3' terminal post-transcriptional processing of miRNAs? - (Jul/16/2007 )

Hello,
I've been using the Bartel protocol for cloning specific miRNAs, and after sequencing, found that several of them contain single nucleotides added to the 3' end that are not encoded in the primary sequence. You would think that there may be some slop with the ligation reaction but this isn't the case because some miRNAs are post-transcriptionally modified while others are not. Additionally no ATP is added to the ligation, only the adenylated 3' adaptor. Other groups have reported these sorts of modifications (such as Tuschl's group, Cell. 2007 Jun 29;129(7):1401-14) but few explanations are offered as to why this is occurring and what the potential functional consequences could be. Any ideas or theories as to what is going on?

Hi Mateo.
In plants miRNAs are methylated on their 3' end by a methyltransferase called HEN1. Methylation prevents the post-transcriptional addition of some (from one to a few) extra nucleotides, mostly Us. Most of the work on this topic has been done by the group of Xuemei Chen. As far as I know, animal miRNAs are not methylated. Maybe there are alternative mechanisms which protect them from 3' end modifications, but I don't know them. Perhaps such mechanisms are not working properly in your system. I think it might be interesting to have a closer look.
Andrea

Here is another reports of Untemplated nucleotide addition at 3 terminus:

J. Graham Ruby, Calvin H. Jan & David P. Bartel Intronic microRNA precursors that bypass
Drosha processing Nature 448, 83-86 (5 July 2007) doi:10.1038/nature05983