We'll discover our own IRES, with blackjack and hookers! :)

Probably I should beg pardon in advance for a mocking way of discussion, it is explicitly expressed that authors of this blog have no intention to offend anybody. Tonight we dissect
Dai, N. et al. mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry. Genes Dev (2011) 25: 1159-1172. [PubMed]

What do we have? Translation of IGF2 mRNA with L4 leader is not affected by rapamycin treatment, while that with L3 leader is (Figures 1A and 1B). Next, overexpression of eIF4E (3-fold over endogenous protein) does not affect translation driven by both leaders (Figure 1C). And, finally, overexpression of nonphosphorylatable 4E-BP1 variant doesn't inhibit both mRNAs (Figure 1D). The authors make a bit unexpected conclusion:
These results indicate that, despite its complex secondary structure, the L3-luciferase mRNA is translated in an eIF-4E-independent manner.

What? English, motherfucker! Do you speak it? (c) Two mRNAs. None is stimulated by eIF4E overexpression. None is inhibited by 4E-BP overexpression. But one of them is translated in an eIF4E-independent fashion, while another is not. Too complex for me to understand...
As a control, authors utilized ODC (ornithine decarboxylase). Rather unwise (or sneaky) control - and again lights are on but no referee is at home. Overexpression of eIF4E stimulates endogenous ODC expression. But it also stimulates ODC mRNA export (1) so cumulative effect is observed and ODC simply can not be used as a positive control for eIF4E and/or 4EBP effects on translation, unless it is transfected as an RNA.
After this striking finding what could be more logical than to look for an IRES? Indeed authors made bicistronic plasmids with L3 leader (L4 leaders would be an instructive control) in the intercistronic position. Authors found that L3 leader promotes internal translation 15 times better that b-globin leader, "expression of the latter being negligible". 15 multiplied by "negligible" equals to negligible, from math's point of view. If we compare b-globin to any conventional IRES, say, EMCV, the latter works 100-1000 times better, depending on cell line.
The acid test for any "IRES" is whether expression from an artificial bicistronic mRNA could be physiologically relevant. And since all those cellular IRESs are naturally capped and monocistronic, the idea is to compare expression levels from m7G-capped monocistronic and bicistronic mRNAs (2,3). So, authors employed RNA transfection to show that 24 hours after transfection... Stop! Does anybody know how many RNA remains in cell a day(!) after transfection. Look at Figure 3C from (4). There's about 85-90% of initially transfected mRNA being lost. So what do you measure when almost none of the input mRNA remains? I don't know. But it's not the only trouble. mRNAs were transfected with Lipofectamine (it's not the antiadvertising, I use it myself), but in the course of lipofection most of the transfected mRNAs are confined in cellular compartments (not reaching cytoplasm!) and their apparent identical stability (see Suppl. Figure 3) is just an artifact (see ref. 5). 
But maybe luciferase values are decisive and unambiguous? In bicistronic mRNA with "L3 IRES" Fluc/Rluc ratio is ~0,15. Is it good or bad? In the absence of negative and positive controls there's no answer. But I can give you a hint: in HEK293T cells the same ratio for bicistronic EMCV mRNA is ~0,05 (note that relative activities of both luciferases - light units per molecule - are distinct). So "L3 IRES" is 3-fold more active than EMCV IRES. Does it correlate with the results of DNA transfection? Ask the referees...
There's one more tricky thing. Much more tricky. L3-Fluc mRNA translation is not stimulated by the cap (Figure 2D and Suppl. Figure 3) and is equally effective in mono- and bicistronic mRNAs. This would suggest an IRES, but 24 h is too much. I would believe in IRES if the same results can be demonstrated 2-4 hours post transfection with A-cap as a control (see below).  
The next part of the manuscript is more profound. Authors find that:

1. Rapamycin inhibits binding of IMP2 to L3, but not L4 leader
2. Overexpression of IMP2 stimulates - and RNAi against IMP2 inhibits - expression of L3-IGF2, but not that of L4-IGF2. Since DNA transfection was performed, we can only say expression, but not translation.
3. Overexpression of IMP2 relieves inhibition by rapamycin.
4. IMP2 is phosphorylated by mTORC1.
5. Mutation of Ser162 and Ser164, which are phosphorylated by mTORC1, to alanines inhibits binding of IMP2 to L3 leader. This fact should be taken with caution. Well-known story is mutation of allegedly phosphorylated Ser53 of eIF4E to alanine which appeared to fuck overall eIF4E structure up, rather than mimic unphosphorylated eIF4E.
6. Expression of phosphomimetic Ser162/164Asp, however, resulted in inability of rapamycin to inhibit L3-driven translation.

So what do we have?
mTORC1-dependent binding of IMP2 stimulates translation of L3-IGF2 mRNA. But that's all, folks.
To prove the title of the paper "mTOR phosphorylates IMP2 to promote IGF2 mRNA translation by internal ribosomal entry" it would be nice to:

1.  Perform RNA transfections to compare efficiencies of monocistronic vs. bicistronic mRNA translation, but to assay luciferases' activity 4 hours post transfection.
2. Compare not methylated vs. unmethylated mRNAs, but m7GpppG-capped vs. ApppG-capped ones. I'm not aware of effective cytoplasmic guanylyl-transferase activity, but in cytoplasmic extracts there's plenty of it (probably it' a contamination)!
3. Include bicistronic L4 mRNA in all tests and provide adequate positive and negative coctrols.
4. If all described in the manuscript is true, it should also work in vitro, HeLa extracts are easy to purchase or even to make by yourself. Invariable DNA transfections are a bit suspicious.
5. Effect of rapamycin/IMP2 on bicistronic mRNA, bearing L3 leader between luciferases.

In fact, forget the IRES (for a while)...


1. Rousseau, D., Kaspar, R.L., Rosenwald, I., Gehrke, L. & Sonenberg, N. Translation initiation of ornithine decarboxylase and nucleocytoplasmic transport of cyclin D1 mRNA are increased in cells overexpressing eukaryotic initiation factor 4E. Proc Natl Acad Sci USA 93, 1065–1070 (1996).
2. Elango, N., Li, Y., Shivshankar, P. & Katz, M.S. Expression of RUNX2 isoforms: involvement of cap-dependent and cap-independent mechanisms of translation. J Cell Biochem 99, 1108–1121 (2006).
3. Andreev, D.E. et al. Differential contribution of the m7G-cap to the 5' end-dependent translation initiation of mammalian mRNAs. Nucleic Acids Res 37, 6135–6147 (2009).
4. Lourenço, S., Costa, F., Débarges, B., Andrieu, T. & Cahour, A. Hepatitis C virus internal ribosome entry site-mediated translation is stimulated by cis-acting RNA elements and trans-acting viral factors. FEBS J 275, 4179–4197 (2008).
5. Barreau, C., Dutertre, S., Paillard, L. & Osborne, H.B. Liposome-mediated RNA transfection should be used with caution. RNA 12, 1790–1793 (2006).