What's in the name? That which we call a rose by any other name would smell as sweet.

de Breyne, Sylvain, Nathalie Chamond, Didier Décimo, Mary-Anne Trabaud, Patrice Andre, Bruno Sargueil, and Théophile Ohlmann. 2012. “In Vitro Studies Reveal That Different Modes of Initiation on HIV-1 mRNA Have Different Levels of Requirement for eIF4F.” The FEBS Journal (July 3). doi:10.1111/j.1742-4658.2012.08689.x.

What I would like to say about this manuscript is that it has nothing to do with HIV-1 mRNA translation. It's just irrelevant and reproduces old data. Bold statement indeed, but I'll try to convince you. What do the authors think they study? HIV-1 translation. As far as I know, there exists a set of HIV-1 mRNAs, all of them possessing 5'UTRs (1). However, the authors continue to use an artificial mRNA without any 5'UTR, thereby strating directly with gag AUG-codon. Somehow they do not bother if this is physiologically relevant or no. So shall we.

Prior to reading this manuscript I highly recommend reading two undeservedly forgotten researches, which were remotely mentioned by the authors. Here they are: (2) and (3). In the former translation of an artificial mRNA with two separated AUG-codons was studied in RRL. And the latter research deals with a behavior of leaderless mRNA in RRL.

Testing different concentrations of an mRNA with 5'UTR and two AUG codons.

de Breyne et al., Figure 1B: increasing mRNA (HIV1 5'UTR-AUG1-AUG2) concentration in RRL results in a continuous increase of initiation frequency at the second AUG codon and, starting from a certain concentration, decrease in initiation frequency at the first AUG codon.

Dasso et al., Figure 4A: increasing mRNA concentration (irrelevant 5'UTR-AUG1-AUG2) in RRL results in a continuous increase of initiation frequency at the second AUG codon and, starting from a certain concentration, decrease in initiation frequency at the first AUG codon.

Addition of a competitor to an mRNA with 5'UTR and two AUG codons.

de Breyne et al., Figure 1C: addition of a competitor (HIV-1 5'UTR) results in an increase of initiation frequency at the second AUG.
Dasso et al., Figure 5: addition of a competitor (E.coli 16S rRNA) results in an increase of initiation frequency at the second AUG.

Effect of 5'-terminal m7G-cap on AUG1 and AUG2

de Breyne et al., Figure 2 and S2: capping simulates translation at AUG1 and does not affect initiation at AUG2.
Dasso et al., Figure 7: initiation frequency at AUG2 is not affected by mRNA capping

Requirements for eIF4A

de Breyne et al., Figure 4: eIF4A R362Q mutant does not inhibit translation initiation at AUG1 of leaderless mRNA
Andreev et al., Figure 3: 48S complex is efficiently formed on the leaderless mRNA in the absence of eIF4F or eIF4A.

In the absence of proper control (any other mRNA with two AUG codons or any other leaderless mRNA which both would behave essentially similar to the corresponding mRNAs used in this study) all the authors say is totally irrelevant to HIV-1. And, sadly, there is nothing new in these data. Is a corpus of published papers so huge that those referees have forgotten these papers (2,3) or are they just unfamiliar with them?

1. Yilmaz, Alper, Cheryl Bolinger, and Kathleen Boris-Lawrie. 2006. “Retrovirus Translation Initiation: Issues and Hypotheses Derived From Study of HIV-1..” Current HIV Research 4 (2): 131–139.

2. Dasso, M C, S C Milburn, J W Hershey, and R J Jackson. 1990. “Selection of the 5'-Proximal Translation Initiation Site Is Influenced by mRNA and eIF-2 Concentrations.” European Journal of Biochemistry / FEBS 187 (2): 361–371.

3. Andreev, Dmitri E, Ilya M Terenin, Yan E Dunaevsky, Sergei E Dmitriev, and Ivan N Shatsky. 2006. “A Leaderless mRNA Can Bind to Mammalian 80S Ribosomes and Direct Polypeptide Synthesis in the Absence of Translation Initiation Factors.” Molecular and Cellular Biology 26 (8): 3164–3169.

Less is more

Pan, Meng, Xiaorong Yang, Lei Zhou, Xinna Ge, Xin Guo, Jinhua Liu, Dabing Zhang, and Hanchun Yang. 2012. “Duck Hepatitis a Virus Possesses a Distinct Type IV Internal Ribosome Entry Site Element of Picornavirus..” Journal of Virology 86 (2): 1129–1144.

In this study the authors have characterized IRES found in 5'UTR of DAHV. Almost all the data presented here are in line with earlier suggestion that this IRES belongs to wide group of hepacivirus/pestivirus (HP) IRESs (1). Not in line with this suggestion is the finding that translation of a bicistronic mRNA with DAHV IRES in the intercistronic position is highly sensitive to eIF4G cleavage by SVDV 2A protease.

