If this were play'd upon a stage now, I could condemn it as an improbable fiction.

One more manuscript made me itching recently...


Thakor, N. & Holcik, M. IRES-mediated translation of cellular messenger RNA operates in eIF2α-independent manner during stress. Nucleic Acids Res (2011). doi:10.1093/nar/gkr701[PubMed]


XIAP IRES is studied in rabbit reticulocyte lysate. RRL is known to produce artifacts when it comes to internal initiation study so a lot of fun was anticipated.
A few questions to warm up. WTFhat on Earth is "ORF (35 nt)" (Figure 1A)? Does it encode not integer number of aminoacids or maybe it encodes frame-shift site? Promega users would be happy to know that now they can purchase a new product: RNAsein.


First, authors wanted us to believe that RRL is suitable to study XIAP translation. They utilized toeprinting approach. And indeed toe-prints were seen. But then come controls.

First, the initiation codon AUG was mutated to AAC (highlighted by a circle in Figure 2A; referred to as Start Codon, SC, mutant). The 40S leading edge toeprints were not observed with the SC mutant (Figure 2B, lane 4).

Well... Although I have to wear spectacles (or maybe due to this fact) I can see some toe-prints in the Figure 2B, line 4, though they are weaker, I have to admit. The very fact that the toe-prints are nevertheless observed on ACC codon is alarming. And even more alarming is the fact that reviewers of this manuscript have not bothered themselves to inspect a figure.

Second, the substitution of UU to AA in the polypyrimidine tract (PPT) of XIAP IRES (highlighted by a rectangle in Figure 2A) <revealed> the inability of the XIAP IRES PPT mutant to form an initiation complex. Strikingly, the PPT mutant was able to form an initiation complex once a m⁷G-cap was added to the 5'-end of the in vitro transcribed RNA.

One can see spots, which correspond to the full-length reverse transcription products (the bottom of Figure 2B). And the spot in lane 3 is much thicker than those in neighbor lanes. This fact has rather simple explanation: there's more cDNA synthesized in this reaction. And thicker toe-print reflects not the better 48S-complex assembly, but rather sloppy performance of the scientist. Moreover, on the Figure 2A I can see two uAUGs in the XIAP 5'UTR. And the context of one of them is considerably improved by this mutation: AUGU is transformed to AUGA. Kozak context, you might have heard of this... If translation on XIAP mRNA is initiated via scanning, this would perfectly explain loss of authentic toe-print. Also, this is very nice internal control. If in RRL toe-prints of similar intensity can be found on all AUG-codons, not only on authentic one... Then RRL sucks. Unfortunately, this part of the gel is not shown.

Strikingly, the uncapped wt XIAP IRES RNA was able to form an initiation complex in the poly I:C-treated RRL (Figure 3B, lanes 2 and 3), and in fact the fluorescence intensity of the 40S leading edge toeprints was enhanced by poly I:C treatment (Figure 3B, lanes 2 and 3).

If one compare lane 1 (supposed to be a control) and lanes 2 and 3 (where poly I:C was added), indeed there are pronounced toeprints after poly I:C addition. However, in the control reaction no ATP was added. This makes such control irrelevant, since two parameters were changed, while only one parameter may be changed. Moreover, if we look in Figure 1C, we can notice that addition of ATP in fact stimulates toe-prints. So was it poly I:C or ATP addition that enhanced toeprint?
One more point. There's more full-length cDNA in "XIAP + poly I:C + GTP" lane (see note above). Therefore, we have no reason to assume that RRL is suitable for XIAP studies. And we have no reason to assume that phosphorylation of eIF2 stimulated XIAP translation in RRL.
Next experiment is not reliable either. Authors tried to prove the identity of toe-prints. Usually, 48S or 80S complexes are purified from sucrose gradient, then reverse transcription is performed (1). Such approach can show us that:

1. One ribosome is bound to mRNA (in from of 48S or 80S).
2. This ribosome is positioned exactly on the authentic AUG-codon. 

The authors, however, purified something via streptotag introduced into the XIAP mRNA, then confirmed presence of ribosomes on this mRNA, and again performed toe-printing. Contrary to the approach described above, this only demonstrates that:

1. There are ribosomes on XIAP mRNA.
2. Some of these ribosomes are positioned on the authentic AUG-codon.

Feel the difference. Moreover, authors supplemented RRL with ribosomes from HeLa. What was the source of the ribosomes? Can one be sure that those ribosomes are devoid of initiation factors contaminations?

At the moment we know two alternative pathways of the initiator tRNA delivery to the ribosome: eIF5B-mediated delivery on HCV-like IRESs and eIF2D-mediated one in this and some other cases. Quite logically, therefore, the authors tried to address the possibility that one of these mechanisms could operate on XIAP mRNA. They show that depletion of eIF2 and eIF5B strongly inhibits 48S-complex formation on the XIAP mRNA. But no control is provided. How would other mRNAs behave? Thus from this experiment we may not deduce any specific translational properties of XIAP mRNA.

Next, the authors performed RNAi against eIF5B and found no decline in endogenous XIAP or b-actin levels. So they added poly I:C to the cells treated with anti-eIF5B siRNA and found that XIAP level is decreased.

It is known, however, that XIAP stability is regulated during apoptosis (see e.g. ref. 2,3). And apoptosis can be induced by poly I:C (see e.g. ref. 4). This issue is not addressed. Therefore, authors' thesis is not proven.

Next point is ridiculous. The authors wrote:

Pestova et al. (5) reported that eIF5B is dispensable to form elongation competent 80S initiation complex on CSFV IRES.

Read the paper (5). eIF5B is strictly required for both eIF2-dependent and - obviously - for eIF2-independent mechanisms. Just read the paper.


So the bottom line is:

1. Control experiment may only differ from test point by one parameter.
2. When addressing toe-prints, it is important to make sure that all other RT-stops except stops from a ribosome are identical both quantitatively and qualitatively.
3. Read thoroughly and think permanently.

I would not even say that this is not the level of NAR. Such manuscripts should not be published at all, because in the absence of proper controls no experiment should be published. And the reviewers (if there were any) are simply unqualified. Sad but true. The referees must be expelled to Saudi Arabia where they will have their hands chopped off. And then decapitated.

1. Anthony, D.D. & Merrick, W.C. Analysis of 40S and 80S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J Biol Chem 267, 1554–1562 (1992).
2. Wen-Hsien Liu, Huey-Wen Hsiao, Wen-I Tsou and Ming-Zong Lai. Notch inhibits apoptosis by direct interference with XIAP ubiquitination and degradation. The EMBO Journal (2007) 26, 1660 - 1669.

3. Dan, H.C. et al. Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP). J Biol Chem 279, 5405–5412 (2004).

4. Dufour, F., Bertrand, L., Pearson, A., Grandvaux, N. & Langelier, Y. The ribonucleotide reductase R1 subunits of herpes simplex virus 1 and 2 protect cells against poly(I · C)-induced apoptosis. J Virol 85, 8689–8701 (2011).

5. Pestova, T.V., de Breyne, S., Pisarev, A.V., Abaeva, I.S. & Hellen, C.U.T. eIF2-dependent and eIF2-independent modes of initiation on the CSFV IRES: a common role of domain II. EMBO J 27, 1060–1072 (2008).