Reader Comments
Post a new comment on this article
Post Your Discussion Comment
Please follow our guidelines for comments and review our competing interests policy. Comments that do not conform to our guidelines will be promptly removed and the user account disabled. The following must be avoided:
- Remarks that could be interpreted as allegations of misconduct
- Unsupported assertions or statements
- Inflammatory or insulting language
Thank You!
Thank you for taking the time to flag this posting; we review flagged postings on a regular basis.
closeReferee Comments: Referee 1
Posted by PLOS_ONE_Group on 31 Mar 2008 at 18:31 GMT
Referee 1's Review:
The manuscript by Lal et al., describes their study of the translational regulation of the endogenous p16 mRNA by the microRNA miR-24. The mechanism of translational repression by miRNAs is still an open question. Current data support a silencing role for miRNA at both initiation and post-initiation steps of translation. Although earlier studies tend to support either of the two possibilities, here the authors claim that miR-24 functions by repressing translation of p16 mRNA at both initiation and elongation steps.
The authors study the regulation of an endogenous mRNA in two different cell lines, where miR-24 appears to regulate translation without affecting target mRNA levels. The target p16 mRNA (which has two binding sites for miR-24) is present in active polysomes even under conditions when the protein product does not accumulate. This suggests that miR-24 acts by inhibiting translation elongation. The miRNA, miR-24 is also associated with active polysomes, as demonstrated by puromycin shift experiments. They further demonstrate that miR-24 associates with exogenous Ago1 and the target p16 mRNA in HeLa cell cultures.
Two major comments:
Significantly, there is a shift (by 1 fraction) to heavier polysomes in the absence of the miRNA (either in a specific cell line or by use of antisense oligonucleoties). The authors claim this as evidence that translation initiation is also affected. However, this shift is essentially from lighter polysomes to heavier polysomes, so would rather go with the explanation that elongation rate is affected and not initiation. It might mean that miRNAs put continuous pressure on translating ribosomes to abort and "drop-off", as suggested by Petersen et al 2006. Also, there is no great movement of RNA from the fractions 1-4 (unbound RNA) into polysomes in these experiments. The protein stability of p16 is not affected under these conditions.
The use of heterologous reporters with miR-binding site mutations add strength to their findings. However, it is not clear to this reviewer when transcription was shut off (if it was ever)? Was transcription shut off after 16 hours? Then analysis should be done at a later time-point. A bit more detail for the HeLa Tet-off experimental protocol would be good. It would be interesting to know if antisense oligonucleotides can derepress already repressed reporter mRNA.
The manuscript will definitely add to the ongoing debate on translational repression by miRNAs. It is also refreshing to see mechanistic study of endogenous mRNA targets, rather than just reporters.
Minor points:
1. The figure legends for Fig. 1A and B are interchanged.
2. Cite q-PCR quantification of miR-24 of Fig. 3A (bottom left panel) in page 6, line 6.
3. Confusion over role of translation initiation page 8, 2nd para. Page 9, 1st para. This should be clarified.
**********
N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.