not much is done nowadays with yeast or mammals. Or at least it is not easy to find. Second, no one tried systematically comparing UTRs from different organisms.

What I have dug out by now is this: in plants 5'UTRs are long, and there is a lot of experimental material here using 5'-RACE (Froner 2007 PIMD 17488843, Kuhn 2005 PIMD 15653634). In yeast - long 5'UTRs as well (review Costanzo 1990 PIMD 2088182), though much less studied experimentally. In mammals 5'UTRs are claimed to be short, at least in humans (Montoya 1981 PIMD 7219535). Here signals for mRNA-specific initiation enhancers are located within the ORF.

Is plant and yeast mitochondria translation radically different? Why did mammalian mitochondrial mRNA loose 5'UTR regulation? If yes, where is the watershed? What are the differences in the yeast+plants mitochondirial machinery vs mammalian?

Mammalian mitochondrial genome is super-streamlined, cutting corners where possible (review Attardi 1985 PIMD 3891661), so that could be a reason for the loss of 3' and 5'UTR. But what drives this minimization? Why trying THAT hard?

Experiments on mitochondrial translation seem to be done on different systems in different areas of research: yeast are used for identifying initiation enhancers and studying genetics and molecular biology of translation regulation, in plants 3' and 5' UTRs are extensively mapped, and in mammals using very, very simplified translational system (tRNA, IF2, IF3, EF-Tu and EF-G) some rudimentary biochemistry is done. This does scitsofrenic devision of labor is in keeping with the mitochondrial spirit indeed.

PS: mitochondrial ribosomes are also very, very strange.

Reviews:

Mitochondrial evolution: Karlberg 2003 PIMD 12728281
Mitochondrial and chloroplast translation: Gillham 1994 PIMD 7893142
Mitochondrial translation and desease: Perez-Martinez 2008 18991722
Plant mitochondrial translation: Binder 2003 PIMD 12594926, Hoffmann 2001 PIMD 11642360
Yeast mitochondrial translation: Costanzo 1990 PIMD 2088182, Dieckmann 1994 PIMD 8206703
Mammalian mitochondrial translation: Spremulli 2004 PIMD 15196894

mitochondrial translation - complete mess

Mitochondria have their own genome, their own translational machinery and their own mRNAs which code a handful of proteins. Most of the proteins come from the cytoplasm, but some get translated inside the mitochondria. And this is done in amazingly weid way...

Bacteria - mitochondrial ancestors - have 3 initiation factors, IF1, 2 and 3, and all of these are absolutely necessary for the bacterial viability.

Mitochondria do not have IF1 (all mitochondria), and unlike mammals, yeast ones do not have IF3! Also they seem to use mitochondria-specific initiation factor AEP3. Do mammals have AEP3 homologue? Worth checking... To make things more complicated, mitochondria have their own special mRNA-specific factors... and again, there is a catch. Yeast mRNA have long 5' UTRs (untranslated regions), and mammals have short, they basically have leaderless mRNAs (I'd love to see a good reference for that! THIS is a little bit out of date...) - therefore I would expect that these mRNA-specific IFs work differently in yeast in mammals. So yeast and mammalian mitochondrial translation seems to have very, very different translational apparatus. Isn't is weird?

Another amazing thing is that translation and protein localization are tightly linked in mitochondria. mRNA-specific initiation factors (i.e. Pet111p) and above-mentioned AEP3 interact with the mitochondrial membrane (most of the mitochondrially-translated proteins are membrane proteins), so that translation is localized where the proteins should go.