5 replies to this topic

[luc]

If I injection pri-mirna into Cytoplasm directly .The pri can be processed into a mature mirna ？

[dpo]

I wouldn't think so, the pri-miRNA is normally processed by Drosha in the nucleus, and then exported as pre-miRNA. I haven't tried it myself, so it might work anyway, but I would suggest you inject the pre-miRNA (or the mature miR) instead of the pri-miRNA if you do want to inject something in the cytoplasm.

Edited by dpo, 19 October 2010 - 10:59 PM.

[luc]

dpo, on 19 October 2010 - 10:58 PM, said:

I wouldn't think so, the pri-miRNA is normally processed by Drosha in the nucleus, and then exported as pre-miRNA. I haven't tried it myself, so it might work anyway, but I would suggest you inject the pre-miRNA (or the mature miR) instead of the pri-miRNA if you do want to inject something in the cytoplasm.

Thank you for you reply . The reason I want to do this is because I recently read an paper, Heritable and Lineage-Specific Gene Knockdown in Zebrafish Embryo . In this papper they cloned both zebrafish pri-miR-30e(409 bp) and pri-miR-155 (447 bp) genomic precursor sequences into the pCS2+ vector .Coinjection of in vitro synthesized capped pri-miR-30e mRNAs and sensor EGFP mRNAs containing two tandem perfectly complementary target sites (26PT for miR-30e binding) in its 39UTR (EGFP-26PTmir30e ) into one-cell stage embryos,can resulted in a striking decrease of both EGFP fluorescence and proteins .

[dpo]

I don't know anything about zebrafish, apart from the fact that it's a small fish

Anyway, I quickly checked the paper you are referring to. Since I don't know anything about zebrafish, I may be wrong here, but if you inject the animal in the one cell stage and you wait 24h, this means there are a lot of cell divisions going on. Now, if a cell divides, the nuclear membrane is disrupted, and reestablished after cell division. If you flood a cell with the synthetic pri-miRNA, some of it will end up in the nucleus after cell division, after which it can be processed by Drosha.

I'm just trying to find an explanation why my hypothesis (no Drosha-mediated processing in the cytoplasm) could be explained, but of course another explanation is that pri-miRNAs can be processed in the cytoplasm. Although this does not correspond to the text book picture of miRNAs, text books are not necessarily correct.

[Jon Moulton]

dpo,

I like your hypothesis that that cell division, with disruption of the nuclear membrane, allows the pre-miRNA into the nucleus from the cytoplasm. Zebrafish embryos go through very active division. I am somewhat surprised that the injected RNA is persistent enough to give a useful knockdown though embryonic development, though loading onto RISC stabilizes an miRNA against degradation.

[dpo]

My hypothesis is based on the fact that you need dividing cells if you want to transduce cells with a retrovirus. Also in this case, the viral RNA cannot enter the nucleus in an active way, but during cell division, it gets 'trapped' in the reformed nucleus. But that's what it is of course, just a hypothesis.