However, this test was performed in vivo by means of DNA transfection. This approach suffers from a plenty of possible artifacts, for example cryptic promotor activity, e.g. present in the HCV IRES cDNA (2). One might argue that using T7 RNA polymerase vaccinia virus system overcomes these shortcomings, but the transfected DNA goes to the nucleus anyway and can be transcribed there, generating short capped monocistronic Fluc containing mRNAs, which translation would be perfectly inhibited by 2A protease. Therefore, RNA controls should have been performed: Nothern, RT-PCR, or RNAi against the first cistron (3). Also, a simple way to show that the intact eIF4G is required is translation in vitro with the addition of recombinant protease.

Funny is the authors' statement about "~0.5-fold stimulation" (and it was stimulated indeed) of the EMCV IRES which normally means 2-fold inhibition. :) Finally, the authors draw their conclusion:

These data indicate that the intact eIF4G is required for DHAV internal initiation of translation and that the HAV IRES element is no longer the only one abolished by cleavage of eIF4G.

Well, not exactly. Translation of the DAHV IRES was stimulated ~0.4-fold inhibited ~2.5 fold upon eIF4G cleavage. (Where is a control with HCV IRES, by the way?) This is not what one would call abolished. If a factor is required, there's no translation in it's absence: EMCV IRES translation is abolished by eIF4A R362Q mutant. Therefore, you can't contend that translation is eIF4G-dependent until you perform a set of in vitro translations with addition of hippuristanol (4) or eIF4A R36Q mutant or anything of this kind.

That's surprising, because this fact is the only one in the whole manuscript that makes a researcher blink at. But there is a single experiment lacking controls. And you can be sure that in a forthcoming review this IRES will be cited as a novel. Which is not proved yet. 

1. Hellen, Christopher U T, and Sylvain de Breyne. 2007. “A Distinct Group of Hepacivirus/Pestivirus-Like Internal Ribosomal Entry Sites in Members of Diverse Picornavirus Genera: Evidence for Modular Exchange of Functional Noncoding RNA Elements by Recombination..” Journal of Virology 8 (11): 5850–5863.

2. Dumas, Estelle, Cathy Staedel, Marie Colombat, Sandrine Reigadas, Sandrine Chabas, Thérèse Astier-Gin, Annie Cahour, Simon Litvak, and Michel Ventura. 2003. “A Promoter Activity Is Present in the DNA Sequence Corresponding to the Hepatitis C Virus 5' UTR..” Nucleic Acids Research 3 (4): 1275–1281.

3. van Eden, Marc E, Marshall P Byrd, Kyle W Sherrill, and Richard E Lloyd. 2004. “Demonstrating Internal Ribosome Entry Sites in Eukaryotic mRNAs Using Stringent RNA Test Procedures..” RNA 10 (4): 720–730.

4. Bordeleau, Marie-Eve, Ayaka Mori, Monika Oberer, Lisa Lindqvist, Louisa S Chard, Tatsuo Higa, Graham J Belsham, Gerhard Wagner, Junichi Tanaka, and Jerry Pelletier. 2006. “Functional Characterization of IRESes by an Inhibitor of the RNA Helicase eIF4A.” Nature Chemical Biology 2 (4): 213–220.

...neither cast ye your pearls before swine...

Another one incredibly stupid experiment from M.Holcik's lab. We have ravished one of their papers already. Now let's look at a new one:

Liwak, U. et al. Tumour Suppressor PDCD4 Represses IRES-Mediated Translation of Anti-Apoptotic Proteins and is Regulated by S6 Kinase 2. Mol Cell Biol (2012).doi:10.1128/MCB.06317-11

What authors claim is that PDCD4 specifically binds to XIAP mRNA 5'UTR and inhibits translation of the latter. Apart from in vivo data, which could seem to be consistent (and will be discussed elsewhere), there are some in vitro experiments presented. Bad luck.

The intention of the very last figure was to demonstrate that PDCD4 inhibits XIAP translation in RRL. So they added PDCD4 to RRL and 

observed that the ability of the XIAP IRES to recruit ribosomes (as determined by a toeprint +17 to +19 nt downstream of AUG) was severely impaired in the presence of His-PDCD4 (Fig. 5B, compare lanes 1, 2 and 4, 5). In contrast, addition of GST had no impact on the formation of the XIAP 48S complex (lanes 3, and 4, 5).

1.  PDCD4 is a homolog of eIF4G. It possesses a binding site for translational RNA helicase eIF4A, but contrary to eIF4G, which enhances helicase activity, PDCD4 inhibits one (1). Conformably, it inhibits cap-dependent and EMCV IRES-dependent translation, the both being eIF4A-dependent. And -  surprise! - PDCD4 must inhibit XIAP translation, since it is also eIF4A-dependent. So this experiment lacks control: any other eIF4A-dependent mRNA. Otherwise, one can add dominant-negative eIF4A mutant (R362Q, for example) to XIAP mRNA and claim that eIF4A is specifically required for XIAP translation. That's stupid.

2. Again, the authors don't give a fuck to differences of intensities of the full-length bands in RT assay (see comment here).

That's ridiculous.

1. Yang, H.-S. et al. The transformation suppressor Pdcd4 is a novel eukaryotic translation initiation factor 4A binding protein that inhibits translation. Mol Cell Biol 23, 26–37 (2003).

