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.
- PKR reduces ferritin mRNA contents in heavy polysomes. This is in line with translational repression.
- 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.
- 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.
- 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.
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.
