microRNA and 3' UTR - (Jan/15/2012 )
We would like to knock down candidate genes with second generation shRNAs using mir-30 backbone. As I read, the guide strand of a miRNAs is against the 3' UTR of the target mRNAs. Can I silence the target gene if I use sequences against coding region or the miRISC activity confine to 3' UTR?
An answer would be appreciated
Yes, you can design sequences to target the coding region because even the vector uses a miRNA backbone, if the guide matches perfectly with a target mRNA (no matter the target is in the coding region or 3UTR), classic RNAi will incur leading to mRNA degradation. This also answers your 2nd question of whether miRISC activity confines to 3UTR?. In addition, miRISC activity can also occur if a guide has targets in coding region or 5UTR with imperfect complementarirty. It is not very clear how general this phenomenon is because only a few papers have reported such observation. Mostly likely, it is.
Thanks for the answer!