Fine words butter no parsnips

One more cellular IRES has been discovered, so I have to abandon my duties and waste my precious time...

Daba, A., Koromilas, A. E. & Pantopoulos, K. Alternative ferritin mRNA translation via internal initiation. RNA (2012).doi:10.1261/rna.029322.111 [can be found ahead of print here].

H-ferrtin expression is known to be transcriptionally up-regulated under a variety of conditions, including oxidative stress. Inhibition of translation under the latter case is well-documented and is attributed to eIF2 phosphorylation. Authors addressed whether H-ferritin mRNA translation could be resistant to translation inhibition.
First, they utilized inducible PKR to inhibit eIF2-dependent translation. Addition of coumermycin induced fusion PKR-GyrB dimerization and autophosphorylation of PKR which, in turn, phosphorylated eIF2 (Figure 1A). This resulted in profound inhibition of protein synthesis (polysomes in Figure 1B and [35S]-Met incorporation in Figure 1C, compare lanes 1 and 2). De novo translation of H-ferritin was also dramatically inhibited! But the authors prefer to express this in other words:

nevertheless, newly synthesized [35

S]-ferritin could be partially recovered in the immunoprecipitate.

OK, and what were the controls, you may well ask? The sad thing is that no control is provided (or was performed). How β-actin is inhibited? To a similar extent or stronger? Or maybe less stronger? Treatment with hemin enhanced ferritin [35S]-labeling with or without coumermycin. Optically (no quantification is provided) there is similar inhibition in both cases. But the authors summarize:

These data suggest that ferritin mRNA possesses the capacity to, at least partially, bypass the translational blockade imposed by eIF2a phosphorylation.

But without controls this can’t be said. 1 to 0. Referees suck.

Next, pulse-labeled [35S]-ferritin was precipitated from cells infected with attenuated poliovirus. The latter is known to express 2Apro that cleaves eIF4G. Indeed, eIF4GI was cleaved, eIF2 was phosphorylated (Figure 2A), and less ferritin were synthesized (Figure 2B). If anyone (but referee) compares how strong is stimulation of ferritin expression by hemin in figures 1C and 2B, he will be definitely surprised. In the former case there’s 2-3-fold stimulation, but in the latter one the stimulation extent is huge, to say the least. Where’s the truth? Fig.2B looks more similar to what these authors published before, but, anyway, it’s minor point. And again authors say:

These findings provide additional evidence that de novo ferritin synthesis is possible under conditions where global protein synthesis is shut down.

No control is provided for sure. Neither we know if eIF4GII is cleaved under these experimental conditions (time-course of eIF4GI/II cleavage is well-known for w/t poliovirus, but not for Sabin I strain), nor how other proteins’ synthesis is affected (quantitatively). No controls. 2 to 0. Referees suck.

Next, mRNA distribution in polysomes was addressed under normal conditions and after PKR induction.

1. PKR reduces ferritin mRNA contents in heavy polysomes. This is in line with translational repression.
2. Hemin treatment shifts this mRNA into heavier fractions which suggests translational upregulation by the way, but the authors did not pay attention to this fact.
3. Combined action does not have any pronounced effect, which is not surprising, you know. If you have two diametrically opposed actions, their mutual cancellation is not unexpected.
4. β-actin mRNA distribution is only affected by PKR activation, and inasmuch as β-actin transcription is not affected by hemin, the combined treatment is inhibitory.

But the authors say:

These results indicate an enhanced capacity of ferritin mRNA to bypass a translational blockade under stress conditions.

To say this you must be either cheating, or stupid, or both. 3 to 0. Referees suck.

Needless to say, Figure 4 fails to impress either. Authors used tet-responsive FLAG-IRP mutant which is constitutively active but not able to inhibit ferritin translation at high cells densities (Wang, J. & Pantopoulos, K. Conditional derepression of ferritin synthesis in cells expressing a constitutive IRP1 mutant. Mol Cell Biol 22, 4638–4651 (2002)). TfR1 or GADPH were supposed to represent controls. TfR1 is known to be inhibited by this IRP mutant. Authors pelleted polysomes in w/t or IRP-overexpressing cells, then immunoprecipitated them with anti-FLAG antibodies and addressed whether ferritin/TfR1/GADPH mRNA was present in the precipitate. Indeed, ferritin and TfR1 mRNAs could be detected. But does it extend our knowledge? Very subtly. And again, no controls.

Demonstration of “bona fide IRES” in the end of the manuscript is methodologically OK in terms of XX century. Unfortunately, these good ol’ times are gone. And in the XXI century we know that without comparison of monocistronic vs. bicistronic, or m7G-capped vs. A-capped mRNAs any IRES research is incomplete. No IRES is demonstrated. In figure 7 there must be Fluc/Rluc ratio presented, rather than Rluc/Fluc one, and 16 hours is too long for RNA transfection. And no positive control is provided.

Nowadays people seem not to be able to work thoroughly. They do not know what control is and why it is required. Is it something about education? And referees are either stupid, or unobservant, or most likely both. And the same referees publish similarly poorly performed researches. And it’s bad for ya.